Science Magazine 18 February 2011

CONTENTS Volume 331 Issue 6019 EDITORIAL 821 Why Bother? Engage Students in Education Reform M. Torchia Response ...

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CONTENTS

Volume 331 Issue 6019

EDITORIAL 821 Why Bother?

Engage Students in Education Reform

M. Torchia

Response

Alice S. Huang

R. Losick and D. O’Dowd

NEWS OF THE WEEK 829

A roundup of the week’s top stories

NEWS & ANALYSIS 832

Obama’s 2012 Vision Clashes With House Cuts in 2011

834

High-Priced Recruiting of Talent Abroad Raises Hackles

835 836

858

BOOKS ET AL. 859 Conservation Science

P. Kareiva and M. Marvier, reviewed by J. Fanshawe

860

Science in Ivins Case Not Ironclad, NRC Says A Quake May Have Hinted That It Was on the Way

Deﬂating the Genomic Bubble

J. P. Evans et al. >> Genome Anniversary Essays p. 872

Growth Defect Blocks Cancer and Diabetes

PERSPECTIVES 863 A Hand to Support the Implantation Window

Emerging Forensics Field May Hit Legal, Ethical Obstacles >> Genome Anniversary Essays p. 872; Science Podcast

S. C. Hewitt and K. S. Korach >> Report p. 912

864

865

Europe’s Eager Reformer Takes on Framework Funding Goliath

866

SPECIAL FEATURE 847 2010 VISUALIZATION CHALLENGE

868

LETTERS 857 Amazon Science Needs Brazilian Leadership A. C. M. Malhado

Unlocking the Door to Invasion

A. Kereszt and E. Kondorosi >> Report p. 909

A Windfall for Defenders of the Planet

>> For related online content, go to www.sciencemag.org/special/vis2010/; Science Podcast

A New Twist on Spintronics

G. L. J. A. Rikken >> Report p. 894

NASA Weighs Asteroids: Cheaper Than Moon, But Still Not Easy

844

Life on Low Flame in Hibernation

G. Heldmaier >> Report p. 906

Controlling the Flow of Suspensions

H.-J. Butt >> Report p. 897 869

Creating Stable Memories

pages 864 & 894

J. D. Sweatt >> Report p. 924 870

Hitting a Tiny Target in the Dark

Boosting CITES Through Research

R. S. Hawley >> Report p. 916

Response

CONTENTS continued >>

M. J. Smith et al.

page 841

POLICY FORUM 861

NEWS FOCUS 838 Can This DNA Sleuth Help Catch Criminals?

841

Visceral

O. Catts and I. Zurr, curators, reviewed by D. Dixon et al.

>> Research Article p. 877

837

CORRECTIONS AND CLARIFICATIONS

D. Bickford et al.

COVER False-colored atomic force micrograph of two types of molecules forming a mixed self-assembled monolayer on a gold surface. The two molecules differ in height by 0.2 nanometers, giving the appearance of ripples. This image was awarded ﬁrst place in the photography category in the 2010 Science/NSF International Science & Engineering Visualization Challenge. The winning entries are featured in a special section starting on page 847 and at www.sciencemag.org/special/vis2010/.

DEPARTMENTS 817 823 826 956 957

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

Image: Seth B. Darling, Argonne National Laboratory; and Steven J. Sibener, University of Chicago

www.sciencemag.org

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CONTENTS

ESSAYS 872 Genome-Sequencing Anniversary: A Celebration of the Genome, Part III A Living Constitution

892

B. S. Zhao et al. The helium molecule can interact at large distances with the evanescent repulsion potential of the surface.

S. Jasanoff

What Does a “Normal” Human Genome Look Like?

894

M. V. Olson

Of Mice and Humans Human Genome Sequencing: Celebrating 10 Years L. Al-Gazali

Gene Patents: The Shadow of Uncertainty

897

R. Cook-Deegan

The Genomic Foundation Is Shifting J. S. Mattick

Famine in the Presence of the Genomic Data Feast

900

E. Hoal >> News story p. 838; Policy Forum p. 861

Synthetic Clonal Reproduction Through Seeds

903

M. P. A. Marimuthu et al. Clonal reproduction is engineered in a sexual plant by manipulating conserved genes controlling meiosis.

881

906

Extended Nucleation of the 1999 Mw 7.6 Izmit Earthquake

909

912

889

Time-Reversed Lasing and Interferometric Control of Absorption W. Wan et al. Tuning the amplitude and phase of incident light can induce an enhancement of the optical absorption process.

Hibernation in Black Bears: Independence of Metabolic Suppression from Body Temperature

LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia

The Antiproliferative Action of Progesterone in Uterine Epithelium Is Mediated by Hand2

page 928

920

916

Distinct Properties of the XY Pseudoautosomal Region Crucial for Male Meiosis

924

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Early Tagging of Cortical Networks Is Required for the Formation of Enduring Associative Memory

E. Lesburguères et al. Tagging of cortical networks at the time of encoding is crucial for long-lasting associative memories. >> Perspective p. 869 928

L. Kauppi et al. Recombination between the sex chromosomes during sperm formation is controlled by a splicing isoform of the SPO11 protein. >> Perspective p. 870

Classic Selective Sweeps Were Rare in Recent Human Evolution

R. D. Hernandez et al. Much human genetic variation is likely due to purifying selection against deleterious mutations.

Q. Li et al. A progesterone-regulated transcription factor regulates stromal-epithelial communication in early pregnancy. >> Perspective p. 863

Metallic and Insulating Oxide Interfaces Controlled by Electronic Correlations

H. W. Jang et al. The strength of electronic correlations dictates the transport properties of oxide interfaces.

The Magnitude and Duration of Late Ordovician–Early Silurian Glaciation

R. Op den Camp et al. Parasponia uses a mycorrhizal signaling receptor essential for arbuscle formation to control rhizobium nodule symbiosis. >> Perspective p. 865

REPORTS 886

Steric Control of the Reaction of CH Stretch–Excited CHD3 with Chlorine Atom

Ø. Tøien et al. Hibernating black bears suppress their metabolic rate to 25% of normal, but only slightly reduce their body temperature. >> Perspective p. 866

The Crystal Structure of the Signal Recognition Particle in Complex with Its Receptor

S. F. Ataide et al. Guanine triphosphate controls changes in the signal recognition particle that facilitate transfer of the signal sequence to the translocon.

pages 868 & 897

S. Finnegan et al. Carbonate isotopes reveal a link between past ocean temperatures and mass extinction.

RESEARCH ARTICLES

M. Bouchon et al. Low-frequency seismic events may have been part of slip accumulation before a large earthquake. >> News & Analysis p. 836

Capillary Forces in Suspension Rheology

E. Koos and N. Willenbacher The addition of a small amount of a nonwetting immiscible ﬂuid to a suspension can drastically alter its rheological properties. >> Perspective p. 868

F. Wang et al. Spectroscopy elucidates the complex interplay between orientational and vibrational effects in a simple chemical reaction.

BREVIA

877

Spin Selectivity in Electron Transmission Through Self-Assembled Monolayers of Double-Stranded DNA

B. Göhler et al. Photoelectrons emitted from a DNA-covered gold surface can have an unbalanced spin population of up to 60%. >> Perspective p. 864

S. Yamanaka

876

Quantum Reﬂection of He2 Several Nanometers Above a Grating Surface

Microtubule Stabilization Reduces Scarring and Causes Axon Regeneration After Spinal Cord Injury

F. Hellal et al. Taxol stimulates the capacity of axons to grow after spinal cord injury.

CONTENTS continued >>

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CONTENTS

SCIENCEONLINE SCIENCESIGNALING SPECIAL FEATURE

Slideshow: 2010 Visualization Challenge Browse the striking sights, sounds, and motion that represent the contest’s winning entries and honorable mentions. www.sciencemag.org/special/vis2010/

SCIENCEXPRESS

www.sciencexpress.org

Crystal Structure of the Dynein Motor Domain A. P. Carter et al. The structure reveals the functional elements of the dynein motor and suggests how they change conformation during motility. 10.1126/science.1202393

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

I. Wirgin et al. Chronic pollution of the Hudson River, New York, results in rapid evolution of resistance to the pollutants. 10.1126/science.1197296 >> Science Podcast

The Biodiversity and Ecosystem Services Science-Policy Interface C. Perrings et al. 10.1126/science.1202400

Atmospheric pCO2 Perturbations Associated with the Central Atlantic Magmatic Province

M. F. Schaller et al. Emplacement of the Central Atlantic Magmatic Province 200 million years ago greatly elevated atmospheric CO2 concentrations. 10.1126/science.1199011

Revealing Extraordinary Intrinsic Tensile Plasticity in Gradient Nano-Grained Copper

www.sciencesignaling.org The Signal Transduction Knowledge Environment 15 February issue: http://scim.ag/ss15Feb11

COMMENTARY: Thinking Outside the Box— Fostering Innovation and Non–Hypothesis-Driven Research at NIH

EDITORIAL GUIDE: Focus Issue— Conquering the Data Mountain

RESEARCH ARTICLE: Growth Hormone Receptor Deﬁciency Is Associated with a Major Reduction in Pro-Aging Signaling, Cancer, and Diabetes in Humans

R. Ahmad et al. An inﬂammatory response of epithelial cells may be co-opted to promote cancer cell survival.

N. R. Gough and M. B. Yaffe Massive data sets create challenges for annotation, sharing, and presentation.

PERSPECTIVE: Effective Representation and Storage of Mass Spectrometry–Based Proteomic Data Sets for the Scientiﬁc Community

PERSPECTIVE: The Potential Cost of High-Throughput Proteomics

PERSPECTIVE: For Placebo Effects in Medicine, Seeing Is Believing

PERSPECTIVE: Integrating Multiple Types of Data for Signaling Research—Challenges and Opportunities

SCIENCEPODCAST

H. S. Wiley Easy integration of biological data has enormous potential to drive advances in biology and medicine.

On the 18 February Science Podcast: pollutant-resistant ﬁsh, forensic genetics, the 2010 Visualization Challenge, and more.

PERSPECTIVE: Setting the Standards for Signal Transduction Research

SCIENCEINSIDER

F. M. White The production of false leads by automated curation of high-throughput proteomic data sets exerts a toll on biological research and advancement.

J. Saez-Rodriguez et al. Standardized methods to link large, complex data sets to models are needed.

SCIENCECAREERS

Do Vibrating Molecules Give Us Our Sense of Smell?

A controversial new study ﬂies in the face of a century of physiological research. http://scim.ag/sense-smell

The Mystery of the Stone Scorpion

Chemical bonds hold clues to how ﬂexible arthropod exoskeletons can turn into fossils. http://scim.ag/stone-scorpion

J. Guevara-Aguirre et al. Ecuadorians who have a genetic mutation in the growth hormone receptor almost never die of cancer or diabetes complications.

RESEARCH ARTICLE: The Effect of Treatment Expectation on Drug Efﬁcacy—Imaging the Analgesic Beneﬁt of the Opioid Remifentanil

SCIENCENOW

Geothermal activity may sustain hidden oceans on worlds ejected from their planetary systems. http://scim.ag/loneplanet

R. Aragon NIH programs that fund the development of high-risk technologies contribute to translational research.

J. V. Olsen and M. Mann Proteomics data need a “reliability” tag to allow scientists to discriminate among data sets in public repositories.

PRESENTATION: Visual Representation of Scientiﬁc Information

Outcast Planets Could Support Life

www.sciencetranslationalmedicine.org Integrating Medicine and Science 16 February issue: http://scim.ag/stm021611

RESEARCH ARTICLE: MUC1-C Onocoprotein Promotes STAT3 Activation in an Auto-Inductive Regulatory Loop

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. 10.1126/science.1200177 www.sciencenow.org Highlights From Our Daily News Coverage

SCIENCETRANSLATIONAL MEDICINE

B. Wong Judicious choice of graphical representations can improve visual communication. www.sciencecareers.org/career_magazine Free Career Resources for Scientists

Tooling Up: Playing the Name Game

D. Jensen Need to ﬁnd out who’s who inside a company? Here’s how the pros do it. http://scim.ag/fhopnC

Seeing Is Believing

J. Carpenter Trained as a chemical engineer, Jon Heras has moved on to become a professional science illustrator and animator. http://scim.ag/i06erj

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U. Bingel et al.

R. L. Gollub and J. Kong Non-invasive whole-brain imaging can enhance our understanding of brain-based placebo effects. www.sciencemag.org/multimedia/podcast Free Weekly Show

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

SPECIAL SERIES

http://scim.ag/genome10 Human Genome 10th Anniversary A special month-long series explores the impacts of the genomics revolution on science and society. 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

Membrane-Docking Complex

CREDITS (TOP TO BOTTOM): ØIVIND TØIEN/INSTITUTE OF ARCTIC BIOLOGY, UNIVERSITY OF ALASKA, FAIRBANKS; BOUCHON ET AL.

Don’t Wake the Bears

In hibernating mammals, temperature and metabolic rate decrease, allowing survival over long winter periods of starvation and cold. In small hibernators, such as hamsters, decreases in metabolic rate and temperature are linked; temperature decreases follow a physiological reduction in metabolic rate. In bears, body temperature remains relatively high during hibernation, which has been assumed to be owing to their large size. Tøien et al. (p. 906; see the Perspective by Heldmaier) undertook the challenging task of maintaining hibernating bears in artiﬁcial conditions and found, surprisingly, that metabolic rate and body temperature were completely unlinked. Instead, the bears cycled through elevated temperatures (between 30° to 36°C) over multiday cycles throughout hibernation, while undergoing a fourfold reduction in heart rate and maintaining a metabolic rate at 25% of normal. Despite a return to active body temperatures, metabolic rate remained depressed for several weeks post-hibernation.

Mixing Sand and Water The ﬂow properties of a suspension of sand in water will be controlled by how much sand is added and will change over time as the sand sediments out. Koos and Willenbacher (p. 897; see the Perspective by Butt) studied the agglomeration of particles suspended in a liquid under the inﬂuence of a small amount of a secondary, immiscible liquid. For the case where

Correct targeting of membrane and secretory proteins involves the binding of the signal recognition particle (SRP), a ribonucleoprotein complex, to the ribosome nascent chain (RNC) complex. This joint complex then binds to a receptor (SR) in the target membrane and delivers the protein cargo to the protein translocation machinery, the translocon, in a guanosine triphosphate (GTP)–dependent process. Ataide et al. (p. 881) now describe the structure of the eubacterial SRP:SR complex bound to a nonhydrolysable GTP analog. The structure shows the arrangement of the SRP proteins relative to the RNA in a conformation that is likely to represent the cargo release state, and gives insight into how GTP hydrolysis may be coupled to conformational changes that facilitate transfer of the signal sequence to the translocon.

Lasing in Reverse Recent theoretical work has shown that the time-reversal symmetry of electromagnetism allows a lasing process to be run backwards, so that photons of the correct amplitude and phase incident on a cavity medium can be coherently absorbed. Wan et al. (p. 889) experimentally demonstrate that such carefully tuned beams incident from either side on a silicon wafer multiply scatter within the wafer so that the total transmitted and reﬂected beams destructively interfere, leading to the predicted enhanced absorption within the silicon cavity. The effect provides a route to control absorption through coherent illumination, with numerous potential applications in optical circuitry.

Turned Away at a Distance When a gas-phase molecule scatters off a solid surface, the interaction potential attracts at a distance because of favorable alignment of dipoles in the molecule and the surface. At a certain point, the molecule gets close enough to the surface so that electron repulsions dominate and the molecule is repelled. In many ways, the scattering of molecules can largely be described as classical scattering off these potential surfaces. Zhao et al. (p. 892) now show that the

www.sciencemag.org SCIENCE VOL 331 Published by AAAS

helium dimer, an extremely weak molecule, can scatter off a grating intact, but does so several nanometers above the surface, when it is still in the attractive part of the interaction potential. The scattering is quantum mechanical in nature, with the wave functions of the repulsive potential and the dimer extending evanescently and interacting at a distance.

Spin Filtering with DNA Monolayers Spin ﬁlters can create an excess of one electron spin population over another with applications in spin-based electronics. Typically, such devices use magnetic materials. Göhler et al. (p. 894; see the Perspective by Rikken) measured the spin distribution of photoelectrons emitted from layers of double-stranded DNA (up to 80 base pairs in length) adsorbed on gold. Excitation with unpolarized ultraviolet light at room temperature led to a 60% increase in spin polarization.

Downloaded from www.sciencemag.org on February 17, 2011

the second liquid preferentially wets the solid particles, one would expect the ﬂow properties to change, but signiﬁcant changes were also observed even when a small amount of a nonwetting ﬂuid was added to the mix.

Prelude to an Earthquake? To improve warning systems and minimize damage, it is important to understand the moments leading up to large earthquakes. Bouchon et al. (p. 877) observed a persistent and repeating low-frequency seismic signal in the hour before the 1999 magnitude 7.6 Izmit earthquake in Turkey that may have been the beginning stages of the major slip along the fault. The signal originated from the base of the brittle crust near the hypocenter, suggesting that slip accumulated leading up to the earthquake. The increase of seismic noise over this time frame indicates possible movement along the fault before the main quake. It is not yet clear whether similar patterns are likely to have occurred before other large ruptures, or whether such patterns occur in the absence of any subsequent rupture.

Frozen to Death Global biodiversity throughout geologic time has been punctuated by mass extinction events, the causes of which are often controversial. Major swings in climate are often accompanied by variations in temperature, sea level, and glacial coverage. Finnegan et al. (p. 903, published

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This Week in Science Continued from page 817

online 27 January) used an isotope-based paleothermometry technique to tease out ocean temperatures during one of the most devastating losses of marine life around 440 to 450 million years ago. Ocean temperatures cooled by up to 5°C in the tropics at the same time that the mass extinction was in full swing, which also coincides with a disruption of the marine carbon cycle.

Many plants form symbiotic relationships with mycorrhizal fungi. A much smaller group of plants, mostly the legumes, form symbiotic relationships with rhizobial bacteria. Op den Camp et al. (p. 909, published online 23 December; see the Perspective by Kereszt and Kondorosi) analyzed the molecular pathways underlying the early stages of mycorrhizal and rhizobial symbioses. The nonleguminous small tree Parasponia can form symbiotic relationships with rhizobia. Comparisons of the nodulation pathways in legumes with that in Parasponia suggest that the rhizobial pathway is derived from the mycorrhizal pathway.

Progesterone-Estrogen Antagonism In reproductive biology, the steroid hormone progesterone counteracts estrogen-induced uterine growth. The antiproliferative action of progesterone in the uterus relates to female infertility as well as to estrogen-driven hyperplasia and endometrial cancer. Using targeted genetic mutation in mice, Li et al. (p. 912; see the Perspective by Hewitt and Korach) demonstrate that the transcription factor Hand2 is a target of progesterone regulation in the uterine stroma and that Hand2 controls estrogen-induced epithelial proliferation via a paracrine mechanism involving ﬁbroblast growth factors.

Minding Your Xs and Ys The double (or “reduction”) division of meiosis generates haploid gametes from diploid cells. Meiotic recombination is required for pairing of homologous chromosomes, which ensures correct segregation of the chromosomes. The X and Y chromosomes of mammals have only a small segment of homology, the pseudoautosomal region, where recombination must take place to hold the chromosomes together. Although sex chromosomes missegregate more frequently than autosomes, X-Y nondisjunction is rare, which suggests that mechanisms exist to ensure X-Y recombination. Kauppi et al. (p. 916; see the Perspective by Hawley) show that, in mice, a distinct chromosome structure renders the PAR more conducive to recombination-initiating double-strand break formation, which occur at a 10- to-20-fold higher frequency than the genome average.

Mastering Memory Maintenance The mechanisms involved in the formation of enduring memories are still largely unknown. Lesburguères et al. (p. 924; see the Perspective by Sweatt) investigated the hippocampal-cortical dialogue during memory consolidation. Neurons in the rat orbitofrontal cortex appeared to be “tagged” at the time of initial encoding, even though the initial learning was hippocampal. Inactivating the orbitofrontal cortex at the time of encoding affected late memory while not affecting memory during the period when it was still supported by the hippocampus.

CREDIT: OP DEN CAMP ET AL.

Taxol and Spinal Cord Repair Because neuron-intrinsic factors and the lesion site itself impose obstacles to axon regeneration, a combination of therapeutic approaches is required to promote axonal regeneration after spinal injury. Hellal et al. (p. 928, published online 27 January) now provide evidence from spinal cord injury experiments on rats that the licensed anticancer drug Taxol can induce axon regeneration by altering microtubule dynamics. Moderate microtubule stabilization reduced scarring and decreased the deposition of factors that prevent axonal regeneration after spinal cord injury so that axons could regenerate through the Taxol-treated lesion site. www.sciencemag.org SCIENCE VOL 331

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Two Ways to Plant Symbiosis

EDITORIAL

Why Bother?

CREDITS: (TOP) AAAS; (RIGHT) ROBERT LEHMANN/GETTY IMAGES

WHEN WE MENTION WOMEN IN SCIENCE AND ENGINEERING, IT IS OFTEN ABOUT THE DIMINISHING

numbers, the lower pay, the many difﬁculties for women, and the personal sacriﬁces that women necessarily make. Perhaps, by focusing on the negatives, we are unwittingly persuading young women that science and engineering may not be the right careers for them. Why bother to join this profession? There are many rewards. The wealthy parents of John Enders, a Nobel Laureate of the 1950s, could not believe that he was being paid to play in the laboratory. He had the freedom to be curious. Carol Reiss, a New York University professor, described this freedom to me: “It is the ability to ask questions and develop experiments to answer them, learning something new which no one else has previously found. Yes, I would deﬁnitely do it again. Science is a very rewarding and gratifying profession, not a job.” Moreover, scientiﬁc work contributes to furthering our understanding of the universe and our place in it. Scientists feel uniquely relevant. In the past, women often received friendly advice to accept less demanding positions, such as research assistant or associate rather than that of professor. It was argued that taking such positions would give women the ﬂexibility for domestic responsibilities and childbirth. Also, there would be no need to search for funding. However, many professors and directors set their own schedule for getting the job done. Financial responsibility may indeed be a hassle, especially now, but having that responsibility also means deciding how that money is spent. Assistants and technicians can be assigned the routine functions, giving additional ﬂexibility to the laboratory chief. Except for teaching, academic scientists and engineers have great ﬂexibility in scheduling, although this might mean working well into the evening hours and weekends. Few other jobs provide such ﬂexibility. Although time-consuming, teaching has special rewards. Another professor (Amy Cheng Vollmer, Swarthmore College) remarked to me: “Being a mentor—in the classroom [or] laboratory . . . has been the most rewarding—seeing former students become successful . . . and developing balance in their professional and personal lives.” Not often mentioned but taken for granted, scientists and engineers mainly interact with smart, capable individuals, making it a pleasure to work together. Sometimes achieving “balance” is elusive for women. Many women feel they have to decide between a career and family. Rosalind Franklin and Barbara McClintock chose not to have children in favor of science. However, for women who can juggle the demands of a profession and a family, there are special rewards of motherhood. As a young investigator (Theresa Chang, Public Health Research Institute, University of Medicine and Dentistry, New Jersey) told me, “many female colleagues . . . chose not to have children to be competitive. Having children is the best thing that I have done. I am more effective at work and integrate my skill and knowledge into the society through my involvement in the school.” To be a mother and a scientist is well worth the effort. And should it be that after training, a woman decides that academic life is not for her, that training provides an open door to a wide variety of options. Scientists are prepared to solve problems, almost any kind. She can analyze new situations and immerse herself in learning about them. She is able to provide solutions and leadership. There are jobs in industry, on Wall Street, in government, in philanthropy, in administration, in national parks, in advocacy, in defense, and numerous others areas. There is nothing to lose and everything to gain. Yes, it is worth the bother!

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Alice S. Huang is the president of AAAS, Senior Faculty Associate at the California Institute of Technology, former Dean for Science at New York University, and former professor of Microbiology and Molecular Genetics at Harvard Medical School. E-mail: alice. [email protected].

– Alice S. Huang

10.1126/science.1203124

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EDITORS’CHOICE EDITED BY KRISTEN MUELLER AND JAKE YESTON

ENGINEERING

The Pros (and Cons) of Plugging In

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The adage out of sight, out of mind has some resonance as the ﬁrst big crop of plug-in hybrid cars hits the road in the United States. People see and sometimes smell gasoline; plugging a car into a socket may make it seem like the energy is conjured from the ether. Of course, power plants actually bear the burden, and Peterson et al. are among the growing number of researchers gauging the implications. They have examined the net effect on carbon, nitrogen, and sulfur emissions of replacing a fraction of the cars in a number of Eastern and Midwestern U.S. states with plug-in hybrids. They modeled several different scenarios, such as when cars were charged and whether carbon dioxide emissions were priced or captured and sequestered. For a 10% hybrid ﬂeet scenario, they found signiﬁcant reductions in CO2 emissions across the board, and NOx reductions in most cases. The principal drawback was an increase in sulfur dioxide emissions as demand for coal combustion rose. — JSY Environ. Sci. Technol. 45, 10.1021/es102464y (2011).

APPLIED PHYSICS

CREDITS (TOP TO BOTTOM):RAMIN TALAIE/CORBIS; DAVID LINDENMAYER

A Sharper Focus Imaging techniques are rarely applied in ideal environments. Ultrasound imaging, for instance, is hindered by scattering from tissue, bone, and other surrounding organs, which often blur the images. The same is true for terahertz body scanners, wherein scattering from clothing or designed concealers may obscure detection. Scattering from random scatterers creates speckle noise in the backscattered signal that results in an overall deterioration in image quality. Montaldo et al. show that this speckle noise can be controlled and mitigated by a time-reversed scattering process using a time-domain array of transmitters and receivers. By sending in pulses of ultrasound from the array and tweaking the timing at each array element, the signals at the receivers are fed back to the transmitters, and the timing is iteratively adjusted to effectively steer the pulses to focus on a desired point. The improved imaging capability is demonstrated in a phantom for ultrasound but should be extendable to other frequency regimes for radar and communication applications. — ISO Phys. Rev. Lett. 106, 54301 (2011). ECOLOGY

Brother, Can You Share a Hollow? Tree hollows are a key resource for numerous forest-dwelling species. Hollow reductions that result from forest harvest and management

practices are a signiﬁcant threat to forest species worldwide. Although the reduction of hollows has generally been followed by a decline in dependent species, two species of Australian possum (Gymnobelidus leadbeateri and Trichosurus cunninghami) have not followed this trend. By tracking the denning behavior of individuals of known genetic relatedness, Banks et al. show that this resilience is largely due to a change in social behavior. When hollows are plentiful, possums use a large number of dens, have overlapping home ranges, and prefer to share hollows with unrelated individuals. When hollows are scarce, the animals show increased aggression and defense of hollows and instead prefer to nest with relatives. Thus, when resources are scarce, animals obtain an inclusive ﬁtness beneﬁt of sharing with relatives and excluding nonrelatives, whereas when resources are plentiful, inclusive ﬁtness is less important than other considerations, such as inbreeding or pathogen avoidance. — SNV

CHEMISTRY

Happy Couple In principle, dimeric molecules may seem easier to synthesize than lower-symmetry compounds of comparable size. After all, both halves can be generated together and then conveniently coupled at the end. In practice, the coupling step often poses a tremendous challenge—all the more frustrating because it comes after long and meticulous preparation of the penultimate monomer. Such was the case with the polycyclic core of lomaiviticin, an organic natural product of exploratory pharmaceutical interest. The compound comprises two identical tetracyclic frameworks linked by a C-C bond and two C-O bonds; an especially unusual feature is the presence in each half of a diazo group, ordinarily a highly reactive substituent. Herzon et al. have now succeeded in assembling the lomaiviticin framework (without the pendant sugars in the natural product) using 11 steps, perhaps the hardest of which proved to be the formation of that central C-C bond. After an extensive search, the authors discovered that a trivalent manganese complex bearing three ﬂuorinated acetylacetonate ligands was a uniquely selec-

Proc. R. Soc. London Ser. B 278, 10.1098/ rspb.2010.2657 (2011).

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Continued on page 825

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EDITORS’CHOICE Continued from page 823

J. Am. Chem. Soc. 133, 10.1021/ja200034b (2011). PLANT SCIENCES

Food Fight During reproduction, some genes in the offspring are “imprinted”: They contain inherited epigenetic markings that promote speciﬁc expression of either the maternal or paternal allele. Genetic imprinting can be viewed within the parental conﬂict theory, which postulates that females allocate resources equally to all offspring, whereas males favor the expression of genes that maximize resource use by individual offspring. In Arabidopsis, a few imprinted genes are known to be expressed in the endoderm, which is the portion of the seed that will nourish the growing embryo. Hsieh et al. have now surveyed the gene expression landscape of the Arabidopsis endoderm and found 43 imprinted genes in the endoderm, 34 maternally expressed and 9 paternally expressed. The genes that maternal and paternal sources disagreed over encode transcription factors, hormone signaling components, and regulators of chromatin modiﬁcation and small RNA pathways. The Arabidopsis embryo, on the other hand, reﬂected an inner peace, with no imprinted genes identiﬁed. — PJH Proc. Natl. Acad. Sci. U.S.A. 108, 1755 (2011). DEVELOPMENT

CREDIT: STSCI

No Damaged Daddies Cells have evolved mechanisms to protect against damaged DNA, including the induction of apoptotic cell death. Such protection is especially important in germline cells in order to ensure the evolutionary stability of a species. The p53 homolog, p63, functions to protect the female germ line by promoting apoptosis of oocytes with damaged DNA. Beyer et al. sought to determine whether p63 also functions in the male germ line and identiﬁed p63 isoforms that are expressed in the human testis. Male germ cell–associated transcriptionally active p63 (GTAp63) is encoded by the p63 gene with a long terminal repeat (LTR), the result of an integration event of the endogenous retrovirus ERV9 LTR, inserted upstream. Spermatogenic precursors, but not mature spermatozoa, expressed GTAp63, which induced www.sciencemag.org

the expression of proapoptotic genes upon DNA damage. Analysis of primate DNA showed conservation of the LTR insertion in great apes and humans, which suggests that the insertion occurred recently in evolution. Besides ensuring germline genomic integrity, p63 may also act as a tumor suppressor: Examination of tissue from human testicular cancers revealed a loss of p63 expression. — BAP Proc. Natl. Acad. Sci. U.S.A. 108, 10.1073/ pnas.1016201108 (2011). ASTROPHYSICS

Pulsing Once Again Just as seismic waves allow scientists to study Earth’s interior, stellar oscillations can tell us about the interior of stars. Cataclysmic variables are binary systems in which an extremely dense star, or white dwarf, accretes material from

another star; in a dozen such cases so far, the white dwarf is known to pulsate. As their name suggests, cataclysmic variables can experience sudden outbursts—with the brightness of the entire system sometimes increasing by several orders of magnitude—when the accreted material undergoes thermonuclear fusion on the white dwarf’s surface. The extent to which these events affect the white dwarf’s interior is not completely understood. The white dwarf in the cataclysmic variable SDSS J074531.92+453829.6 was shown to have stopped pulsating 1 year after its ﬁrst recorded outburst, suggesting that it was heated to temperatures beyond those at which pulsations can occur. Now, 3 years after the outburst, Mukadam et al. show that the pulsations have resumed and that their properties, which depend on fundamental parameters such as the density, pressure, and temperature in the stellar interior, are similar to those of the pre-outburst pulsations. This white dwarf has thus cooled down to its previous state, with no signs of its interior having been perturbed by the outburst. — MJC

SCIENCE

Astrophys. J. 728, L33 (2011).

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tive oxidant for the coupling reaction (though the bacterial solution to the same challenge remains somewhat mysterious). — JSY

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NEWS OF THE WEEK AROUND THE WORLD 3

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Dhaka, Bangladesh 4 Washington, D.C. 1

CREDITS (TOP TO BOTTOM): FRITZ GELLER-GRIMM/WIKIMEDIA COMMONS; ASOCIACIÓN ANDES

Dueling Science Budgets President Barack Obama sent the U.S. science community a valentine this week, submitting a 2012 budget request to Congress that would increase federal support for basic and applied research by 11% over current spending levels, to $66 billion. It ﬂeshes out his promise in last month’s State of the Union speech to “out-innovate, out-educate, and out-build” the rest of the world. The plan is a long way from becoming reality, however. In addition to the usual hurdles facing any presidential budget, there’s a new wrinkle this year: Congress has yet to ﬁnish work on a 2011 budget for the ﬁscal year that ends in September. Toward that end, Republicans in the House of Representatives have proposed cutting $3.3 billion in research from current spending at various federal agencies, including $1.6 billion from the National Institutes of Health, nearly $900 million from the Department of Energy’s Ofﬁce of Science, and $388 million from the National Science Foundation. Those cuts are part of a campaign to shrink the federal deﬁcit, which both political parties agree is a necessity. But they disagree strongly on how to do it. For details, see page 832. Bilaspur, Chhattisgarh, India 2

Court Ignores Nobelists’ Plea to Release Physician-Activist

An appeal by 45 of the world’s top scientists to release an Indian doctor accused of helping Maoist insurgents has failed to sway the High Court in India’s central state of Chhattisgarh. Binayak Sen, the vice president of India’s People’s Union for Civil Liberties, was arrested in 2007 and accused of aiding

local Maoist rebels, including smuggling notes from one of them who was held in jail. He was initially released but was convicted of sedition and sentenced to life in prison on 24 December 2010. In a petition authored by chemistry Nobel laureate and Rice University professor emeritus Robert Curl and released to Indian media, the Nobelists ask that Sen be released on bail, stating that he is an “exceptional, courageous, and selﬂess colleague, dedicated to helping those in India who are least able to help themselves.” Sen’s lawyers have said that they will appeal to India’s Supreme Court. http://scim.ag/sen-appeal Cusco, Peru, and Svalbard, Norway 3

Tubers to Seek Safety on Ice “Potato preservationists” with the Global Crop Diversity Trust plan to send seeds of more than 1500 types of Andean potato to the Svalbard Global Seed Vault, 1300 kilometers north of the Arctic Circle. The seeds come from Potato Park, a 10,000-hectare reserve in Peru established by indigenous groups to preserve potatoes and their role in local culture. In the Andes, potatoes are a cultural symbol and dietary staple that come in thousands of varieties, including the red moro boli (pictured) and the “bride’s potato,” which is so bumpy it’s considered a test of a newlywed’s peeling skills. But global warming and a declining number of varieties under cultivation may threaten the tuber’s diversity. “Sending this collection to Svalbard is like sending our family members to a distant place for safekeeping, in case the rest of us need to be rescued by them in the future,” Potato Park agronomist Alejandro Argumedo said in a statement.

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Emerging Virus Strikes Again A killer is back with a vengeance in South Asia. Over the past 10 years, Nipah virus has haunted villages in western Bangladesh almost every winter and early spring, killing approximately 70% of the people it infects. The virus doesn’t seem to spread easily to humans, so the toll has been low—usually less than a dozen deaths each year nationwide. But this season is shaping up to be bad: So far, Bangladesh health authorities have attributed 24 deaths to the virus, whose hallmark symptoms are fever and encephalitis. Fruit bats are thought to be the viral reservoir, and in Bangladesh authorities suspect that the virus is transmitted mainly when people drink raw date palm sap tainted with bat saliva or feces. There is no vaccine against the virus. Cusco, Peru 5

Yale to Return Machu Picchu Artifacts

Ending a bitter dispute, Yale University will return to Peru thousands of items excavated from Machu Picchu by 20th century explorer Hiram Bingham. In an agreement signed 11 February, Yale and the Universidad Nacional de San Antonio Abad del Cusco said they would create a new museum and research center in an Inca palace in Cusco, Peru, to display and house thousands of artifacts, including pottery, stone tools, and human bones. Peru had sued the university, and last November Peruvian President Alan Garcia called Yale’s refusal to return the artifacts a “global crime.” “Yale has done the right thing probably if they want a quiet life,” said archaeologist Colin Renfrew of the University of Cambridge in the United Kingdom. http://scim.ag/yale-peru

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NEWS OF THE WEEK FINDINGS

Fetal Surgery Success Fetal surgery can help unborn babies with spina biﬁda—but at a price. Although most fetal surgeries focus on fatal malformations, spina biﬁda is an exception: Children with it often have trouble walking and controlling bladder and bowel functions, but they rarely die. Because of the risks to mother and baby, surgeons felt it was especially important to test spina biﬁda fetal surgery in a big clinical trial. Last week, a team led by N. Scott Adzick of Children’s Hospital of Philadelphia reported online in The New England Journal of Medicine that among 158 moth-

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New Stem Cell Lab Designed to Inspire A stem cell research building opened 9 February at the University of California, San Francisco (UCSF), is generating oohs and ahhs from scientists and architecture buffs alike. The $123 million Ray and Dagmar Dolby Regeneration Medicine Building, which will headquarter the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, snakes along a 65˚ slope and features four split-level ﬂoors with terraced roofs planted with native grasses. Labs with open ﬂoor plans, interspersed with ofﬁces and lounge areas, are meant to foster interaction among its 300 researchers. It seems to be working, says the center’s director, Arnold Kriegstein: “People are already congregating in the lunchrooms and in the hallways and striking up collaborations.” ers and their babies, those who were operated on before birth fared better than those who got surgery immediately after. Forty percent of the fetal surgery group received shunts to drain ﬂuid from the brain, and 42% could walk without help from devices. That compared with 82% in the control group who got shunts, and 21% who could walk without assistance. But nearly half of the babies who experienced fetal surgery were born quite prematurely, and there were other risks to mom and baby, too. Some say it may take years, tracking the children as they grow, to really understand when and whether the surgery is worth it. http://scim.ag/fetal-surgery

Probing the Secrets of Prostate Tumors A group of researchers has unveiled the ﬁrst whole genome sequences of prostate tumors. Their results, published online last week in Nature, may lead to the development of more efﬁcient, less invasive ways to diagnose and treat this cancer.

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Sequencing the whole genome of a prostate tumor, says co-author Levi Garraway of Harvard Medical School in Boston, allowed the researchers to see “biology that would have been invisible with any other method.” Most genetic analyses of tumors have focused on ﬁguring out which mutations turn normal cells cancerous and how these “spelling errors” foster tumor growth. But by sequencing the entire genomes of seven prostate tumors and comparing them with genomes from the patients’

NOTED >>The U.S. Department of Health and

Human Services’ Office of Research Integrity has produced an interactive online movie called The Lab. The chooseyour-own-adventure-style film allows viewers to play as one of four characters, including an insecure postdoc and a promising young graduate student, to avert a case of scientiﬁc misconduct. http://scim.ag/labmovie

www.sciencemag.org

Downloaded from www.sciencemag.org on February 17, 2011

The century-old surgical practice of removing lymph nodes from breast cancer patients is likely unnecessary, says a new study in the 9 February issue of The Journal of the American Medical Association. Conventional wisdom has been that after a biopsy from a sentinel lymph node ﬁnds cancer, removal of all the armpit lymph nodes before they can spread the disease throughout the body would increase survival. But researchers at John Wayne Cancer Institute in Santa Monica, California, and other institutions found no difference in survival or clinical outcomes in a randomized trial of 891 women with early-stage breast cancer who were undergoing chemotherapy or radiation. Half of the women had only the sentinel node removed, whereas the others had all their lymph nodes removed. The authors credit the results to aggressive therapy that wiped out the cancer before it could spread from the lymph nodes. They recommend that physicians reconsider the need for the painful surgery, which can have complications such as swelling and infection—although persuading the ﬁeld to change could be tough because the practice is so ingrained. Even so, several cancer clinics such as Memorial Sloan-Kettering, where some of the trials took place, have already started to implement the new standard.

CREDITS (TOP TO BOTTOM): © BRUCE DAMONTE/UCSF; ILLUSTRATION COURTESY OF THE CENTER FOR FETAL DIAGNOSIS AND TREATMENT AT THE CHILDREN’S HOSPITAL OF PHILADELPHIA

Lymph Node Surgery Unnecessary for Early Breast Cancer

NEWS planet between 1/10 and 10 times the size of Earth. They calculated that a planet with Earth’s composition of rock and water but three times as big would, in spite of losing heat through its icy shell, generate enough warmth to maintain a hidden ocean. A planet with much more water, they say, would only need to be one-third Earth’s size. David Ehrenreich, a planetary scientist at the Joseph Fourier University in Grenoble, France, calls the study interesting but notes that life buried under an ice sheet would be hard to detect. So would the planet itself, admits Switzer: Rogue planets are currently visible only within about 100 billion miles of Earth, where the probability of one existing is just one in a billion. http://scim.ag/lone-planet

Outcast Planets Could Support Life

Computer scientist Geoffrey Hinton of the University of Toronto has carried off Canada’s most prestigious science prize—along with a $1 million grant to support his research over the next 5 years. Hinton was awarded the Gerhard Herzberg Canada Gold Medal for Science and Engineering on 14 February for his work on machine learning. British-born Hinton works

CREDITS (TOP TO BOTTOM): (SOURCE) AAAS R&D BUDGET AND POLICY PROGRAM; NSERC; ANDREW ELLISON/BUMC

http://scim.ag/tumor-genes

Many astronomers assume a planet that harbors life would have to be warm and wet like Earth. Now, in a paper submitted to The Astrophysical Journal Letters, Dorian Abbot and Eric Switzer of the University of Chicago in Illinois suggest that a planet that has been slingshotted into the cold of outer space could also support life—in a hidden ocean under a blanket of ice, kept warm by geothermal activity. Abbot and Switzer simulated a lone

NEWSMAKER

Machine Learning Nets Canada’s Top Science Prize

BY THE NUMBERS

The budget for U.S. science got more complicated this week when President Barack Obama submitted his 2012 spending plan. Here are three starting points for the coming debate over how much the country should invest in nondefense research:

$66.8 billion The amount

President Barack Obama has requested for 2012, a 6.5% increase over current spending levels.

Downloaded from www.sciencemag.org on February 17, 2011

normal cells, the researchers discovered an unexpected phenomenon. Rather than single spelling errors, the tumors had long “paragraphs” of DNA that seem to have broken off and moved to another part of the genome. The chunks of DNA that broke off all contained genes that help drive cancer progression, and they moved to prime locations in the genome where they would be most active. The authors note that they need to sequence more prostate tumors to learn how frequently different mutations occur and which of the rearranged genes drive cancer. Once researchers learn which genes may be markers for cancer, says co-author Mark Rubin, a pathologist at Weill Cornell Medical College in New York City, sequencing technology might be able to detect them in a blood or urine test, replacing an invasive prostate exam or biopsy.

$62.7 billion Current spend-

ing under a so-called continuing resolution for 2011 that expires on 4 March.

$58.3 billion The amount

Republicans have budgeted in 2011 as part of a government-wide spending plan being debated this week by the House of Representatives. on neural networks and their application to processes such as monitoring industrial plants for improved safety and systems for voice recognition. The prize, named after the Nobel Prize-winning chemist, is awarded by the Natural Sciences and Engineering Research Council of Canada.

Random Sample

Art From Produce Using the same MRI machines with which he conducts brain scans by day, Andrew Ellison, a technologist at Boston University School of Medicine, spends his evenings scanning fruit and vegetables. The resulting ethereal videos are produced as the scanner passes through the skin, pith, and ﬂesh of everyday edibles. Ellison initially used an orange as a quality control. “A problem with the scanner would show itself with most fruits and veggies,” he explains. But, fascinated by the orange’s ﬂeshy insides, Ellison began to scour the markets for other fruit and vegetables to scan. Encouraged by his colleagues’ enthusiasm for his new art form, he started to post his videos to a blog (http://insideinsides.blogspot.com) that has received more than 500,000 hits since July 2010. Ellison has now racked up 36 different scans and is looking for more-exotic produce. “No one seems to be as amazed and moved by the artichoke as I am,” he says, “but everyone has a favorite.” (See also the Visualization section of this special issue on p. 848.)

www.sciencemag.org

SCIENCE

MRI scans of peapods, a watermelon, black raspberries, and a persimmon (clockwise, from top left).

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NEWS OF THE WEEK FINDINGS

Fetal Surgery Success Fetal surgery can help unborn babies with spina biﬁda—but at a price. Although most fetal surgeries focus on fatal malformations, spina biﬁda is an exception: Children with it often have trouble walking and controlling bladder and bowel functions, but they rarely die. Because of the risks to mother and baby, surgeons felt it was especially important to test spina biﬁda fetal surgery in a big clinical trial. Last week, a team led by N. Scott Adzick of Children’s Hospital of Philadelphia reported online in The New England Journal of Medicine that among 158 moth-

830

New Stem Cell Lab Designed to Inspire A stem cell research building opened 9 February at the University of California, San Francisco (UCSF), is generating oohs and ahhs from scientists and architecture buffs alike. The $123 million Ray and Dagmar Dolby Regeneration Medicine Building, which will headquarter the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, snakes along a 65˚ slope and features four split-level ﬂoors with terraced roofs planted with native grasses. Labs with open ﬂoor plans, interspersed with ofﬁces and lounge areas, are meant to foster interaction among its 300 researchers. It seems to be working, says the center’s director, Arnold Kriegstein: “People are already congregating in the lunchrooms and in the hallways and striking up collaborations.” ers and their babies, those who were operated on before birth fared better than those who got surgery immediately after. Forty percent of the fetal surgery group received shunts to drain ﬂuid from the brain, and 42% could walk without help from devices. That compared with 82% in the control group who got shunts, and 21% who could walk without assistance. But nearly half of the babies who experienced fetal surgery were born quite prematurely, and there were other risks to mom and baby, too. Some say it may take years, tracking the children as they grow, to really understand when and whether the surgery is worth it. http://scim.ag/fetal-surgery

Probing the Secrets of Prostate Tumors A group of researchers has unveiled the ﬁrst whole genome sequences of prostate tumors. Their results, published online last week in Nature, may lead to the development of more efﬁcient, less invasive ways to diagnose and treat this cancer.

18 FEBRUARY 2011

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SCIENCE

Published by AAAS

Sequencing the whole genome of a prostate tumor, says co-author Levi Garraway of Harvard Medical School in Boston, allowed the researchers to see “biology that would have been invisible with any other method.” Most genetic analyses of tumors have focused on ﬁguring out which mutations turn normal cells cancerous and how these “spelling errors” foster tumor growth. But by sequencing the entire genomes of seven prostate tumors and comparing them with genomes from the patients’

NOTED >>The U.S. Department of Health and

Human Services’ Office of Research Integrity has produced an interactive online movie called The Lab. The chooseyour-own-adventure-style film allows viewers to play as one of four characters, including an insecure postdoc and a promising young graduate student, to avert a case of scientiﬁc misconduct. http://scim.ag/labmovie

www.sciencemag.org

Downloaded from www.sciencemag.org on February 17, 2011

The century-old surgical practice of removing lymph nodes from breast cancer patients is likely unnecessary, says a new study in the 9 February issue of The Journal of the American Medical Association. Conventional wisdom has been that after a biopsy from a sentinel lymph node ﬁnds cancer, removal of all the armpit lymph nodes before they can spread the disease throughout the body would increase survival. But researchers at John Wayne Cancer Institute in Santa Monica, California, and other institutions found no difference in survival or clinical outcomes in a randomized trial of 891 women with early-stage breast cancer who were undergoing chemotherapy or radiation. Half of the women had only the sentinel node removed, whereas the others had all their lymph nodes removed. The authors credit the results to aggressive therapy that wiped out the cancer before it could spread from the lymph nodes. They recommend that physicians reconsider the need for the painful surgery, which can have complications such as swelling and infection—although persuading the ﬁeld to change could be tough because the practice is so ingrained. Even so, several cancer clinics such as Memorial Sloan-Kettering, where some of the trials took place, have already started to implement the new standard.

CREDITS (TOP TO BOTTOM): © BRUCE DAMONTE/UCSF; ILLUSTRATION COURTESY OF THE CENTER FOR FETAL DIAGNOSIS AND TREATMENT AT THE CHILDREN’S HOSPITAL OF PHILADELPHIA

Lymph Node Surgery Unnecessary for Early Breast Cancer

Obama’s 2012 Vision Clashes With House Cuts in 2011 It’s a tale of two budgets, and the U.S. science community is applauding one while deploring the other. The twin story lines involve the president’s vision for 2012, submitted to Congress on Valentine’s Day, along with a spending plan by House Republicans for the rest of this ﬁscal year. The latter is needed because Congress still hasn’t ﬁnished work on the budget for the 2011 ﬁscal year that began last October. And while budgets are always political statements, this pair goes further by painting starkly contrasting visions of where the country should be headed—and the role of research in helping it to get there. All this is a recipe for confusion and uncertainty. The ﬁrst order of business is the 2011 budget. Three days before the president unveiled his 2012 budget, Republicans in the House of Representatives offered a plan to cut $62 billion in current spending as a ﬁrst step in erasing the nation’s $1.5 trillion budget deﬁcit. They argue that a bankrupt government can’t fund any type of research, and that what’s needed now is ﬁscal belt-tightening. Representative Harold Rogers (R–KY), chair of the House appropriations committee, acknowledges that his panel has crafted a 2011 spending plan with cuts that “will affect every community in the nation. I know many people will not be happy with everything we’ve proposed in this package.” Republicans have proposed reductions of more than $3.3 billion in current research

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spending by various federal agencies. The list is long and the cuts are deep, including an $893 million reduction below current spending at the Department of Energy’s (DOE’s) Ofﬁce of Science, to $4.1 billion, and a cutback of $1.6 billion at the National Institutes of Health (NIH), to $29.5 billion, bringing both agencies to 2008 spending levels. The National Science Foundation (NSF), one of the favored children of the Obama Administration, would see its current budget pared by $388 million to a level—$6.57 billion—that would be more than $1 billion below the president’s request of $7.77 billion in 2012. The new Republican majority in the House was given the chance to craft a 2011 budget after Democrats gave up trying to pass a spending bill in the lame-duck session after the November elections. Debate was expected this week on the so-called continuing resolution (CR) to ﬁnish out the ﬁscal year, which ends on 30 September. The plan, once passed by the House, would then go to the Democrat-controlled Senate, which is expected to oppose many of the cuts with backing from the White House. The CR expires on 4 March, so Congress must reach a compromise by that date or extend the CR to avoid a government shutdown. With the 2011 budget still up in the air, Obama’s 2012 budget request appears to be more a statement of principle than a blueprint for what might actually be spent. The $3.73 trillion in planned spending for all fed-

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eral agencies ﬂeshes out his promise in last month’s State of the Union speech to “outinnovate, out-educate, and out-build” the rest of the world by providing healthy increases for research and education. Among other things, it keeps his promise for a 10-year budget doubling at three important research agencies, proposes a significant expansion of clean-energy and climate research, including $550 million for a ﬂedgling blue-sky energy research agency, and offers a slew of initiatives to improve U.S. science education by training more and better teachers and helping those already in the classroom. The growth would occur despite a promised 5-year freeze in overall domestic discretionary spending that would put the squeeze on a vast array of government programs, including some research efforts that the Administration likes but says the country can’t afford. “This is a budget that the nation can be proud of,” says the president’s science adviser, John Holdren, speaking about the R&D portfolio. But Holdren said the proposed 11% growth in basic and applied research (all comparisons are with 2010 spending levels since there is no 2011 budget) also exempliﬁes “tough love,” with sharp cuts to environmental programs at the Environmental Protection Agency and within the “development” portfolio of defense research. Ofﬁcials at NASA, which would face a stagnant budget under Obama’s plan, said that the agency will kill plans for a space telescope to explore dark energy, the top priority of a recent decadal survey by the astrophysics community, in the hope that it can collaborate more cheaply with the European Space Agency on a similar mission. NSF Director Subra Suresh says he sees “a lot of love” in the president’s pro-

www.sciencemag.org

CREDIT: M. HICKS/SCIENCE

U.S. SCIENCE BUDGET

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NEWS & ANALYSIS

NEWS

SOURCE: OMB, CONGRESS

posal for a 13% increase for NSF, and he hopes that Congress reciprocates. Even biomedical researchers, reacting to a 2.4% boost for NIH that would fall short of what the Administration requested last year in its 2011 budget, say they are grateful for any increase at a time when so many federal agencies are looking at reductions. Not surprisingly, Republicans have little love for Obama’s Valentine’s Day budget. “The president’s budget will destroy jobs by spending too much, taxing too much, and borrowing too much,” declared House Speaker John Boehner (R–OH). Rogers expressed his disdain for the 2012 proposal by saying that the country “must go much further than this anemic effort of symbolic reductions and additional spending proposed under the guise of funding ‘freezes’ if we are truly to get our nation’s ﬁnances on a sustainable course.” And Representative John Kline (R–MN), chair of the Education and the Workforce Committee that will pass judgment on many of the education initiatives, declared that “the president’s budget denies the reality of the ﬁscal crisis we face.” What’s at stake The dueling budgets offer a sampler of sweets and sourballs for the palates of scientists of various stripes. Obama’s 2012 proposals include: • Interdisciplinary research at NSF would ﬂourish, reﬂecting Suresh’s effort to break down disciplinary walls. He’s proposed a $30 million National Robotics Initiative in collaboration with other federal agencies, for example, as well as a solicitation for a new program supporting all manner of interdisciplinary proposals that he promises “will change the way NSF does business.” • A 24% boost for competitive research within the Department of Agriculture, to $325 million, for grants ranging from improving food safety to mitigating the impact of climate change on agriculture. Obama had requested $104 million more than that for the program in 2011, while the Republican proposal would result in a cut of 13% from 2010 levels. • Climate change research across the government would rise by 20%, to $2.6 billion, including a 25% boost for earth sciences at NASA. A related collection of programs that NSF began in 2010, called

Science, Engineering, and Education for Sustainability, would jump by nearly onethird, to $998 million. • The Advanced Research Projects Agency–Energy, which Energy Secretary Steven Chu sees as the ﬂagship for the Administration’s push to discover, scale up, and disseminate clean-energy technologies, would receive $550 million in 2012. It would be the ﬁrst annual budget for the 2-year-old agency that was launched with money from the 2009 stimulus act. On the other hand,

NIH

$31.8 billion • $ 31.1 billion • $29.5 billion •

Obama’s 2012 request Current funding House plan for 2011 CR

NSF

$7.8 billion • Obama’s 2012 request $ 6.9 billion • Current funding $ 6.5 billion • House plan for 2011 CR DOE Science

$5.4 billion • Obama’s 2012 request $ 4.9 billion • Current funding $ 4.0 billion • House plan for 2011 CR House Republicans would keep it on a starvation diet, with $50 million in 2011. • A new National Center for Advancing Translational Sciences (NCATS) at NIH to “reengineer” the drug-development pipeline. NCATS would include a $100 million Cures Acceleration Network, created by last year’s health care law. Although creating NCATS would entail abolishing the National Center for Research Resources, NCRR remains in the 2012 budget and NCATS does not appear because NIH is still trying to assign various pieces of NCRR.

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• DOE science programs would pony up $15 million to keep alive a project to turn an abandoned gold mine in South Dakota into a vast Deep Underground Science and Engineering Laboratory (DUSEL). NSF’s oversight board decided recently that the proposed $875 million lab didn’t ﬁt its mission, and the agency zeroed out funding for DUSEL in its 2012 request. • A 32% increase in funding for the in-house laboratories and other research initiatives at the National Institute of Standards and Technology, to $679 million, would expand efforts in nanoscale manufacturing, advanced energy materials, and novel time and electrical measurement capabilities. • Research on solar photovoltaics would grow from $126 million to $337 million at DOE as part of Chu’s SunShot Initiative to spur commercial use of the renewable energy source by lowering its cost. In contrast, House Republicans would prefer that private industry take on the task. • Termination of two NSF programs to strengthen precollege science education, one for graduate students to spend time in the classroom and another to support research on the science of learning. They would be replaced by more targeted efforts to improve the teacher workforce through improved training and research on best practices. The fate of these and other budget proposals hinges on whether the White House and Congress can ﬁnd common ground. Senate Democrats, who have so far kept their powder dry, cobbled together a plan last December that would have provided increases for many science agencies while still coming in under the president’s 2011 request. But it was withdrawn after failing to win enough support from Republicans. In the meantime, the science community has lined up behind Obama’s version of the future. “His budget, while freezing discretionary spending and taking other steps to reduce deﬁcits, invests in research that will help us grow our economy, conquer disease, achieve greater energy independence, and strengthen our national security,” says Robert Berdahl, president of the Association of American Universities in Washington, D.C. The House CR, he notes, “would truly harm this nation’s capacity for innovation by slashing research spending for nearly every agency that sponsors scientiﬁc research.” The fate of many research projects—for this year and well beyond—hinges on which vision prevails. –JEFFREY MERVIS

Downloaded from www.sciencemag.org on February 17, 2011

Presidential vision. Science adviser John Holdren is ﬂanked by the president’s numbers and words as he rolls out the 2012 science budget.

With reporting by the Science News staff.

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NEWS&ANALYSIS CHINA

834

“[The current policy tells] the best and the brightest to spend their most productive years abroad.” —MU-MING POO, INSTITUTE OF NEUROSCIENCE, CHINESE ACADEMY OF SCIENCES

“It is better to invest in a whole new generation of talent than to buy reputation.” —DAVID HO, ADARC

p. 1732), a phenomenon that many scientists dismiss out of hand. University President Zhang Jie and Montagnier did not respond to messages before Science went to press. In the original Qianren Jihua, the program’s sponsor, the Organization Department of the Chinese Communist Party’s Central Committee (Zhongzubu), provides each recruit with a $150,000 tax-free relocation allowance. Beyond this subsidy, awardees must negotiate salaries and start-up packages with recruiters. Eager to please Zhongzubu, university and institute officials routinely dangle start-up funds of $1.5 million or more

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to entice candidates—then fail to deliver, says Xu Ruiming, a structural biologist at CAS’s Institute of Biophysics in Beijing. Xiao-Fan Wang, a cancer researcher at Duke University in Durham, North Carolina, says some Qianren recipients “sincerely wanted to go back but have to reconsider when they see no research funding.” However, the $150,000 incentive, sometimes doubled or tripled by local governments, entices opportunists, Wang and others say. Problems that dogged previous recruitment programs have tainted Qianren Jihua as well, such as recruits padding their resumés and professors with full-time jobs overseas double-dipping in China. Some Qianren recruits, Science has learned, are postdocs masquerading as faculty members or are not full professors as the program announcement requires. That doesn’t appear to perturb Zhongzubu. “Coming from overseas is the most important qualiﬁcation of Qianren Jihua. All other factors, such as age, degree, or one’s professional title, are not that important,” says Zhongzubu ofﬁcial Zhang Dong. The perception that a tide of opportunists is cashing in and that many Qianren awardees are holding out for even bigger carrots riles many Chinese scientists. In recent years, CAS has established a merit-based promotion and compensation system, but “now you suddenly have people coming from the outside getting paid four to ﬁve times more,” says Poo, who also holds a full-time faculty position at the University of California, Berkeley, and does not receive a salary from CAS. Qianren packages make “people really jealous,” says Poo. Qianren may also inadvertently worsen a long-standing problem faced by Chinese science: the paucity of talented young researchers. It is difficult to recruit good assistant professors in China already, says Poo, and Qianren Jihua, by requiring recipients to be tenured full professors or the equivalent at an overseas institution, essentially tells “the best and the brightest to spend their most productive years abroad.” Buffeted by complaints and misinformation, Zhongzubu has chosen to keep the names of most awardees under wraps. The agency “does not want recipients subjected to criticism before they even begin working” in China, says Zhang. But Internet users have collected some 450 Qianren names gleaned from Chinese university announcements. Of known awardees, more than 300 have full-

www.sciencemag.org

CREDITS (TOP TO BOTTOM): COURTESY OF MU-MING POO; ADARC

How much would it take to get you to relocate to China? Would 150 million yuan ($23 million) do the trick? If so, pack your bags—if you are a Nobel laureate, that is. Science has learned that the Chinese government will soon announce a new initiative to lure up to 10 winners of prestigious international science prizes—including the Nobel Prize—to China each year by offering what may be the heftiest reward ever paid to individual researchers. Some prizewinners may be salivating, but at least one prominent Chinese-American scientist aware of the new program blasts it as a massive waste of resources. “It is better to invest in a whole new generation of talent than to buy reputation,” says David Ho, director of the Aaron Diamond AIDS Research Center (ADARC) in New York City. “Someone should step up and put an end to this folly.” The initiative will be a new component of the Chinese government’s Recruitment Program of Global Experts, commonly known as Qianren Jihua. Launched in 2008 with the goal of recruiting up to 2000 experts from abroad over 5 to 10 years (Science, 31 July 2009, p. 534), the program so far has tallied 1143 recipients, including 880 “innovative talents” to work at universities and research institutes. The rest are “entrepreneurial talents” recruited to run high-tech companies. Despite Qianren Jihua’s impressive numbers, many observers say the program is foundering. Initially, it mandated that awardees spend 6 to 9 months a year in China for a minimum of 3 years. Most recruits, however, have not signed contracts or moved to China, says Li Ning, a public policy researcher at the University of Guam. The main reason awardees give for not having signed contracts is that they haven’t received start-up funds. Qianren Jihua “is a huge disaster right now,” claims Mu-ming Poo, part-time director of the Institute of Neuroscience of the Chinese Academy of Sciences (CAS) in Shanghai. That sentiment is shared by many contributors to blog sites on Chinese science. The new component dubbed “Top Qianren” is likely to make the program even more controversial. Its first catch, sources say, is 78-year-old French virologist Luc Montagnier, who won the 2008 Nobel Prize in physiology or medicine for co-discovering HIV. Shanghai Jiaotong University recruited Montagnier, who intends to lead research into electromagnetic radiation from highly diluted pathogen DNA (Science, 24 December 2010,

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High-Priced Recruiting of Talent Abroad Raises Hackles

NEWS&ANALYSIS time faculty positions outside China, judging by information on their employers’ Web sites. More than 100 have research projects sponsored by the U.S. National Institutes of Health or the National Science Foundation, with funding for several years down the line. Most of these awardees cannot spend six or more months in China, as the program envisioned, without reducing the commitment to their current employers. To accommodate them, Zhongzubu created a Qianren

category last year that allows recipients to spend as little as 2 months a year in China. The government expects fewer than 100 awardees each year to choose the short-term category, says Zhang. The failure to land many big fish has prompted Zhongzubu to cast for small fry. Last month, the government launched Young Qianren Jihua, which over the next 5 years aims to recruit from overseas 2000 researchers under age 40. The government will pro-

vide young recipients with a relocation allowance of about $75,000 and research funds ranging from $152,000 to $456,000 over 3 years. Requirements include Ph.D.s obtained from foreign universities and overseas work experience of 3 years or more. At 1000 or so pay grades higher will be the Nobel laureates and other elites who accept a Top Qianren award. They’ll never have to worry about funding again.

–HAO XIN

A N T H R A X I N V E S T I G AT I O N

CREDIT: NEWSCOM

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Science in Ivins Case Not Ironclad, NRC Says The scientific evidence behind the U.S. The FBI, working with scientists inside and logical weapon. U.S. intelligence ofﬁcials colgovernment’s implication of U.S. Army outside the bureau, used assays to screen lected environmental samples from this undisresearcher Bruce Ivins as the perpetrator of anthrax samples at labs around the United closed site in May 2004 as part of the anthrax the 2001 anthrax letter attacks is not as strong States and overseas for the presence of four investigation, swabbing “the outside of an as claimed by the Federal Bureau of Inves- morphological types of the Ames strain of unopened medicine dropper package, a sink tigation (FBI), according to a long-awaited Bacillus anthracis that were present in the and a sink drain hose.” The swabs tested posreview by the National Research Council attack material. The results of this analysis itive for anthrax and contained certain DNA (NRC) released this week. Although the led the FBI to conclude that the spores were sequences of the Ames strain. However, when report agrees with the FBI on most of the con- derived from ﬂask RMR-1029, to which Ivins the paraphernalia from this site was brought to clusions drawn from the analysis of anthrax had access. the United States in November 2004, no traces spores used in the mailings, it cautions that Although the review panel agreed that of anthrax could be found. “The committee the science by itself does not deﬁnitively link the spores could have originated from believes that the complete set of data and conthe attack material to a ﬂask under Ivins’s RMR-1029, it did not agree with the inves- clusions concerning these samples, including control at the U.S. Army Medical Research tigative claim that the spores could only all relevant classiﬁed documents, deserves a Institute for Infectious Diseases more thorough scientiﬁc review,” in Frederick, Maryland, as the the report says. FBI asserted. The panel also suggests that the The FBI asked NRC to conFBI could have made use of more duct the review in September advanced tools for genetic analy2008, weeks after the FBI and sis that became available as the the Department of Justice (DOJ) investigation progressed. “Using claimed Ivins was behind the tools such as high-throughput, deadly mailings that killed five ‘next generation’ DNA sequencpeople and terrorized the nation. ing could have strengthened or Ivins committed suicide just days weakened the association between before the FBI went public with spores found in the mailed letters the charges, so the case never and spores from RMR-1029,” went to trial. Many of Ivins’s col- Inconclusive science. The methods the FBI used to investigate the anthrax let- says Stanford University microleagues alleged that the FBI had ters were sound, says a new report, but not sufﬁcient to establish their source. biologist David Relman, vice chair hounded an innocent man to of the committee. death, whereas the FBI and DOJ laid out their have come from RMR-1029. “Some of the The review is bound to stir up controversy case against Ivins at press conferences and mutations identified in the spores of the and provide fodder for critics of the FBI investhrough the release of hundreds of pages from attack letters … might have arisen by paral- tigation. However, Paul Keim, an anthrax the case ﬁles. A cornerstone of the investiga- lel evolution rather than by derivation from researcher at Northern Arizona University in tion, FBI ofﬁcials said, was the use of genetic RMR-1029,” the review concludes. “This Flagstaff who helped the FBI conduct some analysis to trace the source of the anthrax. possible explanation of genetic similarity of the analysis, says the ﬂaws pointed out by Although not intended to be a proxy trial, between spores in the letters and in RMR- the NRC panel do not undermine the evidence NRC’s review of the FBI’s scientiﬁc evidence 1029 was not rigorously explored during in any signiﬁcant way. That said, he agrees in the case has been the closest thing to an the course of the investigation.” that studies of how the Ames strain evolves independent assessment of the investigation. The review also questions whether inves- in the lab would have strengthened the forenThe review found that the methods used tigators had sufﬁcient evidence to rule out sic side of the investigation, and such studies for the genetic and chemical analyses were the possibility, however remote, that the are now under way, he says, but overall “the generally sound but that the FBI and DOJ had spores originated at an overseas site where review seems like a cautious endorsement of overstated the scientiﬁc case against Ivins. Al Qaeda might have been developing a bio- the investigation.” –YUDHIJIT BHATTACHARJEE www.sciencemag.org

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NEWS&ANALYSIS time faculty positions outside China, judging by information on their employers’ Web sites. More than 100 have research projects sponsored by the U.S. National Institutes of Health or the National Science Foundation, with funding for several years down the line. Most of these awardees cannot spend six or more months in China, as the program envisioned, without reducing the commitment to their current employers. To accommodate them, Zhongzubu created a Qianren

category last year that allows recipients to spend as little as 2 months a year in China. The government expects fewer than 100 awardees each year to choose the short-term category, says Zhang. The failure to land many big fish has prompted Zhongzubu to cast for small fry. Last month, the government launched Young Qianren Jihua, which over the next 5 years aims to recruit from overseas 2000 researchers under age 40. The government will pro-

vide young recipients with a relocation allowance of about $75,000 and research funds ranging from $152,000 to $456,000 over 3 years. Requirements include Ph.D.s obtained from foreign universities and overseas work experience of 3 years or more. At 1000 or so pay grades higher will be the Nobel laureates and other elites who accept a Top Qianren award. They’ll never have to worry about funding again.

–HAO XIN

A N T H R A X I N V E S T I G AT I O N

CREDIT: NEWSCOM

Downloaded from www.sciencemag.org on February 17, 2011

Science in Ivins Case Not Ironclad, NRC Says The scientific evidence behind the U.S. The FBI, working with scientists inside and logical weapon. U.S. intelligence ofﬁcials colgovernment’s implication of U.S. Army outside the bureau, used assays to screen lected environmental samples from this undisresearcher Bruce Ivins as the perpetrator of anthrax samples at labs around the United closed site in May 2004 as part of the anthrax the 2001 anthrax letter attacks is not as strong States and overseas for the presence of four investigation, swabbing “the outside of an as claimed by the Federal Bureau of Inves- morphological types of the Ames strain of unopened medicine dropper package, a sink tigation (FBI), according to a long-awaited Bacillus anthracis that were present in the and a sink drain hose.” The swabs tested posreview by the National Research Council attack material. The results of this analysis itive for anthrax and contained certain DNA (NRC) released this week. Although the led the FBI to conclude that the spores were sequences of the Ames strain. However, when report agrees with the FBI on most of the con- derived from ﬂask RMR-1029, to which Ivins the paraphernalia from this site was brought to clusions drawn from the analysis of anthrax had access. the United States in November 2004, no traces spores used in the mailings, it cautions that Although the review panel agreed that of anthrax could be found. “The committee the science by itself does not deﬁnitively link the spores could have originated from believes that the complete set of data and conthe attack material to a ﬂask under Ivins’s RMR-1029, it did not agree with the inves- clusions concerning these samples, including control at the U.S. Army Medical Research tigative claim that the spores could only all relevant classiﬁed documents, deserves a Institute for Infectious Diseases more thorough scientiﬁc review,” in Frederick, Maryland, as the the report says. FBI asserted. The panel also suggests that the The FBI asked NRC to conFBI could have made use of more duct the review in September advanced tools for genetic analy2008, weeks after the FBI and sis that became available as the the Department of Justice (DOJ) investigation progressed. “Using claimed Ivins was behind the tools such as high-throughput, deadly mailings that killed five ‘next generation’ DNA sequencpeople and terrorized the nation. ing could have strengthened or Ivins committed suicide just days weakened the association between before the FBI went public with spores found in the mailed letters the charges, so the case never and spores from RMR-1029,” went to trial. Many of Ivins’s col- Inconclusive science. The methods the FBI used to investigate the anthrax let- says Stanford University microleagues alleged that the FBI had ters were sound, says a new report, but not sufﬁcient to establish their source. biologist David Relman, vice chair hounded an innocent man to of the committee. death, whereas the FBI and DOJ laid out their have come from RMR-1029. “Some of the The review is bound to stir up controversy case against Ivins at press conferences and mutations identified in the spores of the and provide fodder for critics of the FBI investhrough the release of hundreds of pages from attack letters … might have arisen by paral- tigation. However, Paul Keim, an anthrax the case ﬁles. A cornerstone of the investiga- lel evolution rather than by derivation from researcher at Northern Arizona University in tion, FBI ofﬁcials said, was the use of genetic RMR-1029,” the review concludes. “This Flagstaff who helped the FBI conduct some analysis to trace the source of the anthrax. possible explanation of genetic similarity of the analysis, says the ﬂaws pointed out by Although not intended to be a proxy trial, between spores in the letters and in RMR- the NRC panel do not undermine the evidence NRC’s review of the FBI’s scientiﬁc evidence 1029 was not rigorously explored during in any signiﬁcant way. That said, he agrees in the case has been the closest thing to an the course of the investigation.” that studies of how the Ames strain evolves independent assessment of the investigation. The review also questions whether inves- in the lab would have strengthened the forenThe review found that the methods used tigators had sufﬁcient evidence to rule out sic side of the investigation, and such studies for the genetic and chemical analyses were the possibility, however remote, that the are now under way, he says, but overall “the generally sound but that the FBI and DOJ had spores originated at an overseas site where review seems like a cautious endorsement of overstated the scientiﬁc case against Ivins. Al Qaeda might have been developing a bio- the investigation.” –YUDHIJIT BHATTACHARJEE www.sciencemag.org

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A Quake May Have Hinted That It Was on the Way All this discouraged seismologists, including Michel Bouchon of Joseph Fourier University in Grenoble, France, who had intensively studied the Izmit quake and its aftershocks. But about a year ago, Bouchon and ﬁve Turkish and French colleagues got around to analyzing the skimpy foreshock record using new techniques for extracting information from seismic recordings that look like mostly noise. The methods were developed in the past 10 years to understand seismic tremor, the newly recognized continuous seismic “hum” that strikes some faults. Using these and other techniques, the group found some 22 foreshocks in addition to the 18 obvious ones, all of them impercep-

Earthquake

Storms at sea gave sailors of old fair warning: a falling barometer, a shift in the wind, ominous harbingers in the sky. Seismologists have not been so lucky. Decades of monitoring have failed to turn up any warning sign of an imminent quake, and most seismologists have moved on to other problems. Now, however, a new analysis of decadeold, low-quality data from a large Turkish earthquake has revealed tantalizing signs of a quake precursor. “The jury’s out, but we should be in court,” says seismologist Ross Stein of the U.S. Geological Survey (USGS) in Menlo Park, California, who was not involved in the new study. “This is too important to dismiss. It’s what we should be doing.”

Cross−correlation

17

0

17a 17b 17c 17d

5

18

10 15 20 25 30 35 Time (seconds)

Harbingers? Tiny earthquakes uncovered in a decade-old seismic record (labeled spikes, right) may have signaled slow fault slip that triggered the 1999 quake near Izmit, Turkey.

The encouraging development, reported on page 877, comes from records of the magnitude-7.6 Izmit earthquake that struck just 100 km from Istanbul in 1999. Having ruptured 150 km of the well-studied North Anatolian fault and killed about 17,000 people, the quake got seismologists’ attention. The tiny foreshocks that ticked off in the minutes leading up to it garnered far less notice, for good reason: Only one seismometer near the quake’s starting point recorded more than one foreshock. (Complete records from multiple instrument sites enable seismologists in effect to triangulate quakes and multiply the information that can be extracted about them.) And even the single seismograph was in the geometrically least helpful location, directly opposite the spot on the fault where the large quake got started.

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tible at the surface. In some cases, the foreshocks’ squiggly seismogram traces showed a striking similarity, which suggested that most or all of the foreshocks resulted from the rupture of the same patch of fault. From tiny differences in how long different sorts of seismic waves took to reach the seismograph, Bouchon and colleagues concluded that the foreshock ruptures were striking the same spot on the fault where the main shock would get started. And they found that the whole process was accelerating, from the time of the ﬁrst foreshock to one 44 minutes later, just 0.07 second before the main shock. To Bouchon and his colleagues, the Izmit foreshocks signify far more than a run-ofthe-mill swarm of microearthquakes. They are a clear sign that the fault was slipping— not just feeling the increasing strain—during

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a buildup to the magnitude-7.6 shock. In the group’s interpretation, a patch of the fault a few hundred meters across located about 15 km beneath the eventual epicenter repeatedly ruptured, letting the fault patch slip 1 millimeter to 1 centimeter each time. Bouchon thinks the fault slipped at the same spot where the big quake eventually started. So it was the accelerating slow slip that led to the runaway rupture of the big quake. “Izmit is the ﬁrst time we see the fault slipping at depth before the earthquake,” says Bouchon. And, he says, the distinctive nature of the ﬁrst couple of foreshocks tells a seismologist—or a computer program monitoring a fault—that “there’s a probability that something big is coming.” Researchers are impressed, up to a point. Bouchon “has wrung as much as anyone possibly could out of such crappy data,” says Stein. And, says seismologist William Ellsworth of the USGS in Menlo Park, “there is a very clear sequence of some foreshocks from a small region that leads to eventual initiation” of the large quake. But the view from the single seismic record is still fuzzy, Ellsworth says. All the foreshocks “may be at the same spot, maybe not.” More fundamentally, seismologists wonder how far the Izmit sequence of accelerating foreshocks can be generalized. “We know there’s a range of behavior” among large quakes, says Ellsworth. More than half of large earthquakes have no foreshocks at all. “Many earthquakes simply come out of the blue,” he notes. Researchers were able to take particularly close looks at the starting points on the San Andreas fault of both the 2004 magnitude-6.0 Parkﬁeld earthquake and the 1989 magnitude-6.9 Loma Prieta earthquake. “We simply did not see anything,” says Ellsworth. And for all researchers know, says seismologist David Shelly of the USGS in Menlo Park, fault slip before a quake may “happen relatively frequently and then may or may not trigger an earthquake.” The Izmit observations are both tantalizing and frustrating, says seismologist Gregory Beroza of Stanford University in Palo Alto, California. Tantalizing, he says, because “this is the sort of thing you’d expect to see” based on theory. “The frustrating part is they don’t have enough data to show exactly what’s going on. But this is going to cause people to redouble their efforts to closely examine the beginning of other earthquakes.” The problem with that, he notes, will be snaring big quakes in dense nets of geophysical monitors, because “we don’t know where earthquakes are going to start.”

www.sciencemag.org

–RICHARD A. KERR

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EARTHQUAKE PREDICTION

CREDITS (TOP TO BOTTOM): NEWSCOM; ADAPTED FROM M. BOUCHON ET AL., SCIENCE 331, 6019 (18 FEBRUARY 2011)

NEWS&ANALYSIS

NEWS&ANALYSIS GENETICS AND DISEASE

Life can be treacherous for Ecuadorians with Laron syndrome, a rare type of dwarﬁsm. As children, they are vulnerable to infectious diseases. As adults, they are prone to fatal accidents, such as falls on stairs that aren’t sized for their short legs. But a new study shows that these people, who carry a genetic defect that prevents them from responding to growth hormone (GH), are almost exempt from cancer and diabetes. The paper solidiﬁes a link researchers have long suspected from animal studies and suggests that dialing down the growth-controlling molecular pathways might protect healthy adults from these diseases. “The strength [of this paper] is that finally someone has made this connection in humans,” says physiologist Holly BrownBorg of the University of North Dakota School of Medicine and Health Sciences in Grand Forks. “The fact that they found such a striking absence of cancer and diabetes in such a large cohort makes it a very valuable study,” says mammalian endocrinologist Andrzej Bartke of the Southern Illinois University School of Medicine in Springﬁeld. Numerous studies show that mice gain from short circuits in the GH pathways. Minimice that can’t make GH or lack the receptor that relays its message are less susceptible to cancer than are their huskier counterparts. They also have low quantities of blood insulin, and their cells are sensitive to the hormone—high insulin levels and insulin resistance can be harbingers of diabetes. Although some data support the notion that GH-deﬁcient people derive similar benefits, the evidence has been “anecdotal,” says Brown-Borg. Enter diabetologist Jaime GuevaraAguirre of the Institute of Endocrinology, Metabolism and Reproduction in Quito, Ecuador. In the late 1980s, he was studying body composition when he chanced on several people with Laron syndrome who lived in small, isolated villages in Ecuador. He has been tracking the group—which now numbers almost 100 people—ever since. They carry mutations that cripple the GH receptor and stunt growth. The tallest men top out at about 140 centimeters (4.5 feet), the tallest women at about 124 centimeters (4 feet). In 1994, Guevara-Aguirre noticed that none of the subjects had cancer. To investi-

gate, he and his colleagues teamed up with researchers led by molecular geneticist Valter Longo of the University of Southern California in Los Angeles. The scientists analyzed medical information for the 99 subjects Guevara-Aguirre has been tracking, for another 53 Laron patients who died before 1988, and for more than 1600 relatives who didn’t have the condition. Only one of the Laron subjects developed cancer, an ovarian tumor that didn’t

Cancer free. Jaime Guevara-Aguirre with several of his Laron syndrome subjects in 1988 (top) and in 2009 (bottom).

recur after chemotherapy. By contrast, cancer killed 20% of the normal-sized relatives. The Laron syndrome individuals were also free of type 2 diabetes, the cause of death for 5% of their taller kin, the team reported in the 16 February issue of Science Translational Medicine. The diabetes result was surprising because Laron syndrome often causes obesity, a risk factor for diabetes. But the researchers found that the subjects, like the minimice, had lower blood insulin levels and much higher sensitivity to the hormone than did controls.

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Scientists haven’t worked out exactly how GH prompts diabetes. But Longo and colleagues did identify a possible mechanism for the Laron subjects’ cancer resistance. They immersed human cells in blood serum from either the Laron subjects or the control group and added DNA-breaking hydrogen peroxide. Cells basting in the control serum accumulated more DNA breaks that can spur tumors and were less likely to commit suicide, a mechanism for weeding out potentially cancerous cells. GH rouses another hormone, insulin-like growth factor-1 (IGF-1), and researchers think that both molecules promote cancer. Adding IGF-1 to the serum from the Laron group cut the amount of cell suicide to control levels, possibly increasing cancer susceptibility. Unlike the dwarf mice with similar genetic defects, the Laron subjects didn’t live longer than normal, despite their lack of mortality from cancer and diabetes. Along with higher death rates from accidents, they also fell victim to epilepsy and similar conditions and to other causes. The study shows that “two major diseases are prevented by a single point mutation in the GH receptor,” says Longo. Does that mean that comparable changes in healthy people could prevent cancer and diabetes? It’s possible, Longo says. In the future, doctors might prescribe drugs that interfere with the GH pathways for adults who have abovenormal levels of IGF-1, much like they currently prescribe statins for people who have high cholesterol, he says. Other researchers suggest that we should be cautious about intervening in the growthcontrolling pathways to forestall disease. “Manipulating this in a healthy person as prevention is a very delicate issue and very complicated,” says Bartke. Although it’s not necessary for growth in adults, GH might still perform important functions, such as preventing obesity, he says. Reducing insulin levels throughout the body might have a downside, adds geriatrician William Banks of the University of Washington School of Medicine in Seattle. “There could be unexpected consequences on the central nervous system” because insulin in the brain improves mental acuity.

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CREDITS: JAIME GUEVARA-AGUIRRE ET AL., SCIENCE TRANSLATIONAL MEDICINE 3, 70 (16 FEBRUARY 2011)

Growth Defect Blocks Cancer and Diabetes

–MITCH LESLIE

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Forensic geneticist Manfred Kayser is exploring whether DNA found at a crime scene can predict what a suspect looks like 838

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Can This DNA Sleuth Help Catch Criminals?

ROTTERDAM, THE NETHERLANDS—The murder was heinous, there were no witnesses, and the police had few clues—except for some skin found under the ﬁngernails of the victim that might belong to the killer. And that was all it took. From a few nanograms of DNA in the skin cells, a police lab determined that the murderer was a man of European origin with brown eyes and straight, dark-brown hair, approximately 45 years old and balding, and likely 1.90 meters in height. Within a few more hours, a police computer spewed out a sketch of the man’s face seen from three different angles, which was all over the evening news. Soon, calls started pouring in from people who recognized the suspect. Sure, that’s science ﬁction. But according to German gene sleuth Manfred Kayser, the scenario might come true one day. Kayser, who leads the forensic molecular biology department at Erasmus University Medical Center here, is at the forefront of an emerging research area that seeks to predict people’s looks, age, and geographic ancestry from their DNA. If successful, the endeavor, sped along by the genomic revolution, could provide crime ﬁghters with a powerful new toolbox. Kayser’s group made headlines around the world last year with a paper showing how the DNA in a blood sample can give away someone’s age—albeit with a margin of error of at least 9 years. His group has developed a DNA test to predict someone’s eye color; work on hair color, skin color, and other traits is in progress. Kayser is “an upcoming star” in forensic DNA phenotyping, as the ﬁeld is called, says Bruce Budowle, a geneticist with 40 years of experience with the FBI who is now at the University of North Texas Health Science Center in Fort Worth. He’s “a clear leader,” adds Christopher Phillips, a forensic geneticist at the University of Santiago de Compostela in Spain. The genetic clues that Kayser and others are trying to glean from minuscule amounts of blood, semen, saliva, and hair are unlikely to be introduced as evidence in a courtroom. After all, when someone is suspected of a crime, or charged, a conventional DNA fingerprinting test can determine if his or her DNA matches traces found at the crime scene. Instead, forensic DNA phenotyping could be useful during an investigation, when predicting a criminal’s looks can help the police focus their search. Forensic DNA phenotyping raises new ethical and legal issues, and the Netherlands is the only country so far to regulate the practice in a new law (see sidebar, p. 840). But Kayser doesn’t anticipate that the concerns will stop

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NEWSFOCUS

HUMAN GENOME 10TH ANNIVERSARY | NEWSFOCUS and stayed in his old college town until he was offered the leadership of a new forensics department at Erasmus University Medical Center in 2004. In an unusual arrangement, his department is co-funded by the NetherMusic and animals lands Forensic Institute (NFI), a governmenKayser, 43, isn’t fascinated by crime. He tal lab in The Hague. NFI management recdoesn’t read John Grisham novels or watch ognized that an academic setting would be CSI, the sleek TV hit show centered on foren- a more fertile place to invest in science and sic research. His lab looks as unexciting as development than a lab that’s loaded with any other molecular biology casework, according to Kayser. lab in the world. In fact, Kayser “When I saw the business entered the entire ﬁeld more or plan, I realized this departless by chance. ment was really going to He was born and raised in be a unique place,” he says, East Berlin in the communist because it would run “the full era. As a teenager, he developed monty” from basic science passions for both music and the to developing and validating animal world, neither of which tests. Kayser works closely Find a podcast on this topic were particularly appreciated with NFI researchers and and other genome stories at in the German Democratic with Peter de Knijff, who runs http://scim.ag/genome10. Republic, he says. After high a well-established forensic school, he worked in the frog genetics lab at Leiden Univercollection of the Natural History Museum in sity Medical Center where Kayser did part Berlin; he was accepted as an accordion stu- of his Ph.D. work. The three groups have dent at the Weimar conservatory but eventu- formed a consortium that in 2008 bagged ally chose to study biology at the University of a €6.5 million grant from the Netherlands Leipzig in 1989. Two months later, the Berlin Organisation for Scientiﬁc Research. Wall came down. Kayser said it was relatively easy to setHis first experiences with forensic sci- tle in Holland—although he misses sourence left him frustrated. Between 1994 and dough bread and other things German. From 1998, at Humboldt University in Berlin, Kay- his 10th-ﬂoor ofﬁce, he has a view of Rotser studied microsatellites—short, repetitive terdam’s ultramodern skyline. And although DNA sequences—on the human Y chromo- he would have preferred a city with more hissome. The work, carried out mostly at the uni- toric character, he feels he can’t complain: versity’s Institute of Legal Medicine’s genetic After all, the Germans ﬂattened Rotterdam’s research lab, had obvious forensic applica- ancient center in 1940. tions; in rape cases, for instance, evidence often comes from vaginal swabs that contain Eyes, hair, and skin a mix of DNA from perpetrator and victim. The division of labor within Kayser’s Zooming in on the Y chromosome ensures 15-person lab reﬂects the stages within the that you’re looking at the rapist’s DNA. full monty. Some researchers are primarKayser’s work helped lay the groundwork ily focused on ﬁnding the genes and genetic for male identiﬁcation using the Y chromo- markers that underlie phenotypic differences, some, a now widespread method. But Kayser utilizing so-called genome-wide association says it took a long time for the technique to studies, for instance. Others are working on become accepted in forensics. “Forensic sci- turning what’s found into actual forensics entists aren’t the most open-minded people tests and validating them—a laborious prowhen it comes to innovative science,” he says. cess governed by international standards. “It was discouraging.” He decided to join Eye color was low-hanging fruit. Over the anthropological geneticist Mark Stoneking’s past 2 years, his group has developed a test, lab at Pennsylvania State University, Univer- called Irisplex, based on the identity of just six sity Park. Stoneking was piecing together the so-called single-nucleotide polymorphisms. human history of Polynesia with mitochon- It can now predict with over 90% accuracy drial DNA, which is inherited via the maternal whether someone has blue or brown eyes— line. Kayser’s expertise with the Y chromo- not perfect, says Kayser, but a lot better than some—which tells the male story—complemented this work perfectly, Stoneking says. Predictable? Many visible traits are at least in part Less than a year later, Stoneking moved genetically determined and may be predictable to the Max Planck Institute for Evolutionary from a person’s DNA—but the only validated test Anthropology in Leipzig. Kayser joined him so far is for eye color. www.sciencemag.org SCIENCE VOL 331 Published by AAAS

eyewitness testimony, which research has shown to be off the mark at alarming rates. Last month, Kayser’s team published a paper in Human Genetics that indicated hair color, too, can be predicted fairly accurately, at roughly 90% for red or black hair and 80% for blond or brown. Those ﬁndings are now being translated in a test kit too—most likely combined with eye color, so as to save precious DNA. Skin color is the next candidate. Although his team and others have identiﬁed some of the genes involved—they overlap with those for eye and hair color—the picture isn’t complete yet. Beyond that, forensic DNA phenotyping quickly gets complicated. Height, for

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the ﬁeld. “We are not doing anything else than what police are doing with eyewitnesses,” he says. Except for one thing, he asserts: DNA will prove a more reliable witness.

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instance, is known to have a high degree of heritability: Although diet plays a role in how fast and tall people grow, much of the variation between people is caused by genes. Indeed, a genome-wide association study among 180,000 people published last year revealed more than 180 genetic loci that appear to inﬂuence adult height—but together, they account for only 10% of the variation between individuals. De Knijff believes height to be so complex that a useful DNA test is “a bridge too far.” The age test offered a different challenge, as it’s not something one can easily read in a person’s genome. People have tried to predict age by counting the number of mutations in a person’s mitochondria, or measuring the length of their telomeres— the protective caps on the ends of chromosomes, which fray as we age—but both pose practical problems. Kayser’s method instead relies on circles of leftover DNA stored in T cells whose amount decreases as people get older. The ﬁeld could also target traits like hair structure, baldness, handedness, and earlobe attachment. Its greatest triumph, however, would be that computer-generated, DNA-based facial sketch. Kayser’s group is working on it as part of a consortium of labs

called VisiGen that Kayser founded with Tim Spector of King’s College London. Fan Liu, a genetic epidemiologist in Kayser’s lab, is trying to link genome data to key facial morphology traits, such as facial width or nose size. Kayser is cautious about the prospects. “On paper, it’s possible,” he says. The extreme resemblance between monozygotic twins suggests that facial features are mostly genetic. “But we have no idea yet what genes they are or how complex it is.” Ingrained racism Kayser’s department is also working on tests that can determine someone’s biogeographical ancestry. The technique appears to have been used in hundreds of crime investigations in the United States, but modesty is still in order, says Kayser. Although several companies offer detailed ancestry tests to the public, scientists don’t have a strong basis to go much beyond the continental level—that is, predicting whether someone is, say, European, sub-Saharan African, East Asian, or Native American. Distinguishing a Norwegian from a Swede, for example, is not usually possible, and Kayser rejects as “totally baseless” a controversial U.K. program to use DNA to determine asylum seekers’ nationality (Science, 2 October 2009, p. 30).

This application of forensic DNA phenotyping is an area ﬁlled with explosive issues about race and crime about which the debate hasn’t fully begun. Most forensic DNA phenotyping predictions will likely come with a signiﬁcant level of uncertainty, as opposed to conventional DNA fingerprinting matches, and police ofﬁcers may have trouble interpreting them. Moreover, genetic ancestry does not equal race, a concept that most scientists shun because it has no well-deﬁned meaning, and does not necessarily predict someone’s appearance. “Ancestry and appearance overlap, but they’re not the same,” Kayser says. Just how important caution is was driven home to Kayser by a series of psychology studies, published in 2004, that showed how deeply ingrained stereotypes about black men and crime are among U.S. law enforcement ofﬁcers. “It was an eye-opener to me,” he says. It will be important for scientists like him to explain the uncertainties in DNA-based phenotyping carefully, he stresses. Fortunately, says Budowle, Kayser is unlikely to oversell his science to overzealous cops. “Manfred knows the molecular biology but also the population genetics and the statistics,” he says. “He won’t overstate the evidence, and he’ll make clear what the limitations are.” –MARTIN ENSERINK

Emerging Forensics Field May Hit Legal, Ethical Obstacles A year after a 16-year-old girl was brutally raped and murdered in the Netherlands in 1999, forensic geneticist Peter de Knijff broke the law himself. At the police’s request, he set out to determine the geographic ancestry of the murderer from DNA in his semen. That was, he later admitted, “completely illegal” under Dutch law, which at the time allowed using DNA for traditional DNA identiﬁcation but not for determining race, looks, or disease risk. De Knijff has no remorse. The police were unable to solve the case, and tensions were escalating in the rural community where the girl lived. Many pointed ﬁngers at a nearby hostel for Kurdish, Iraqi, and Afghan asylum seekers. De Knijff’s analysis showed that the killer was most likely from northwest Europe, which helped cooler heads prevail. His dilemma could arise any day in many countries. In the wake of the murder—still unsolved today—the Dutch parliament adopted a law in 2003 regulating forensic DNA phenotyping, the use of DNA samples to predict a suspect’s ancestry or physical characteristics (see main text). But the Netherlands is still the only country to have done so. Countries such as Belgium and Germany—and the U.S. states of Indiana, Rhode Island, and Wyoming—explicitly ban the practice, says BertJaap Koops, a professor at Tilburg University Law School who studies the issue. In the United Kingdom and most U.S. states, forensic DNA pheno-typing isn’t explicitly regulated but is allowed under existing laws for forensic DNA. Geographical ancestry tests appear to have been used in several hundred U.S. investigations, says Pamela Sankar, a bioethicist at the University of Pennsylvania. Most were done by a company called DNAPrint Genomics, she says, which ceased operations in 2009. Sankar, who is tracking the ﬁeld with funding from the U.S. National

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Human Genome Research Institute, says forensic DNA phenotyping raises ethical and legal issues that need more debate. Her own research, for instance, suggests that police ofﬁcers may not realize that—as opposed to the high degree of certainty in DNA ﬁngerprinting—phenotyping is probabilistic in nature. Cops could also use ancestry predictions, however imprecise, as an excuse to target minority populations, she says. Other dilemmas loom. In theory, knowing that a criminal is, say, a hemophiliac or a diabetic could help nab him. But the 2003 Dutch law categorically bans looking for disease-related genes because it would violate a suspect’s privacy and the “right not to know,” a basic tenet of medical ethics. (The law allows only the prediction of visible traits and “race.”) Koops argues that these restrictions are overly protective; if genetic sleuthing can reveal that a killer has a very mild or curable disease, or is likely to be a chain smoker, solving the crime might well trump individual rights, he says. The distinction between visible traits and disease is not always tenable anyway, says forensic geneticist Christopher Phillips of the University of Santiago de Compostela in Spain. A mutation in a gene called MC1R can cause pale skin, for instance—which increases the risk of melanoma. “Predictive tests should conﬁne themselves to the visible,” he says, “but sometimes this encroaches on the private at the same time.” The paradox is that conventional DNA fingerprinting has been introduced with the reassurance that it can never reveal any personal information—but now, it’s personal information that forensic geneticists are after. That’s why caution is important, says Phillips. “If we move too quickly, the whole ediﬁce of public conﬁdence in DNA proﬁling could erode.” –M.E.

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NASA Weighs Asteroids: Cheaper Than Moon, But Still Not Easy

CREDITS (TOP TO BOTTOM): ©DANIEL DURDA/FIAAA; NASA/JHUAPL/ISAS/JAXA/EMILY LAKDAWALLA

Budgetary realities are driving U.S. astronauts away from the moon toward near-Earth asteroids, but the new goal on the way to Mars should prove uniquely challenging

gravity-free asteroid would be like taking a spacewalk rather than landing on the moon, but without a space station’s handholds.

knows of even one NEA that would clearly serve as a practical ﬁrst target for astronauts. And no one knows what the first visitors will ﬁnd at a tiny, nearly gravity-free body, except that it could be both bizarre and dangerous. From what they do know, planetary scientists say, meeting the president’s 2025 schedule would be a heck of a rush requiring considerable investment.

Asteroids are about to get a new reputation. appointed lunar scientists, naturally enough, For decades, those that pass near Earth on but planetary scientists who study the small their circuits about the sun—the so-called bodies of the solar system are delighted. For near-Earth asteroids (NEAs)—have played them, the redirection promises a bonanza of the heavy. A huge NEA 10 kilometers across new data that had seemed beyond their reach killed off the dinosaurs, after all. And there for decades to come. And astronomers look- A bridge too costly was that tiny, perhaps 40-meter-diameter ing for that next catastrophic impacter would Mars has long been the ultimate goal of NEA that leveled 2000 square kilometers of get a boost just as their ongoing search gets a U.S. space exploration. Presidents George Siberian forest in 1908. Earth will inevitably lot harder (see sidebar, p. 843). H. W. Bush and George W. Bush promulcollide with more, both huge and small. But the same planetary scientists who gated national space policies that would But NEAs could soon be redeemed. would most beneﬁt from an NEA-studded have humans setting foot on Mars in the They recently became the next prized des- “flexible path” to Mars are warning that coming decades. In the past decade, NASA tination in the U.S. human exploration of the new route looks bumpy. So far, no one took up George W. Bush’s plan, spending space. President Barack $9.1 billion on the developObama has declared that the ment of a giant rocket that path to landing astronauts on could get astronauts to the Mars is dotted with NEAs. moon by 2020, paving the Rather than f irst returnway for a permanent base. ing astronauts to the moon Only then would NASA turn before heading off to Mars, toward Mars. NASA will be aiming for But this moon-ﬁrst stratasteroids as steppingstones egy wasn’t going to fly, in the human exploration of according to a committee set deep space. Smaller still. Spacecraft have visited two near-Earth asteroids, big Eros (left) and up by Obama and headed by The new approach has dis- little Itokawa. The ﬁrst target for astronauts would likely be just 100 meters across. Norman Augustine, former www.sciencemag.org SCIENCE VOL 331 Published by AAAS

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Where do I stand? Visiting a small, nearly

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But wait Extending humans’ reach into deep space within 15 years is all well and good, NEA researchers say, but there are some costly and time-consuming hurdles to be cleared. First, someone needs to ﬁnd a suitable asteroid. “There just aren’t very many targets out there,” says NEA researcher Alan Harris of the Space Science Institute in La Cañada, California. “Even though there are thousands and thousands of objects out there, there are only a few tens that will ever ﬁt the bill.” Some NEAs are too small, little larger than the craft that would carry astronauts to them. Some spin so fast—with a “day” of minutes or an hour or two—that they couldn’t be approached and would fling away any astronaut touching down on them. The toughest requirement is finding an asteroid with an orbit enough like Earth’s that astronauts could catch up with it using a practical amount of fuel and get back to Earth in a reasonable amount of time—say, 6 months. On to an asteroid A NASA study of possible The Obama Administration asteroid targets conducted last went for the ﬂexible path, aimfall came up empty-handed. ing at visiting an asteroid, which “We’ve found only a handcan have nearly no gravity well ful of objects accessible in the at all. Going to an asteroid 2025 to 2030 time frame,” says would not require any construcLindley Johnson, NASA protion on the surface, but NASA gram executive for near-Earth would acquire experience in object observations, “all of long-duration, deep-space operthem quite small and not parations needed for the trip from ticularly attractive targets.” All Earth to Mars. By 2025, “we’ll were smaller than 50 meters start by sending astronauts to an in diameter; objects about the asteroid for the ﬁrst time in his- A matter of scale. Asteroid Itokawa dwarfs the international space station (lower length of a football field or tory,” Obama told an audience right) and the Orion crew exploration vehicle (upper right). The 50-meter-long pitch are thought more desirat Kennedy Space Center last boulder Yoshinodai on Itokawa is about the size object astronauts would visit. able. So right now, Johnson April. As for the moon, he said, says, there’s no steppingstone “I just have to say pretty bluntly here: We’ve NASA headquarters. The notional schedule known on the way to Mars around the presibeen there before.” through 2018 has major missions to an NEA dent’s target date. Those were harsh words for lunar sci- launching in 2014 and 2017 that would cost They’re out there, everyone agrees; it’s entists, or “lunatics,” as some call them- $640 million to $840 million each and a third just a matter of looking harder. In a report selves. In the past few years, lunar science NEA mission launching 2014 that would run in October on the threat of impacts on Earth, has experienced a renaissance as the Euro- $100 million to $200 million. a National Research Council committee pean Union, Japan, China, India, and the These missions would check out poten- looked at two ways of searching for small United States sent robotic missions into tial targets for human exploration, but NEAs. The cheaper way of ﬁnding 90% of lunar orbit. Sending humans to the moon Jenkins did note the “tremendous poten- objects 140 meters in diameter and larger motivated both U.S. and Chinese efforts, tial for collateral beneﬁt” for NEA science. passing near Earth is using a telescope on the which included NASA’s $504 million Lunar Lunar science would not fare so well. Only ground, the committee concluded. But even Reconnaissance Orbiter (LRO) still orbit- one mission would target the moon, sending for these larger objects, the survey would ing the moon as well as the LCROSS mis- a tiny rover to the surface in 2015 to verify not be completed until late in the 2020s, far sion that “bombed” the moon for water ice some of LRO’s observations. Laurie Leshin, too late for Obama’s goal. A telescope with of potential use to astronauts. NASA deputy associate administrator in the a better vantage point, one orbiting the sun Whatever the motivation, the rush to the Exploration Systems Mission Directorate, inward of Earth’s orbit, would cost more moon has revitalized lunar studies. At the says that even then, most funding early on than a ground-based telescope—perhaps Lunar and Planetary Science Conference would go to developing NEA missions, put- half a billion dollars—and involve greater held each March in Houston, Texas, the num- ting the modest lunar rover mission on the risk, but it could complete the survey faster, ber of moon sessions has more than doubled back burner for the moment. “perhaps as early as 2022.” Given the need

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since 2007 to ﬁve times the number of asteroid sessions and equal to the number of sessions on perennial favorite Mars. Researchers’ attachment to our nearest neighbor is stronger than ever. “It’s not just any moon, it’s our moon, with a shared history with Earth,” notes planetary scientist David Morrison of NASA’s Ames Research Center in Mountain View, California, who has been involved on both the lunar and asteroid sides. Now, in the wake of Obama’s speech, “small NEAs are it,” says Morrison, at least in the United States. The president’s ﬁscal year 2011 budget request (yet to be taken up by Congress) more than triples NASA’s budget for the search for NEAs to $20.3 million. Any newly discovered NEAs could be the targets for a series of robotic missions called for in the budget request, as outlined last September at a NASA meeting in Washington, D.C., by Jason Jenkins of

CREDIT: P. ABELL ET AL., METEORITICS & PLANETARY SCIENCE 44, 12 (DECEMBER 2009)

CEO of Lockheed Martin. “The U.S. human spaceflight program appears to be on an unsustainable trajectory,” its September 2009 report found. The Augustine committee did see options for returning to the moon under increased NASA budgets, but its novel contribution was the “Flexible Path.” This is an approach that leads—at least for years if not decades to come—anywhere but to the moon’s surface: to lunar orbit, to gravitationally balanced holding patterns near Earth called Lagrange points, to NEAs, and to the tiny moons of Mars. The strategy would be to put off the considerable expense of a vehicle able to ease astronauts onto the lunar surface and lift them off again against the moon’s pull, not to mention the cost of building an outpost on the surface. The idea, says Augustine committee member Christopher Chyba of Princeton University, was “let’s avoid going into gravity wells as long as we can.”

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A Windfall for Defenders of the Planet

CREDIT: ©DANIEL DURDA/FIAAA

for robotic scouting missions in the 20-teens and a likely requirement for backup targets, says Morrison, “we really need to get cracking. We all have 2025 looming.” Another challenge unique to the asteroidﬁrst approach is tiny NEAs themselves. On a 100-meter NEA, an astronaut would weigh something like 10 grams, Harris notes, space suit included. Operating around such an object would be like a spacewalk around the international space station, says Morrison, but without the built-in handholds. And making handholds or installing instrumentation on the surface could be a dicey business, says planetary dynamicist Daniel Scheeres of the University of Colorado, Boulder. Hundred-meter NEAs could be “rubble piles” of boulders, cobbles, and pebbles held together, barely, by their own microgravity. Stepping onto the surface could be “like jumping into a pit of Styrofoam ‘peanuts,’ ” he says. Once kicked up, dust and pebbles could take hours or days to settle out. Even NEAs of equal size could

Inbound. Asteroids will pass

near Earth this century, but none are known that will hit. targets before 2020. And the asteroids easiest for astronauts to reach happen to be the ones most likely to strike Earth. So human exploration could pick up the half-billion-dollar-or-more tab for an NEA search that the NASA task force recommended. In addition, the precursor robotic missions needed to inspect potential targets for later astronaut visits would provide information on the physical makeup of NEAs crucial to deciding how to nudge a threatening NEA off its collision course with Earth. Human space exploration could also beneﬁt planetary defense by prodding governments to prepare for the inevitable. “Increasing [NEA] surveys are going to create many worrisome situations” that no one is yet ready for, says asteroid researcher Richard Binzel of the Massachusetts Institute of Technology in Cambridge. A newly discovered NEA may have a small but signiﬁcant chance of hitting Earth that will take years to evaluate. “We should be ready,” says Binzel. There are early signs that we could be. In October, at the direction of Congress, the president’s science adviser, John Holdren, outlined a plan to notify federal agencies and emergency response organizations in case of an impending threat and recommended that NASA be the agency responsible for protecting the United States from a speciﬁc threat. –R.A.K.

have different reactions to the tread of astronauts because NEAs come in rocky, metallic, and crumbly carbonaceous versions. Is the moon in the stars after all? Although Obama’s vision for space exploration has often been said to “bypass the moon,” it may not happen that way. “I still don’t think a rational person would try to go to Mars without going to the moon ﬁrst,” says geochemist David Kring of the Lunar and Planetary Institute in Houston, principal investigator of the institute’s Center for Lunar Science and Exploration. Actually, the Augustine report leaves that possibility wide open. “When we said ‘ﬂexible path,’ we meant it,” says committee member Chyba. The moon “is not the near-term objective,” but a decision to return humans to the moon could be made further down the road, he says. Moon advocates argue that the return should come soon. The moon is, after all, our nearest near-Earth object, they note. It’s easier to get to than an NEA whizzing

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If U.S. astronauts hopscotch from one near-Earth asteroid practicing for their trip to Mars (see main text), it will be a big help to astronomers campaigning to ﬁnd and fend off the rare NEAs that threaten to collide with Earth. With a nudge from Congress, NASA-led astronomers have now reached a major milestone in their search for threatening NEAs. After a decade-long concerted hunt, they have found enough of the largest NEAs to reduce their estimate of the potential hazard by about 90%. That’s because they have now found about 90% of the estimated 1000 NEAs a kilometer across and larger that could have been on a collision course with Earth. It turns out that none actually are, so the risk in this century of a civilization-ending impact is essentially gone. But that leaves the other part of the threat, the one from objects 140 meters in diameter and larger—so-called city killers. Only about 5% of them have been found under NASA’s search program. In 2005, Congress directed NASA to identify 90% of city killers by 2020, but so far, Congress has provided no additional funds for its mandated search. Funding has been about $4 million a year recently, raised to $6.5 million last year. But at current discovery rates, most of the city killers would remain undetected for many decades. In October, an ad hoc task force on planetary defense recommended to the NASA Advisory Council that NASA should meet the 2020 congressional target date using a space-based telescope rather than the slower ground-based option. And the president’s ﬁscal year 2011 budget request for NASA (not yet acted on by Congress) includes a boost in search funding from $6.2 million to $20.3 million. Planetary defense may be in line for a boost, but the present Congress is in a serious budget-cutting mood. That’s where the human exploration of space could come in. If NASA hopes to meet the president’s goal of sending humans to an NEA by 2025, the search for small NEAs will have to be at least as fast as that required to meet the 2020 planetary defense goal. That’s because NASA would need to identify potential exploration

by on its way around the sun. It offers a far more complex setting beﬁtting the problemsolving skills of on-scene astronauts. Much more remains to be learned about the moon chemically, mineralogically, and geologically than about asteroids, they say; samples from asteroids—meteorites picked up on Earth— number over 50,000, whereas there are little more than 2000 samples with which to unravel the moon’s more complex history. The strongest argument, moon advocates say, is gravity: The moon has it and NEAs don’t. Most crucially, the safe landing and return of astronauts could be demonstrated only on the moon, not to mention astronaut activities on a solid surface. But that may not help. The president’s own National Space Policy, released in June after his asteroidﬁrst speech, directs that the NASA administrator shall “by the mid-2030s, send humans to orbit Mars and return them safely to Earth.” His policy for the next 30 years says nothing about landing on Mars.

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–RICHARD A. KERR

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Europe’s Eager Reformer Takes on Framework Funding Goliath An outsider to the scientiﬁc community, the E.U.’s new research commissioner promises to cut red tape, champion basic research, and ﬁght to save ITER For Irish teacher-turned-politician Máire Geogheghan-Quinn, landing one of Europe’s top political jobs in charge of research has meant a steep learning curve. Having served her country in ministerial roles covering Gaelic culture, justice, and European affairs, she then spent 10 years overseeing European Union ﬁnances. But in her ﬁrst year as E.U. commissioner for research, innovation, and science, she has had to come up with plans to reengineer the European Union’s huge €54 billion research-funding program, prepare for a battle over budgets, ﬁnd extra funding for the troubled ITER fusion reactor project, and has managed to raise the proﬁle of science policy at the European Union’s highest levels. She spoke with Science last week in her Brussels ofﬁce. Her remarks have been edited for clarity and brevity. (You can read more of this interview at http://scim.ag/MGQ.)

–GRETCHEN VOGEL

Q: You weren’t known for much involvement with science before taking this job. What’s the most provocative or most interesting bit of science you’ve come across in your ﬁrst year?

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M.G.-Q.: Oh, there have been lots of things, but I suppose at the very beginning when I went to one of my ﬁrst meetings at the ERC [European Research Council], there was a professor from Italy who was getting a large grant to get this driverless truck to go from Europe to Shanghai. And that was just mindboggling. I said to myself, “It’s not going to happen.” And yet it did. Later, I told everybody about it: “I was there when he was telling us about this, and I had a doubt in my mind!” To me it proves that the ERC is about championing researchers with good ideas who would never get that kind of substantial money from a member state. So there are fantastic possibilities in what they do. And it has made me ever since a real strong supporter and probably the ERC’s greatest fan. Q: If that’s the case, ERC leaders are hoping for a big boost in funding … M.G.-Q.: Aren’t we all! Q: They’re hoping for as much as €24 billion between 2013 and 2020. What’s your reaction? M.G.-Q.: My reaction is that we’ve only just

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CREDIT: EUROPEAN COMMISSION

N E W S M A K E R I NT E RV I E W: M Á I R E G E O G H EG H A N - QU I N N

Q: The green paper you issued last week on future E.U. research funding included few concrete proposals, which disappointed some observers. What do you say to them? M.G.-Q.: The green paper is the start of the conversation. I don’t think you should ever start a conversation by laying down in black and white the answers you want. So instead you pose questions, you put it out there. We are very anxious to encourage the scientiﬁc community and the other stakeholders to really get involved and engaged in this conversation, because this is an opportunity to really change the whole landscape of the way we fund research and innovation. I want to help the people who have said to me, “Look, we’re overloaded with the E.U. administrative burden. The bureaucracy has gone mad. There’s so much red tape that if we could ﬁnd the money elsewhere, we wouldn’t come to the European Union.” And to me that’s a tragedy. In looking at all of this, we looked at what does a small research center have to do when they come for the funding? If they’re going to the Framework Programme, they go to one postbox. If they are going to the CIP [Competitiveness and Innovation Framework Programme], they go to another. If it’s contributions to the EIT [European Institute of Innovation and Technology], then it’s another. So let’s bring it all together, under one framework so that there is one postbox: one simple, unique set of rules so that it cuts out all the extra paperwork that people have to go to— and the expense that they have to go to—to put together an application. For the moment we’re calling it the Common Strategic Framework, but that’s not going to be the name.

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started the budget question. No ﬁgures are on the table, and no ﬁgures will be on the table for quite some time. First of all, I have to make a pitch for my two directorates general. And depending on what we get out of that, I hope to be in a position to be able to strengthen the ERC. Unfortunately, we’re in the hands of two other institutions—the Council [of Ministers] and the [European] Parliament—who decide whether or not to give us an increased budget and what that increase might be. I think it will be one of the most difﬁcult budgetary discussions that the E.U. has ever had, mainly because of the ﬁnancial crisis. I think you’ll ﬁnd that a lot of those member states who have had to take deep cuts in their own budgets will try and curtail any increases in the E.U. budget. But within all of those possibilities, the ERC has a very strong supporter in myself.

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Q: Some worry that the new funding program will focus on “innovation” and that basic science will be squeezed. What do you see as a proper balance between the two? M.G.-Q.: You can’t have innovation, as I keep saying, unless you have really excellent basic research and unless you give support to excellent basic research. My problem is that we have had this wonderful, excellent research here. We have delivered the goods, as it were, on the research side. But we have failed to bring that research all the way to the marketplace. It has been brought to the marketplace elsewhere, outside the E.U. And I want to see the excellent research that we do here brought to the market here. But we can’t have innovation of any kind unless you have basic research. So my commitment is total when it comes to basic research. It is so vital and so important.

CREDITS (TOP TO BOTTOM): F4E/ITER; VIAC/VISLAB

Who’s paying and who’s driving? The ITER fusion reactor project under construction in France (top). ERCfunded driverless solar-powered trucks (bottom) at the Shanghai Expo.

Q: Under your proposal, EIT would become part of the new, expanded program. The scientiﬁc community has viewed EIT with a healthy dose of skepticism. What value do you think it will really add to European research? M.G.-Q.: I think it’s very, very early to be judging something that has just been set up. [European Commission] President [José Manuel] Barroso, as I understand it, went to MIT [the Massachusetts Institute of Technology] and was very impressed with what he saw. Now MIT has taken quite some time to become as successful and well-known as it has become. [The EIT] needed to have security, and I think the EIT is very happy to be within the Common Strategic Framework now. I think it sees it has a home. It’s like a sapling; we have to help it to grow and nourish it. I really do believe that once it is up and running, it’s going to be a very, very positive element within the E.U. Q: How is the commission intending to cover the €1.4 billion shortfall in ITER funding for 2012 and 2013, and how will further cost increases be contained in the Common Strategic Framework? M.G.-Q.: ITER. [Laughs] Wonderful. When I came into this ofﬁce almost a year ago now, one of the ﬁrst ﬁles that was put on my

desk was the ITER ﬁle, and I wanted to run out the door and go home. This project was badly managed. There were issues not just in relation to the ﬁnancing of it, there were also issues of governance that needed to be tackled. Those were resolved, at both the European and international level. Then we sat down to discuss with the budget commissioner and the commission as a whole how we might ﬁll this [laughs] “hole,” as it were, for 2012 and 2013. And we agreed that two-thirds of the money would come from unspent funds and one-third from the Framework Programme. We had that package put together, and we went to the parliament and the council. And suddenly you had the commission caught in a row between the parliament and the council. That row had nothing to do with ITER. [The ITER package was cut out of the budget deal.] As a result, we now have to restart the whole thing again. We still have the two-thirds/one-third on the table. [Budget] Commissioner Lewandowski has been strongly supporting that and pushing that. And we have to live in hope and watch what happens with the parliament and the council. It’s very hard for our international partners to understand. When the U.S. government makes a decision, it’s implemented.

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Q: Will there be a European chief science adviser? M.G.-Q.: It will absolutely happen. President Barroso is very committed to a chief scientiﬁc adviser. I know he is looking at a list of people that’s been drawn up and trying to decide what the best solution would be. I’m very anxious for it to happen. I think it strengthens the whole area of research and science, which is what I’m interested in doing. You know, for us to have a European Council meeting just last week that discussed energy and research and innovation was almost a miracle when you consider everything else that was going on. And I think discussing research and innovation proved that the European heads of government realize that it’s an economic policy. And it’s the policy that will bring us the growth, the competitiveness, and the jobs. That shows the tremendous importance that is now attached to the whole area of research and innovation. When [U.S.] President [Barack] Obama gave his State of the Union address, I thought it was fantastic. It happened just before the European Council, and it reinforced once again how important research and innovation is on a world scale. It shows that on both sides of the Atlantic we’re competitors, but there are lots of things I believe we should cooperate on in order to compete with the rest of the world. There are lots of ways that we do cooperate, but we should even intensify our cooperation.

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The E.U. works in a different way. The commission makes a proposal, but then it’s in the hands of the parliament and the council to decide, and they have to agree to do it.

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VISUALIZATION CHALLENGE AN “OCEAN” COMPOSED OF A SINGLE LAYER OF MOLECULES; AN INTRICATE depiction of an HIV particle as a study in orange and gray; a phantasmagoria of fungi; a video tracing the long-distance travels of items dumped in the trash in Seattle: The four ﬁrst-place winners in this year’s International Science & Engineering Visualization Challenge grab your attention and draw you into unseen worlds in very different ways. Researchers are generating mind-boggling volumes of data at exponentially increasing rates. The ability to process that information and display it in ways that enhance understanding is an increasingly important aspect of the way scientists communicate with each other and—especially—with students and the general public. That’s why, for the past 8 years, Science and the U.S. National Science Foundation (NSF) have co-sponsored annual challenges to promote cutting-edge efforts to visualize scientiﬁc data, principles, and ideas. This year’s awardees span scales from nanoparticles to colliding galaxies, and from microseconds to millennia. We received 111 entries from 63 countries (U.S. entries came from 24 states). A committee of staff members from Science and NSF screened the entries, and an outside panel of experts in scientiﬁc visualization reviewed the ﬁnalists and selected the winners. The winning entries are featured on the following pages, in a slideshow at www.sciencemag.org/special/vis2010/, and at www.nsf.gov/ news/scivis. Some entries were put together by large teams, not all of whose members could be listed in print; the online presentations provide full details. Tarri Joyner of NSF and Esther Chon of Crabtree + Company organized this year’s challenge. Kristen Minogue of Science’s News staff wrote the text that accompanies the images in this special section. We encourage you to submit applications for next year’s challenge, details of which will be available on NSF’s Web site, and to join us in celebrating this year’s winners.

Patrice Legro Marian Koshland Science Museum Washington, DC Thomas Lucas Thomas Lucas Productions Ossining, NY Alisa Zapp Machalek National Institute of General Medical Sciences Bethesda, MD Corinne Sandone The Johns Hopkins School of Medicine Baltimore, MD Tom Wagner NASA Headquarters Washington, DC

–JEFF NESBIT, DIRECTOR, OFFICE OF LEGISLATIVE AND PUBLIC AFFAIRS, NSF COLIN NORMAN, NEWS EDITOR, SCIENCE

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PLACE: Ivan Konstantinov Yury Stefanov Aleksander Kovalevsky Yegor Voronin Visual Science Company

At ﬁrst glance, it could pass for a piece of crochet, a ﬂuffy gray and orange ball. But its real-world counterpart is far more destructive: It claims an estimated 2 million lives a year and has wreaked more global havoc than some wars. Ivan Konstantinov’s winning illustration reduces HIV to unnerving simplicity. His team at the Visual Science Company in Moscow spent months combing through the latest research, compiling data from more than 100 papers and assembling the information into a coherent image of a 100-nanometer HIV particle. They depicted the proteins in just two basic colors: Gray equals host, orange equals virus. HIV breaks into immune cells and hijacks their genes. The orange proteins on the outside bind to the immune cell, letting the viral core slip inside. Once in, it fuses with the cell

membrane (gray shell), turns its viral RNA into DNA, and integrates into the cell nucleus. The host cell then starts making viral proteins, turning into a virus factory. The restrained, two-color system worked for the judges. “It uses material from the host to sort of wrap itself in this membrane,” says panel of judges member Corinne Sandone. “That point is brought home much clearer than another example of that same model that might be coded with eight to 10 different colors.” In addition to the stark color scheme, the image of the particle split open to reveal its viral core itself deeply shook the panel, says panel of judges member Tom Wagner: “You have this gaping mouth that almost looks like it’s ready to eat you the way AIDS is eating away at society.”

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(3-way tie)

AraNet: A Genome-wide Gene Function Association Network for Insuk Lee Michael Ahn Edward Marcotte Seung Yon Rhee Carnegie Institution for Science

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Picture DNA on Facebook. The image to the right is a map of links between the genes of the mustard plant Arabidopsis thaliana. Genes involved in the same biological process are connected by lines: red for more certain links, blue for less certain links. “It’s not unlike a social network,” says biologist Seung Yon Rhee.

Enterobacteria Phage T4 Jonathan Heras Equinox Graphics Ltd. One judge compared this illustration of a virus attacking an Escherichia coli bacterium to something out of a 1950s science-ﬁction ﬁlm. That’s not too far from the creator’s view, either. Before creating it, chemical engineer Jonathan Heras says he knew almost nothing about viruses. When he ﬁrst saw a depiction of one in a textbook, he admits not believing it, until he looked at microscopic images: “It really did have these spindly legs and this really alien, weird appearance.”

Proposed Structure of Yeast Mitotic Spindle The Mitotic Spindle Group* University of North Carolina, Chapel Hill This is a depiction of a yeast cell about to divide, a stage biologists call metaphase. Green microtubules prepare to pull apart 16 pairs of chromosomes (yellow) in a process scientists still don’t fully understand. It’s the product of 2 years of collaboration among biologists, physicists, computer scientists, and artists, but it’s still a work in progress. “This is our version 20 of probably 50 we’re going to end up doing,” says computer scientist Russell Taylor. *www.cismm.org/research-collaborations/cell-mechanicscluster/mitotic-spindle

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PLACE: Kandis Elliot Mo Fayyaz University of Wisconsin, Madison

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For senior artist Kandis Elliot, postermaking is one of the best tasks of the job. Her series of educational posters started 4 years ago, when greenhouse and garden director Mo Fayyaz of the University of Wisconsin (UW), Madison, asked for a fruit poster. Introduction to Fungi is just the latest—and one of the hardest, because the botany department lacks a mycologist. And Elliot didn’t want to settle for a simple mushroom poster. “There’s a gazillion of those things,” Elliot says. “We wanted something that shows fungi as mushrooms but something more than mushrooms. Your beer, your wine, and your bread. The stuff on the back of your fridge.” The beer and wine are easy enough to spot in the center of this poster. Other specimens include gourmet delicacies, such as trufﬂes and the mold on bleu cheese, and the less savory stinkhorns, whose stench attracts carrion beetles to disperse their spores. It also features some unfriendly fungi, such as the culprit behind white-nose syndrome, a mysterious white fungus that grows on hibernating bats and seems to kill them by leading to starvation. “The fungi poster was a clear winner. That was just amazing,” says panel of judges member Alisa Zapp Machalek. Besides the imagery, the fact that it was about fungi had an appeal of its own. “There’s sort of an innate intrigue factor. If it was different kinds of apples, even though I love apples, I don’t know that it would hold our attention as much.” “It’s very appealing to the layperson,” says panel of judges member Corinne Sandone. “There’s nothing that hard. It’s very accessible.” That’s valuable praise for Elliot, who says squeezing all the information in was one of the hardest parts. “There’s like 25 pounds of information on a 5-pound poster, and you want to put it in some kind of logical order,” she says. “There is order in there, but you kind of have to search for it.”

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HONORABLE MENTION Everyone Ever in the World Peter Crnokrak The Luxury of Protest

The poster represents roughly what the title says: every person who lived and died on the planet, from 3200 B.C.E. to 2009 C.E. The total paper area represents the 78 billion people who lived over the past 5000 years. The gaping hole in the center represents every person who died in a major war, genocide, or massacre: approximately 969 million people, or 1.25% of the total number of people who have ever lived on the planet. Text is printed in transparent ink on plastic (shown as white on black, right). The circles at

the top represent the number of conﬂicts per millennium with more than 1000 deaths, and the circle of text lists them by name. The bottom circle represents the expected number of conﬂicts in the next millennium if the escalating pattern continues. Human life is one of the few values that’s almost always given as an absolute (1100 died in a ﬂood in Pakistan; 20 million Russians died in World War II), says Peter Crnokrak of The Luxury of Protest design studio in London. Framing deaths as a percent of those who ever lived, he says, might risk degrading the value of individual life. But he says he wanted to create something thought-provoking, and the judges say he succeeded. “People have made the case that we need to bring more artists and poets into the science process to explain the results,” says panel of judges member Tom Wagner. “And I think a poster like that achieves that goal.

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PLACE: Seth B. Darling Argonne National Laboratory Steven J. Sibener University of Chicago

Don’t take the title literally. The ripples Seth Darling of Argonne National Laboratory and Steven Sibener of the University of Chicago, both in Illinois, captured with an atomic force microscope may look like the surface of an ocean, but they are a mere nanometer deep, and there’s not a drop of water in sight. The rich shades of turquoise and indigo are artiﬁcial, but the choppy waves are real. They are formed by millions of molecules arranging themselves on a gold surface. These “self-assembled monolayers” come with a head that clings to the surface and a tail that sticks out into the environment. Darling compares it to dumping a bowl of wet spaghetti on the ﬂoor and “all of a sudden it stands up as if it were uncooked spaghetti on end. That’s kind of a weird thing to happen.”

The ripple effect in the image is caused by two similar molecules, synthesized by Dong-Chan Lee and Luping Yu of the University of Chicago. Each molecule has sulfur at the head, but one has carbon and hydrogen at the tail whereas the other has carbon and ﬂuorine, which leads to their heights differing by about 0.2 nanometers. Darling captured them at the moment they began separating. He hopes to ﬁnd out if surfaces like these will form larger “islands” of short and tall molecules or if they will remain blended together. Eventually, Darling says, scientists hope to use such monolayers to adjust the properties of a surface. Winning entries need good art and good science, says panel of judges member Alisa Zapp Machalek, and this photo had both. “The science was amazing, and the image was—wow.”

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HONORABLE MENTION (tie) TRICHOMES (Hairs) on the Seed of the Common Tomato Robert Rock Belliveau Downloaded from www.sciencemag.org on February 17, 2011

Tomato seeds have hair. Not the ordinary, dead protein that hangs limply off human scalps. These trichomes secrete an insect-repelling, ﬂavor-inducing mucus that helps give tomatoes their signature taste while acting as a natural bug spray. Robert Rock Belliveau took this photo of a 2-mm × 3-mm tomato seed last April. The color contrast comes from the polarizing microscope he uses, which has both transmitted and reflected light capabilities. The thinner parts at the edge of the seed (purple) are viewed with transmitted light while the trichomes on the top of the seed (red) are viewed with reﬂected light. The pathologist has been taking microscopic photos of plants ever since he retired 10 years ago. He started with desert wildﬂowers, but when a drought in Las Vegas temporarily wiped out his subjects, he switched to vegetables. Anything, from ovaries to leaves to pulp, is fair game. “Every once in a while,” he says, “you see something that’s so bizarre, it is startling!”

Centipede Millirobot Katie L. Hoffman Robert J. Wood Harvard University Imitating insects is all the rage in robotics right now. Graduate student Katie Hoffman based this 12-legged, segmented robot on the body morphology of a centipede. The top view shows the actuators that control each leg, the reﬂection shows the ﬂexible connections between the segments, and the penny gives a sense of the robot’s size. Hoffman says most robots that size mimic cockroaches, which have only six legs and much more rigid bodies. By modeling a centipede, she hopes to study how ﬂexibility and body undulations enhance locomotion. www.sciencemag.org SCIENCE VOL 331 18 FEBRUARY 2011 Published by AAAS

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PLACE: SENSEable City Lab Massachusetts Institute of Technology

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Many people know about the supply chain. They care about whether their fruit is organic, whether their chickens are freerange, whether the products they buy are domestically manufactured or imported. But the other end of the consumer chain—where all the used products go after they’re dumped in the trash—is murkier. Dietmar Offenhuber and his colleagues at the SENSEable City Laboratory at the Massachusetts Institute of Technology in Cambridge wanted to bring that side of consumerism to light. So they stuck location sensors onto 3000 pieces of trash to see where the garbage ended up months later. They recruited households in Seattle and gave them wish lists of items they wanted to track: cell phones, ﬂuorescent light bulbs, and other “household hazardous waste” that’s difﬁcult to regulate. The sensors were shielded by an insulating foam to protect them from being crushed. Over the next 2 months, they kept tabs on their garbage and plotted its journey on a U.S. map. While most of it remained in Washington state, after a week some had seeped into Oregon and Idaho. In 2 weeks, some of the cell phones had gone as far as Florida. The end of the 2 months found ﬂuorescent light bulbs in the Midwest, batteries in Michigan, and printer cartridges in Mexico. “It’s one of those knowledge is power things,” says panel of judges member Tom Wagner. “Hey, guess what, if you have complex packaging, this is what’s going to happen to it when it goes in our refuse system.” 18 FEBRUARY 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

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(4-way tie)

GPS and Relativity Damian Pope Greg Dick Sean Bradley Steve Kelley Perimeter Institute for Theoretical Physics

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Damian Pope made this video for any nonscientists who think relativity matters only to theoretical physicists and Star Trek fans who want to know how many laws of physics the series has broken. Millions of people use relativity every day—in their GPS receivers. GPS satellites orbit at an altitude of 20,000 kilometers. The problem: Time moves 38 microseconds a day faster at that altitude than on Earth’s surface. That may not seem like much, but after a month, it could throw the GPS system off by more than 300 kilometers. The solution: Slow down the satellite clocks so they tick 38 microseconds less every day. Just one more reason to thank Einstein.

GlyphSea Amit Chourasia Emmett Mcquinn Bernard Minster Jurgen Schulze San Diego Supercomputer Center, UCSD For earthquake scientists, predicting when the next “big one” will strike is the million-dollar question. But predicting how much damage it will do is just as important—and almost as uncertain. Knowing exactly how seismic waves transform the landscape could offer clues. Seismologists have made numerous attempts to model seismic waves passing through Earth. But depicting their direction is difﬁcult. Arrows or cones are ambiguous because viewed from the very front or the very back, they have the same shape: a circle. Amit Chourasia and his team at the University of California, San Diego, devised a straightforward solution: Use simple glyph shapes, such as spheres or ellipsoids, with a white dot on the end moving toward the observer and a black dot on the end moving away. By varying size and color to show magnitude, the method can display any kind of motion intuitively, from a major earthquake on the San Andreas fault to magnetic turbulence in stars millions of light-years from Earth. “How’s [an earthquake] going to affect people 30 miles away?” asks panel of judges member Tom Wagner. The new model offers an answer. “There has not been a way to do that before that was nearly as sophisticated.” www.sciencemag.org SCIENCE VOL 331 18 FEBRUARY 2011 Published by AAAS

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Thomas J. Cox Carnegie Institution for Science Galaxy mergers rank among the most violent and spectacular events in the cosmos. In a dance that can last billions of years, spiral arms of stars are thrown out into sweeping tails, and clouds of dust and gas are shoved into the center where, according to the prevailing theory, a supermassive black hole waits to consume them. Most astronomers think that all the gas pouring in will excite the black holes into quasars, the brightest objects in the universe, and that many merging galaxies will evolve into “binary quasars,” two quasars circling each other until their host galaxies unite. The Sloan Digital Sky Survey observed a binary quasar, SDSS J1254+0846, 4.6 billion light-years away. In 2009, deep imaging

at the Carnegie Observatories’ Magellan Telescope conﬁrmed that this binary quasar was indeed a pair of interacting spiral galaxies 70,000 light-years apart, one of the ﬁrst clear sightings of a binary quasar being triggered by a galaxy interaction. Thomas Cox modeled the galactic pas de deux over 3.6 billion years; the two partners arrived at a stage similar to that of the SDSS J1254+0846 image after 2.33 billion years. There was no narration and no audio except for background music. But that simplicity made it easy to follow, according to panel of judges member Corinne Sandone: “It was very visual. … You don’t need much narration to understand what you’re viewing.”

Visualization of the Whole Brain Catalog Drew Berry Mark Ellisman François Tétaz The Walter and Eliza Hall Institute of Medical Research

VISUALIZATION CHALLENGE 2010

Animator Drew Berry and his neurobiologist colleagues take you on a journey deep inside the mouse brain. The video brings to life data from the Whole Brain Catalog, a massive database of microscopy and other data sets on the mouse brain, under development at the University of California, San Diego. It opens with a mouse snifﬁng a camera—a ﬁtting image, says Berry, because the part of the brain that stores memory also controls the sense of smell. The video then zooms in on the mouse brain, focusing on the hippocampus, the headquarters of scent and memory. From there it isolates the dentate gyrus (upper right), the region that recognizes smells and creates new memories. Individual brain cells then start to appear. Finally, a new connection forms between two neurons, representing the creation of a new memory. “For a memory, you’d have many, many neurons forming, or connections being broken and new patterns being made,” says Berry. Even so, he hopes this video will inspire a sense of wonder at how the brain works.

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Noninteractive Media cont.

Computer Simulation of a Binary Quasar

COMMENTARY Facing up to vexing problems

A nod to nodules

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LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES

LETTERS edited by Jennifer Sills

A. REGALADO’S NEWS FOCUS STORY “BRAZILIAN SCIENCE: RIDING A GUSHER” (3 DECEMBER 2010, p. 1306) rightly highlights the “dearth of Brazilian-led science” in the Amazon and the “very delicate question” of Brazil’s dependence on foreign knowledge production. However, based on my experience and publication statistics from the Large-Scale Biosphere-Atmosphere experiment in Amazonia (1), I would challenge the statement that the “majority of publications on the Amazon don’t have a Brazilian author.” In fact, many papers have Brazilian co-authors. Brazilians (and Amazonian researchers) are involved in the majority of Amazonian research projects, but often in a subsidiary role as data providers and ﬁeld workers rather than leading the research and developing the scientiﬁc arguments. Clearly, foreign researchers, resources, technology, and expertise have been instrumental in developing Amazonian science and raising Brazilian capacity in this biologically unique and important part of world. However, Brazilian researchers need to stop gratefully receiving ﬁsh and quickly learn how to use the rod and the line.

ANA C. M. MALHADO

Laboratory of Analysis and Processing of Imaging Satellites, LAPIS, Federal University of Alagoas, Maceió, Alagoas, Brazil. E-mail: [email protected]

Reference

Research in Brazil. Many Amazon projects are led by foreign researchers.

CREDIT: DEVA RODRIGUES/EMBRAPA

Boosting CITES Through Research IN THEIR POLICY FORUM “BOOSTING CITES” (24 December 2010, p. 1752), J. Phelps et al. propose improvements to the implementation of the Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES). Most of their recommendations would involve the negotiation of cross-Party agreements and procurement of additional ﬁnancial resources, both of which are likely to delay or prevent their implementation. We suggest a more straightforward improvement to CITES implementation: Scientists should consider choosing CITES-listed taxa as model taxa for their research. At a workshop in 2008, more than 100

1. Ministério de Ciência e Tecnologia, Programa de Grande Escala da Biosfera-Atmosfera na Amazônia (http://lba.inpa.gov.br/lba/) [in Portuguese].

scientists and regulators compiled 60 case studies covering a wide range of CITESlisted taxa. The group outlined how information on the biology, harvesting, and management could be used to determine whether international trade in CITES-listed taxa is detrimental to their survival in the wild. Most of those case studies (78%) mentioned that more basic information on the biology of the taxa in the wild (including taxonomy, biology, and ecology) would improve their ability to make this determination (1). A lot of scientiﬁc research is done on species that are chosen out of convenience. We recommend that, all else being equal, scientists coordinate with national scientific authorities, local communities, and commercial traders to work on CITES-listed taxa instead. Such research could directly address

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Amazon Science Needs Brazilian Leadership

sustainable exploitation practices or could simply aim to generate relevant information as a by-product (for example, by generating additional information on the basic taxonomy, biology, and ecology of taxa). Either way, those implementing CITES would beneﬁt from the additional information and scientists would achieve additional impact from their research.

MATTHEW J. SMITH,* RICHARD J. WILLIAMS, DREW W. PURVES

Computational Science Laboratory, Microsoft Research, 7 J J Thompson Avenue, Cambridge, CB3 0FB, UK. *To whom correspondence should be addressed. E-mail: [email protected]

Reference

1. M. J. Smith et al., Biol. Conserv. 144, 82 (2011).

Response

WE AGREE WITH SMITH ET AL. THAT BASIC BIOlogical information is the cornerstone of CITES effectiveness and sustainable resource management, and that scientists should be encouraged to select CITES-listed species as research taxa. However, many of the most important and delicate issues for CITES go far beyond biology of species under threat. Biological data provide a critical, lowest common denominator for CITES decisionmaking, but strengthening CITES funding, checks and balances, analyses, and accompanying human institutions and capacities are equally critical to conservation. Smith et al. suggest that collecting biological research would be a more straightforward approach to improving CITES implementation than our solutions, many of which would require substantial ﬁnancial support and political will. In fact, even issues of basic biological research on vulnerable species are about Party funding, negotiations, safeguards, and collaboration. Bridging the logistical, ﬁnancial, political, and permit-laden gulf to attract signiﬁcantly more researchers to study these vulnerable species is not a simple process. For example, obtaining research permits for work on CITES-listed or locally threatened (e.g., Red List) species is time-

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LETTERS

DAVID BICKFORD,1* JACOB PHELPS,1 EDWARD L. WEBB,1 VINCENT NIJMAN,2 NAVJOT S. SODHI1

Department of Biological Sciences, National University of Singapore, Singapore, 117543, Singapore. 2School of Social Sciences and Law, Oxford Brookes University, Oxford, OX3 0BP, UK. 1

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

Engage Students in Education Reform

the authors’ seven initiatives. Students— the largest stakeholders in the current landscape of science education reform— should be added to the list of chairs, deans, and presidents as parties to engage in the policy debate (initiative 7). Such an addition would add a fresh perspective on the issue, open valuable communication pipelines between students and policy-makers, and provide opportunities for students interested in educational policy to gain experience in the ﬁeld. Restructuring the culture of education at research universities will require a new paradigm: Students should be considered not as passive consumers but as active participants in their education.

MIKE TORCHIA

University of Minnesota, Minneapolis, MN 55455, USA. E-mail: [email protected]

WE AGREE THAT STUDENTS SHOULD BE ACTIVE participants in their own education, and we have indeed made changes to programs at both of our own institutions based on student feedback from anonymous course evaluations. For example, several years ago one of us (D.O.) began experimenting with in-class demonstrations using large objects to illustrate dynamic microscopic biological processes. Feedback indicated that students felt demonstrations were more useful than any other tools we used, including iClickers and animations, for understanding the course material. Guided by student feedback, we reﬁned existing demonstrations and continue to develop new demonstrations each year (1). We maintain a Web site with video clips of demonstrations, some with additional instructions for faculty, to promote the use of this type of teaching tool both at our own institutions and more broadly (2). We thank Torchia for calling attention to this important aspect of the educational policy debate.

RICHARD LOSICK1 AND DIANE O’DOWD2

Harvard University, Cambridge, MA 02138, USA. E-mail: [email protected] 2University of California, Irvine, Irvine, CA 92697, USA. E-mail: [email protected] 1

References

1. D. K. O’Dowd, N. Aguilar-Roca, CBE-Life Sci. Educ. 8, 118 (2009). 2. Bridging the Divide Between Research and Teaching, Biological Demonstrations (www.researchandteaching. bio.uci.edu/lecture_demo.html).

CORRECTIONS AND CLARIFICATIONS

12,000 Fellowships 10,000

Traineeships Research assistantships

6,000 4,000

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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.

Training the Next Generation

1984

Letters to the Editor

News and Analysis: “NIH report urges greater emphasis on training for all graduate students” by J. Mervis (4 February, p. 525). Two bars were mislabeled in the graph showing modes of support for graduate students. The labels for fellowships and research assistantships were reversed. The correctly labeled graph appears here. Also, the comments attributed to Carolyn Bertozzi regarding the pressures on today’s graduate students actually were expressed by another council member and HHMI investigator, Karolin Luger of Colorado State University. Luger was speaking about high-performing students who are repelled by the cut-throat atmosphere within a laboratory and choose to leave research altogether.

Number of graduate students

AS A SENIOR UNDERGRADUATE STUDENT AT a large public research university, I agree with W. A. Anderson et al. that excellent teaching and research are not mutually exclusive pursuits (“Changing the culture of science education at research universities,” Education Forum, 14 January, p. 152). However, engagement with the students themselves is nowhere to be found in

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consuming and expensive. A streamlined research permit process could facilitate this type of research. Furthermore, studying species with cross-border distributions and trade requires inter-Party collaboration and support; only with this coordination can researchers make efficient use of limited ﬁnancial resources. Whereas many policymakers seek quality data to support sustainable harvests, those research ﬁndings that could lead to increased regulations, monitoring, and management requirements may be viewed as increasing burdens on Party governments. Furthermore, improving wildlife harvest and trade data, a necessary supplement to data on sustainable use, will rely on increased Party funding, strengthened institutions, improved capacity, and greater collaboration. Increasing biological research cannot take place without these related changes. Perhaps one of the best ways to help researchers, CITES policy-makers, and customs agents to increase research collaborations would be to identify the needs of all stakeholders and improve communication among them.

BOOKS ET AL. ENVIRONMENT

of contemporary conservation. The authors confront readers with the reality that conservation raises uncomfortable truths, such as the valuation of endangered species. They also demonstrate that the roles of Conservation Science conservation scientists demand Balancing the Needs ence, Kareiva and Marvier interdisciplinary skills rooted in of People and Nature are, at the very least, putting diverse disciplines ranging across the tools of argument into the ecology, economics, and ethics. by Peter Kareiva and hands of the next generation To satisfy an unashamedly Michelle Marvier of conservation scientists and biocentric readership, the authors Roberts, Greenwood on-the-ground practitioners. have drawn on a diverse range of Village, CO, 2011. 573 pp. $90, £50, €60. The book is aimed at taxa to illustrate their arguments: ISBN 9781936221066. undergraduates and earlyfrom African wild dog (Lycaon career graduates, but the pictus) to wood frog (Rana sylauthors entreat those of us with established vatica), chanterelle mushroom (Cantharelconservation careers to confront our out- lus cibarius) to eelgrass (Zostera marina), of-date learning and assumptions and to and monarch butterﬂy (Danaus plexippus) explore new ideas and analyses. We should. to zebra mussel (Dreissena polymorpha), One of the book’s strengths lies in its mix of as well as, of course, that iconic symbol of scholarship and practice—Kareiva is chief extinction, Raphus cucullatus (which they scientist for The Nature Conservancy—and charmingly index as “dodo bird”). the book’s acknowledgments list a stellar Each chapter concludes with a potentially cast of, albeit U.S.-centric, reviewers from polarizing short essay. These “consider this” both lab and ﬁeld. pieces raise such questions as: What are the Neatly designed—with boxed texts, ques- risks of relying on the economic valuation of tions for discussion, suggestions for group ecosystem services? Who beneﬁts from proprojects, and examples of case studies and tected areas? Does habitat restoration work? sound practice—the book is divided into four Are carbon markets a silver bullet? Are popbroad sections. These cover the need for con- ulation viability assessments useful? When servation; the roles of policy, protected areas, do we give up? Clearly, Kareiva and Marand planning; how science informs conserva- vier do not balk at tough debate, and their tion strategy; and how we face the challenge concluding chapter, “Making conservation a success story,” even opens with a substantial nod to the pundits who have “bemoaned the failure of the environmental and conservation movements.” If the decline of public empathy for environmentalism is to be reversed, the authors argue that, as Nurse proposed, we must all broaden the appeal of conservation science. A failure to do so, as Dowling despairs, renders any scientist-versus-skeptic debate “a mere distraction ... destined to be overtaken by events.” In Conservation Science, Kareiva and Marvier have deﬁnitely crafted a book that combines sound argument with practical examples. While being entirely frank about the complex challenges ahead, their efforts must effectively arm a new wave of environmentalists with the skills needed to face those.

John Fanshawe

CREDIT: AMI VITALE/WWW.AMIVITALE.COM

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mong many great images in Peter Kareiva and Michelle Marvier’s robust and elegant Conservation Science is a striking 1947 advertisement for pesticides from the company Killing Salt Chemicals. In it, a happy chorus of dog, tomato, milkmaid, cow, potato, and hen sing out, “DDT is good for me-e-e!” Fifteen years later, Rachel Carson published Silent Spring (1), so often cited as the dawning inspiration for the modern environment movement. For lifelong conservation scientists and practitioners, the depredations of DDT, and Carson’s response, represent a seminal evidence-based legacy that is characterized by many of the examples that ﬁll Kareiva and Marvier’s book. As indicated by the subtitle Balancing the Needs of People and Nature, the authors have shaped a textbook that makes a robust case for balance and for the role conservation science must play in achieving it. The book is timely, especially at this moment when science faces a regular media siege. In his recent, brilliantly calm Horizon program on BBC2, “Science under attack” (2), Paul Nurse, president of the Royal Society, made a seemingly irrefutable case for rational, evidence-driven decision-making. By challenging several high-profile skeptics—most notably over Climategate—Nurse made, at least to any reasonable observer, a clear case for science. Indeed, Tim Dowling, the Guardian’s critic, lamented in his paper the following day that it was only a television show and not “a fouryear degree course in climatology” (3). This apparent high ground is natural territory for Science’s readership, but it remains the case that the public at large, whose support for conservation (as Kareiva and Marvier acknowledge) is often lacking, is losing faith in science. When combined with a growing disconnect from natural environments— more than half the world’s population now live in urban areas, and the ﬁgure is likely to reach 70% by 2050 (4)—the case for a global strategy that really does balance the needs of people and nature is paramount. As Dowling explained in his review, “Nurse issued a call to scientists to be more politically savvy … and to make more of an effort to put data in the public domain.” With Conservation SciThe reviewer is at BirdLife International, Wellbrook Court, Girton Road, Cambridge CB3 0NA, UK. E-mail: john. [email protected]

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Facing Up to Vexing Choices

References

Humans are the crucial factor. Home to more than three million people, China’s northwest Yunnan Province retains diverse habitats that harbor numerous endangered animals.

1. R. Carson, Silent Spring (Houghton Mifﬂin, Boston, 1962). 2. P. Nurse, on Horizon, BBC 2, 24 January 2011. 3. T. Dowling, review of “Science under attack” (BBC 2), Guardian (London), 25 January 2011, p. G2. 4. U.N. Habitat [United Nations Human Settlements Programme], State of the World’s Cities 2010/11: Cities for All: Bridging the Urban Divide (Earthscan, London, 2010).

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BOOKS ET AL. art-science collaborations. In the wet lab at ise of a more open, inclusive world for others. the University of Western Australia, artists Several exhibits—including Paul Vanouse’s are fully implicated in the biological world Latent Figure Protocol (2008), which uses that they comment on and from which they DNA samples from an industrially produced draw inspiration. Indeed, the organism to create an image in immediate focal point upon reactive gel; Andre Brodyk’s Visceral entering the Dublin gallery Proto-Animate20 (2009–), in Deborah Dixon, Elizabeth Straughan, The Living Art Experiment is a working biosafety level which Escherichia coli and the Harriet Hawkins* Oron Catts and Ionat Zurr, 2 laboratory, in which the artgene APOE (associated with Curators Alzheimer’s disease) are used en years ago SymbioticA, an art- ists grow and maintain their Science Gallery, Trinity as creative agents; and Nigel science collective based within the artworks. All 15 pieces in the College, Dublin, Ireland. Helyer’s Host (2003), which School of Anatomy and Human exhibition deploy “living” Through 25 February 2011. presents 200 live crickets with Biology, University of Western Australia, biomaterials as an expreshttp://sciencegallery.com/ a video presentation on the sex embarked on an ambitious program wherein sive medium. Tissue Culture visceral life of insects—explore quesartists would be invited to enter scientiﬁc lab- and Art Project’s now-iconic tions of how we are to consider oratories as “residents.” There they would Semi-Living Worry Dolls learn various biological techniques and proto- (2000) was crafted from degradable poly- and negotiate both life and agency in a postcols and would be mentored by both scientists mers and surgical sutures and seeded with Darwinian world. The signiﬁcance of the exhibition’s title and other artists as they produced “bioart.” A living cells that are nurtured within the surretrospective of some of the resulting work is rogate body of a microgravity bioreactor. In thus seems to ensue not so much from the Neurotica’s Silent Barrage (2009), a gory spectacle of experimentally manipuset of robotic relays, responding to lated viscera but from the desire to seek audience movement, ampliﬁes neu- within these tissues a vision of things to ral activity in distantly located, cul- come. Collaborators such as Paul Thomas tured nerve cells. Boo Chapple’s and Kevin Raxworthy do not seek to presTransjuicer (2010) takes advantage ent the “truth” of science but rather to place of the piezoelectric nature of bone particular scientific endeavors and techmatrix to make cow-bone audio niques within a format and setting that allows speakers that emit nanosonic vibra- them to be experienced via the senses, thus tions. Elsewhere in the exhibition, encouraging the emergence of a complex works—such as Alicia King’s The set of feelings and thoughts toward them. In Vision Splendid (2009), Svenja Thomas and Raxworthy’s immersive installaKratz’s Afterlife: Immortalisation of tion Midas, this is accomplished by realizing Kira and Rama (2011), and Tagny touch at the nanoscale (otherwise indiscernSemi-Living Worry Dolls. Tissue Culture and Arts Project Duff’s Cryobook Archives (2010)— ible) as a series of atomic vibrations recorded (Oron Catts and Ionat Zurr) (2000). examine technological and ethical via an atomic force microscope. This sonic issues involved in the storage of tis- representation is played back by touching a currently on display in Visceral (curated by sue-based bioart as well as more philosoph- gold-coated model of a skin cell connected to SymbioticA cofounder Oron Catts and mem- ical issues involved in thinking of cells and a digital projection of a cell. A work created especially for the exhibiber Ionat Zurr), at the Science Gallery, Trin- living systems as reservoirs for inheritance tion, Perdita Phillips’s The Summer Flurries ity College, Dublin. The show’s three-day and memory. The embeddedness of the artist within (2011), collapses both scale and space. Wearopening (“birth”) included a symposium that drew together the curators, former residents, the laboratory undercuts any easy reading ing headphones and using a Global Positionother bioartists, and scholars working in the of these works as critical of the “monstrous” ing System receiver, visitors walk the streets humanities and social sciences (1), as well capacity of the biological sciences to reor- of Dublin while listening to the sounds of an as a seminar by students in the SymbioticA der and reform living material. Certainly, Australian wetland. The waves of sound dismasters program. The exhibition will close such works as Abhishek Hazra’s Let a Thou- locate one’s sense of place but also allow for with a “death” ritual, during which the equip- sand Proteins Bloom (2011), which attempts connections to emerge between the human ment nurturing the living tissues that form an to produce ammonium nitrate from human and nonhuman, the city and the environment. Although encompassing a wide range of essential part of several installations will be breast milk, speak to an underlying philosoturned off; essential nutrient solutions will phy of science that privileges utilitarianism. practices and materials, the works appearnot be renewed, and samples will be contami- And the exhibition’s guidebook points out ing in Visceral are drawn together by a sense the squeamishness invoked by the chimeri- of ongoing experimentalism and a delight in nated by exposure to air. It is this emphasis on working with living cal, semi-living creatures on show elsewhere exploring what contemporary experimental tissue (from animals, plants, fungi, protists, within the gallery. But, taken as a collection, systems make possible. Such bioart is at once archaea, and bacteria), as well as viruses, these works harken not to a Faustianism but wondrous and unsettling, spectacular and that marks SymbioticA’s key contribution to to the classical monster (from the Latin mon- deeply felt. stratum, to point to that which is worthy of The reviewers are at the Institute of Geography and Earth References and Notes warning). These singular wonders portend Sciences, Llandinam Building, Aberystwyth University, 1. Deborah Dixon was an invited speaker at this symposium. a radical shift in the social order, prompting Penglais Campus, Aberystwyth SY23 3DB, UK. *Author for correspondence. E-mail: [email protected] 10.1126/science.1203549 horror and anxiety in some but also the promEXHIBITION

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Deﬂating the Genomic Bubble James P. Evans,1* Eric M. Meslin,2 Theresa M. Marteau,3 Timothy Caulﬁeld4

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CREDIT: P. HUEY/SCIENCE

occer is the sport of the future in America … and it always will be.” This oft-quoted epithet poking fun at the promise of the “beautiful game” in the United States can seem uncomfortably apt when applied to genomic medicine. It’s now been 10 years since humans deciphered the digital code that deﬁnes us as a species. Although it may be hard to overestimate the signiﬁcance of that achievement, it is easy to misconstrue its meaning and promise. People argue about whether mapping the human genome was worth the investment (1–3). With global funding for genomics approaching $3 billion/year (4), some wonder what became of all the genomic medicine we were promised (5). It thus seems an appropriate time to take stock of whence the real beneﬁts from genomic research may come and how best to attain a future in which genomics improves human health. Recent methodological progress in genomics has been breathtaking. We now regularly assay genomes at millions of loci (6), and routine whole-genome sequencing may soon be a reality (7). If this trajectory continues, genomic research will illuminate fundamental mechanisms of human disease with a reasonable expectation of practical results (8). But claims of near-term applications are too often unrealistic and ultimately counterproductive. From the South Sea and dot-com “bubbles” to the ongoing housing market crisis, the world has seen its share of inﬂated expectations and attendant dangers. Science is immune to neither. If we fail to evaluate the considerable promise of genomics through a realistic lens, exaggerated expectations will undermine its legitimacy (9), threaten its sustainability, and result in misallocation of resources. Fueling unrealistic expectations for predictive genetic testing and uncritical translation of discoveries may also distract our gaze from other promising approaches to preventing disease and improving health. 1 Departments of Genetics and Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. 2 Center for Bioethics, Indiana University, Indianapolis, IN 46202, USA. 3Health Psychology Section, King’s College London, London SE1 9RT, UK. 4Faculty of Law and School of Public Health, Health Law Institute, University of Alberta, Edmonton, Alberta T6G 2H5, Canada.

*Author for correspondence. E-mail: [email protected]

Impediments and Hyperbole

Substantial impediments to realizing many of the claims most frequently heard include the following: The problem of clinical utility and relative risk. The numerous genetic variants that mediate disease risk typically confer woefully low relative risks (i.e., compared with the much more meaningful absolute risk) and are thus meager in their predictive power (10). Their applicability to patient care shows little promise; studies (11–14) demonstrate that even combining dozens of risk markers provides little clinically meaningful information. In the public health realm, the prospect of effectively stratifying populations as high or low risk, thereby guiding screening, is equally dismal. Given the multifactorial nature of common diseases and the weak predictive properties of genetic-risk alleles, the probability of misclassifying individuals as high or low risk is likely too great to make such an approach feasible in the general population for guiding such things as mammography or colorectal cancer screening (15). The illusion of parsing risk. For common diseases, by deﬁnition, we are all at high levels of absolute risk. In this setting, deﬁning precise relative risk on the basis of individuals’ genetic information is less meaningful; interventions that lower risk will be useful to everyone, regardless of their relative risk. And for rare diseases, shifting an individual’s risk from an already low level may not be very clinically meaningful. For example, the lifetime risk for an individual in the United States to develop Crohn’s disease is about 1/1000. How helpful is it for clinicians and patients if that risk shifts to 1/500 or 1/2000?

Unrealistic expectations and uncritical translation of genetic discoveries may undermine other promising approaches to preventing disease and improving health.

The difficulty of changing behaviors. The idea that genetic information will promote a healthy life-style has emerged as a dominant claim by those who promote genomic medicine (16, 17). However, there is little evidence that simply telling someone they are at a genetically increased risk for heart disease or diabetes, for example, leads to lasting beneﬁcial changes in diet or exercise habits (18, 19). Altering environments is increasingly recognized as a more effective way of changing those counterproductive behaviors that contribute most to poor health in high-income countries—namely, diet, sedentary behavior, smoking, and alcohol use (20). The paradox of risk information. Even if, despite evidence to the contrary, knowledge of one’s genetic status drives behavior change, another problem emerges: for everyone identiﬁed at increased risk of a malady, there will be an equal number at decreased risk. Thus, if genetic information were actually found to be uniquely powerful in changing behavior, it could well promote counterproductive behaviors. The translation of science into the clinic is inherently messy. The public, researchers, and clinicians frequently fail to appreciate that the history of medicine is strewn with ideas once thought promising that did not pan out when scrutinized through the lens of evidencebased medicine (21). Hormone replacement therapy, prostate-speciﬁc antigen screening, peri–myocardial infarction lidocaine, and many other good ideas, when prematurely implemented, created bubbles of expectation and investment, leaving sponsors disappointed and patients ill-served when reality did not live up to theoretical promise. Given these hurdles to practical application, why has genomics been the recipient of such hyperbole? Impatience for practical applications from genetic advances is understandable. To be sure, there is much room for improvement in modern medicine: Screening programs

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POLICYFORUM

Realistic Promises

Harold Varmus observed that the full potential of a DNA-based transformation of medicine will be realized only over the course of decades (8). We agree; the true promise of genomics is to help lay bare the mechanisms of human disease. Genes responsible for most Mendelian disorders will soon be identiﬁed. Genome-wide association studies are illuminating loci that contribute to common disease, and novel drug targets are being identified that will ultimately lead to new therapies. But the timeline for translation of such discoveries will be long. Pharmacogenomics (PGx; the study of influence of genetic variation on drug response) may represent a near-term payoff of genomic research for carefully selected treatments and could enhance the safety and utility of treatments used for serious disorders (26, 27). But it is unrealistic to expect PGx to revolutionize the use of all (or perhaps even most) drugs, given that much variability in efﬁcacy is not genetically determined (28). Indeed, the most powerful predictor of drug efﬁcacy is whether a patient takes the drug, highlighting the importance of human behavior in health outcomes. If properly harnessed and based on evidence, appropriate risk assessment could aid in clinical decision-making (29). The ability to make diagnoses, especially for disorders that result from disruption of a single gene, will provide tangible beneﬁt in the near term. Enhanced diagnostic capacity promises to spare both anxiety and money, ending the cruel “diagnostic odyssey” of families who

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go for years without a deﬁnitive diagnosis. But we should not overestimate the value of diagnosis or risk stratiﬁcation. Without effective interventions, a diagnosis is only a dimly realized, partially fulﬁlled hope. Couples will be empowered to make informed reproductive decisions, as preconceptual screening, augmented by robust genomic analysis, allows them to learn whether they are carriers of disease-related genes. Newborn screening will also beneﬁt as medically actionable conditions are identiﬁed. Such advances hold great promise if the information so gathered is useful, costeffective, and welcome (since not all parents may welcome such information). So how do we avoid inﬂating an unsustainable genomic bubble but still realize the true—and considerable—promise of the “genomic revolution”? Solutions range from the political to the personal, from short term to long term. We offer a short list of recommendations as a starting point for debate, aimed at deﬂating the genomic bubble and realizing the ﬁeld’s long-term promise: 1. Reevaluate funding priorities. A sober assessment of disease etiology suggests that funding priorities may be mismatched to the potential for practical beneﬁt. Much morbidity and premature mortality in high-income countries results from smoking, sedentary behavior, and excessive food and alcohol consumption (30, 31). It is likely that common diseases arising from these behaviors can be reduced by behavioral change (32, 33), but our knowledge of how to effect such change across populations is limited. Yet, U.S. National Institutes of Health and Department of Energy spending on genomics vastly exceeds the budget for behavioral and social science research (4, 34). Given that even a small improvement in our ability to alter behaviors could yield major beneﬁts, we suggest a reappraisal of the apportioning of funds to promote the promise of improved human health. 2. Foster a realistic understanding among the scientific community, the media, and the public of the incremental nature of science and need for statistical rigor. Scientists can start this process by making responsible claims and by advocating that reporters and editors do the same. 3. Maintain focus on developing highquality evidence before integrating good ideas into medical practice. Develop novel ways of assessing evidence so as not to delay implementing promising modalities. We believe that genomic discovery and resultant applications will provide great beneﬁts to human health. Ours is not a call

to gut existing research or too rigidly tie funding to the degree of disease burden. Indeed, the nature of scientiﬁc progress is arguably not optimized by a rigid allocation of resources to purely practical need. But failing a (desirable but unlikely) massive expansion of total funding for all types of research, a realistic view of the promise of genomics and an appropriate prioritization of research funding are vital to realizing that future. The pursuit of our common goal—improved human health—demands that we take a hard look at disease causation and order our priorities accordingly. 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. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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References and Notes

A. Pollack, New York Times, 14 June 2010, p. B5. P. Kraft, D. J. Hunter, N. Engl. J. Med. 360, 1701 (2009). J. N. Hirschhorn, N. Engl. J. Med. 360, 1699 (2009). J. R. Pohlhaus, R. M. Cook-Deegan, BMC Genomics 9, 472 (2008). F. S. Collins, N. Engl. J. Med. 341, 28 (1999). Editors, Nature 464, 649 (2010). E. Pettersson, J. Lundeberg, A. Ahmadian, Genomics 93, 105 (2009). H. Varmus, The Art and Politics of Science (Norton, New York, 2009). A. Petersen, Monash Bioeth. Rev. 28, 5.1 (2009). L. A. Hindorff et al., Proc. Natl. Acad. Sci. U.S.A. 106, 9362 (2009). S. Wacholder et al., N. Engl. J. Med. 362, 986 (2010). S. Kathiresan et al., N. Engl. J. Med. 358, 1240 (2008). V. Lyssenko et al., N. Engl. J. Med. 359, 2220 (2008). N. L. Pedersen, JAMA 303, 1864 (2010). M. S. Pepe, H. Janes, G. Longton, W. Leisenring, P. Newcomb, Am. J. Epidemiol. 159, 882 (2004). F. Collins, Nature 464, 674 (2010). Navigenics Inc., Genetics and health; http://www. navigenics.com/visitor/genetics_and_health/taking_ action/personalized_health/. J. B. McClure, Am. J. Prev. Med. 22, 200 (2002). T. M. Marteau et al., Cochrane Database Syst. Rev. 10, CD007275 (2010). WHO Regional Committee for Europe, “Resolution: Behaviour change strategies & health: The role of health systems” (World Health Organization, Geneva, 2008). K. Goodman, Ethics and Evidence-Based Medicine: Fallibility and Responsibility in Clinical Science (Cambridge Univ. Press, Cambridge, UK, 2003). J. Kimmelman, Gene Transfer and the Ethics of First-inHuman Research: Lost in Translation (Cambridge Univ. Press, Cambridge, UK, 2009). L. P. Garrison Jr. et al., Drug Metab. Rev. 40, 377 (2008). T. Caulﬁeld, PLoS. Med. 1, e38 (2004). B. Carlsson, Z. J. Acs, D. B. Audretsch, P. Braunerhjelm, Ind. Corp. Change 18, 1193 (2009). S. Mallal et al., N. Engl. J. Med. 358, 568 (2008). M. P. Goetz et al., Breast Cancer Res. Treat. 101, 113 (2007). G. Tucker, BMJ 329, 4 (2004). A. J. Vickers, E. Basch, M. W. Kattan, Ann. Intern. Med. 149, 200 (2008). R. L. Keeney, Oper. Res. 56, 1335 (2008). W. C. Willett, Science 296, 695 (2002). W. C. Knowler et al., Lancet 374, 1677 (2009). R. Doll, R. Peto, J. Boreham, I. Sutherland, BMJ 328, 1519 (2004). H. J. Silver, A. L. Sharpe, H. Kelly, P. Kobor, G. Sroufe, in AAAS Report 33: Research and Development 2009 (AAAS, Washington, DC, 2008), chap. 19; www.aaas.org/ spp/rd/09pch19.htm. The authors thank R. Whiting and Genome Alberta.

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are inherently inefﬁcient given the need to test entire populations, drugs have widely variable efﬁcacy, and diseases strike capriciously. But there are other drivers of inﬂated expectations (22). Researchers gain funding, jobs, and fame, while pressure to commercialize their work adds fuel to the tendency to oversell (23, 24). In a distressing way, biomedical research is often viewed by governments as primarily an engine of economic growth (25) and, only secondarily, as an engine of scientiﬁc and medical progress. Further pressure results from retail marketing of genomic information, such as directto-consumer genetic testing. As boundaries between private and public efforts erode, academic endeavors are increasingly subject to market forces that demand quick payoffs. Finally, the press plays an obvious role in creating unrealistic hopes (24). Collectively, these factors contribute to heightened expectations; left unchallenged, they take on a momentum that is hard to unseat.

PERSPECTIVES CELL BIOLOGY

A Hand to Support the Implantation Window

A signaling pathway connects ovarian hormones to preparation of the mammalian uterus for pregnancy.

Sylvia C. Hewitt and Kenneth S. Korach

CREDIT: Y. HAMMOND/SCIENCE

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National Institute of Environmental Health Science, National Institutes of Health, Research Triangle Park, NC 27709, USA. E-mail: [email protected]

Fallopian tube

Epithelium Endometrium (epithelium, stroma)

Uterus lumen

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eproduction in mammals is coordinated by an intricate sequence of events and signals that culminate in implantation of the embryo in the uterus (1–3). To succeed, these signals must be produced at the correct time, in the proper place, and in the amounts needed, and must be received by the appropriate targets. Perturbation of any of these factors can prevent establishment of pregnancy, resulting in infertility. On page 912 of this issue, Li et al. (4) show that the transcription factor Hand2 regulates signals that establish conditions in the mammalian uterus to support pregnancy. Uterine tissue (see the ﬁgure) consists of an outer muscle layer (myometrium) which contracts during labor to facilitate delivery. The inner lumen is lined by an epithelial cell layer from which emanate glandular structures, also lined by epithelial cells. The embryo must attach to, and then invade through, the epithelial layer to establish a pregnancy, and the glands secrete factors essential to the process. Stromal cells between the outer and inner layers expand and differentiate into decidual cells to support fetal and placental development. The ovarian hormones estrogen and progesterone have relatively complementary roles, reﬂecting their temporal and cyclical ﬂuctuations. Estrogen production surges just before ovulation, and induces growth of the uterine epithelial cells that will come into contact with a developing embryo. Progesterone increases after ovulation and further prepares the endometrial tissue (the stromal and epithelial cells) for pregnancy by both dampening the proliferative effect of estrogen on the epithelial cells, and acting with estrogen to stimulate proliferation of stromal decidual tissues. This convergence of hormonal and other signals with the arrival of the embryo at the proper time is called the “implantation window.” The responses of uterine cells to estrogen and progesterone are mediated in part by receptors that are transcription factors. The estrogen receptor (ER) is found in all uter-

Myometrium (muscle) Lumen

Stroma

Cervix Vagina Signaling Epithelium Ovulation

Progesterone

Hand2 (Transcription factor)

Blocks FGF production and secretion

Prevents ERα activation in epithelium

Blocks epithelium growth

Allows embryo implantation

Implantation signals. Stromal cells respond to progesterone during pregnancy by activating the transcription factor Hand2. This triggers a signaling cascade that blocks estrogen receptor (ERα) signaling in uterine epithelial cells, thereby creating an epithelium that is receptive for embryo implantation.

ine cell types, whereas progesterone receptor (PR) expression varies depending on the cell type and phase of the ovarian cycle. As in earlier studies with PR-deficient mice (1, 3), Li et al. show that when progesterone is unable to decrease estrogen’s proliferative effect on the endometrial epithelium, pregnancy cannot be established. Disrupting this effect of progesterone is the basis for the estrogen-containing “morning after pill.” In certain clinical conditions, estrogen in the absence of progesterone (“unopposed estrogen”) may damage the endometrium. Hormone-replacement therapies that lack progesterone, or anovulatory conditions (such as polycystic ovarian syndrome) that lengthen the estrogenic portion of the menstrual cycle, are associated with increased incidence of endometrial cancers (5). However, the signaling pathways by which estrogen and progesterone successfully establish uterine receptivity have not been fully elucidated. Estrogen, acting through ERα in the stromal cells, increases the production of paracrine factors, including insulin-like growth factor 1 (IGF-1), which then trigger the proliferation of epithelial cells (6, 7). Estrogen also maintains the epithelia by preventing

programmed cell death of epithelial cells (8). These mechanisms have been established through numerous approaches including ablation of certain progesterone target genes (Ihh, Couptf2, Fkbp4, and Errﬁ1), leading to loss of uterine receptivity (3, 9, 10). Overall, these ﬁndings emphasize that proper progesterone-mediated regulation of multiple uterine stromal signals is critical for controlling the epithelial estrogenic responses that allow embryo implantation. To identify the progesterone-regulated signaling pathways that underlie implantation, Li et al. used microarray gene proﬁling analysis of progesterone-responsive transcription at the implantation window in the mouse. Comparison of mRNA in receptive uterine tissue to that in tissue treated with the PR antagonist RU486 revealed altered expression of Hand2. Selective ablation of the Hand2 gene in uterine cells showed that the transcription factor plays a key role in establishing a receptive endometrium. Microarray analysis also revealed misregulation of signaling by ﬁbroblast growth factor (FGF) in the uterine tissue of Hand2-deﬁcient mice compared to that in normal mice. Previous studies had indicated a role for FGF signaling in ovine implanta-

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PERSPECTIVES an FGF receptor (FGFR2) are associated with endometrial cancer (12, 13). Whether mutations in the Hand2 gene can explain clinical dysfunction also will require further research. Reproduction and endometrial function involve intricate interplay between different cell types that are too complex to adequately study with cell culture approaches. The ﬁndings of Li et al. relied on the development of elaborate in vivo models. Such approaches promise to open important new avenues of investigation into this critical window of receptivity.

2. F. W. Bazer, T. E. Spencer, G. A. Johnson, R. C. Burghardt, G. Wu, Reproduction 138, 195 (2009). 3. H. J. Lim, H. Wang, J. Clin. Invest. 120, 1004 (2010). 4. Q. Li et al., Science 331, 912 (2011). 5. A. E. Schindler, Maturitas 62, 334 (2009). 6. G. R. Cunha, P. S. Cooke, T. Kurita, Arch. Histol. Cytol. 67, 417 (2004). 7. L. J. Murphy, A. Ghahary, Endocr. Rev. 11, 443 (1990). 8. W. Winuthayanon, S. C. Hewitt, G. D. Orvis, R. R. Behringer, K. S. Korach, Proc. Natl. Acad. Sci. U.S.A. 107, 19272 (2010). 9. D.-K. Lee et al., Mol. Endocrinol. 24, 930 (2010). 10. T. H. Kim, D. K. Lee, H. L. Franco, J. P. Lydon, J. W. Jeong, Biol. Reprod. 82, 706 (2010). 11. B. C. Paria et al., Proc. Natl. Acad. Sci. U.S.A. 98, 1047 (2001). 12. P. M. Pollock et al., Oncogene 26, 7158 (2007). 13. P. Krejci, J. Prochazkova, V. Bryja, A. Kozubik, W. R. Wilcox, Hum. Mutat. 30, 1245 (2009).

References

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tion (2), indicating that the mechanism may have broader application to other mammals. Additionally, expression of Fgf2 transcript in the stroma surrounding an implanting blastocyst was previously reported in mice (11). However, Li et al. link regulation of the FGF signals to progesterone regulation of Hand2, which helps to explain how progesterone establishes uterine receptivity. Whether there is a general role for Hand2 and FGF signaling in establishing receptive endometria of other mammals remains to be determined. It will be important to evaluate whether perturbing this pathway might lead to adverse clinical outcomes, such as infertility, endometriosis, or malignancy. Indeed, mutations in the genes encoding FGF9 and

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1. H. Wang, S. K. Dey, Nat. Rev. Genet. 7, 185 (2006).

PHYSICS

A New Twist on Spintronics

A thin layer of ordered DNA acts as an efﬁcient spin ﬁlter for photoemitted electrons from a gold surface.

Geert L. J. A. Rikken

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Laboratoire National des Champs Magnétiques Intenses, CNRS/INSA/UJF/UPS, 143 Avenue de Rangueil, 31400 Toulouse and 25 Avenue de Martyrs, 38042 Grenoble, France. E-mail: [email protected]

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Twist and spin. Illustrating the spin-ﬁltering effect. Electrons with their spin parallel to their velocity (here represented as rightrotating) are mostly transmitted through an ordered layer of DNA helices, whereas electrons with their spin antiparallel to their velocity (represented as left-rotating) are mostly reﬂected by it.

ily, meaning that this layer acts as a spin ﬁlter, strongly hindering the passage of the other spin type. This ﬁlter effect is observed only if the DNA is assembled on the gold surface as a closely packed ordered array of helices, and is stronger if the helices are longer, reaching selectivities of 60%. For chaotic assemblies of ﬂoppy DNA chains on the gold surface, the spin ﬁlter effect was not observed.

These findings reveal a fascinating and promising link between chirality and spintronics, which at first sight seem unlikely bedfellows. Objects are chiral (from the Greek “cheir,” meaning “hand”) if they exist in two nonsuperimposable forms that are each other’s mirror image, like our hands. Chirality is essential in stereochemistry and plays a vital role in biochemistry, as natural sugars are all righthanded and natural proteins are all left-handed. DNA chains are made of chiral building blocks, and can assemble into helical (chiral) superstructures, like the helices used by Göhler et al. Spin, on the other hand, has nothing to do with spatial symmetries, but is a quantum-mechanical property that is related to rotation, i.e., an absence of timereversal invariance. However, if the spincarrying particle has a velocity along its spin axis, it is chiral, as the combination of velocity and rotation results in a screwlike motion. Spin-polarized currents therefore are chiral, and their handedness depends on

18 FEBRUARY 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

CREDIT: P. HUEY/SCIENCE

reat hopes exist for improving the storage and processing of information by using not only the charge of electrons, as is done in everyday electronics, but by also exploiting a quantum mechanical property of electrons known as spin. In the rapidly growing ﬁeld of spintronics (1), the electron spin is essentially a minute magnet that can be pointing either “up” or “down.” The most efﬁcient way to manipulate spins is by means of a magnetic ﬁeld, either externally applied or existing inside a ferromagnetic material. By sending electrons through ferromagnetic materials, scientists have achieved spin-filtering effects, discriminating between electrons with “up” and “down” spins, with selectivity up to 25% (2). On page 894 of this issue, Göhler et al. (3) describe a surprisingly efﬁcient method for electronic spin ﬁltering. They have studied how electrons emitted by a gold substrate, upon absorption of light, pass through a self-assembled DNA monolayer on the gold surface. In particular, they have studied the spin of the electrons after their passage through the DNA layer and have found that one spin type passes through much more eas-

PERSPECTIVES an FGF receptor (FGFR2) are associated with endometrial cancer (12, 13). Whether mutations in the Hand2 gene can explain clinical dysfunction also will require further research. Reproduction and endometrial function involve intricate interplay between different cell types that are too complex to adequately study with cell culture approaches. The ﬁndings of Li et al. relied on the development of elaborate in vivo models. Such approaches promise to open important new avenues of investigation into this critical window of receptivity.

2. F. W. Bazer, T. E. Spencer, G. A. Johnson, R. C. Burghardt, G. Wu, Reproduction 138, 195 (2009). 3. H. J. Lim, H. Wang, J. Clin. Invest. 120, 1004 (2010). 4. Q. Li et al., Science 331, 912 (2011). 5. A. E. Schindler, Maturitas 62, 334 (2009). 6. G. R. Cunha, P. S. Cooke, T. Kurita, Arch. Histol. Cytol. 67, 417 (2004). 7. L. J. Murphy, A. Ghahary, Endocr. Rev. 11, 443 (1990). 8. W. Winuthayanon, S. C. Hewitt, G. D. Orvis, R. R. Behringer, K. S. Korach, Proc. Natl. Acad. Sci. U.S.A. 107, 19272 (2010). 9. D.-K. Lee et al., Mol. Endocrinol. 24, 930 (2010). 10. T. H. Kim, D. K. Lee, H. L. Franco, J. P. Lydon, J. W. Jeong, Biol. Reprod. 82, 706 (2010). 11. B. C. Paria et al., Proc. Natl. Acad. Sci. U.S.A. 98, 1047 (2001). 12. P. M. Pollock et al., Oncogene 26, 7158 (2007). 13. P. Krejci, J. Prochazkova, V. Bryja, A. Kozubik, W. R. Wilcox, Hum. Mutat. 30, 1245 (2009).

References

Downloaded from www.sciencemag.org on February 17, 2011

tion (2), indicating that the mechanism may have broader application to other mammals. Additionally, expression of Fgf2 transcript in the stroma surrounding an implanting blastocyst was previously reported in mice (11). However, Li et al. link regulation of the FGF signals to progesterone regulation of Hand2, which helps to explain how progesterone establishes uterine receptivity. Whether there is a general role for Hand2 and FGF signaling in establishing receptive endometria of other mammals remains to be determined. It will be important to evaluate whether perturbing this pathway might lead to adverse clinical outcomes, such as infertility, endometriosis, or malignancy. Indeed, mutations in the genes encoding FGF9 and

10.1126/science.1202372

1. H. Wang, S. K. Dey, Nat. Rev. Genet. 7, 185 (2006).

PHYSICS

A New Twist on Spintronics

A thin layer of ordered DNA acts as an efﬁcient spin ﬁlter for photoemitted electrons from a gold surface.

Geert L. J. A. Rikken

G

Laboratoire National des Champs Magnétiques Intenses, CNRS/INSA/UJF/UPS, 143 Avenue de Rangueil, 31400 Toulouse and 25 Avenue de Martyrs, 38042 Grenoble, France. E-mail: [email protected]

864

Twist and spin. Illustrating the spin-ﬁltering effect. Electrons with their spin parallel to their velocity (here represented as rightrotating) are mostly transmitted through an ordered layer of DNA helices, whereas electrons with their spin antiparallel to their velocity (represented as left-rotating) are mostly reﬂected by it.

ily, meaning that this layer acts as a spin ﬁlter, strongly hindering the passage of the other spin type. This ﬁlter effect is observed only if the DNA is assembled on the gold surface as a closely packed ordered array of helices, and is stronger if the helices are longer, reaching selectivities of 60%. For chaotic assemblies of ﬂoppy DNA chains on the gold surface, the spin ﬁlter effect was not observed.

These findings reveal a fascinating and promising link between chirality and spintronics, which at first sight seem unlikely bedfellows. Objects are chiral (from the Greek “cheir,” meaning “hand”) if they exist in two nonsuperimposable forms that are each other’s mirror image, like our hands. Chirality is essential in stereochemistry and plays a vital role in biochemistry, as natural sugars are all righthanded and natural proteins are all left-handed. DNA chains are made of chiral building blocks, and can assemble into helical (chiral) superstructures, like the helices used by Göhler et al. Spin, on the other hand, has nothing to do with spatial symmetries, but is a quantum-mechanical property that is related to rotation, i.e., an absence of timereversal invariance. However, if the spincarrying particle has a velocity along its spin axis, it is chiral, as the combination of velocity and rotation results in a screwlike motion. Spin-polarized currents therefore are chiral, and their handedness depends on

18 FEBRUARY 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

CREDIT: P. HUEY/SCIENCE

reat hopes exist for improving the storage and processing of information by using not only the charge of electrons, as is done in everyday electronics, but by also exploiting a quantum mechanical property of electrons known as spin. In the rapidly growing ﬁeld of spintronics (1), the electron spin is essentially a minute magnet that can be pointing either “up” or “down.” The most efﬁcient way to manipulate spins is by means of a magnetic ﬁeld, either externally applied or existing inside a ferromagnetic material. By sending electrons through ferromagnetic materials, scientists have achieved spin-filtering effects, discriminating between electrons with “up” and “down” spins, with selectivity up to 25% (2). On page 894 of this issue, Göhler et al. (3) describe a surprisingly efﬁcient method for electronic spin ﬁltering. They have studied how electrons emitted by a gold substrate, upon absorption of light, pass through a self-assembled DNA monolayer on the gold surface. In particular, they have studied the spin of the electrons after their passage through the DNA layer and have found that one spin type passes through much more eas-

PERSPECTIVES Mayer et al. (4) observed spin selectivity only if a heavy atom, in their case ytterbium, was incorporated in the chiral molecule. They did not observe any selectivity for molecules consisting only of light atoms, like carbon, oxygen, or hydrogen. Other experiments studying the transmission of electrons polarized by an external magnetic ﬁeld through chiral conductors also showed only very small spinﬁltering effects (5). The spin ﬁlter effect reported by Göhler et al. is almost four orders of magnitude stronger than the one observed by Mayer et al. As there are no heavy atoms in the DNA molecule, spin-orbit coupling alone cannot explain it. The fact that only well-ordered long DNA helices show a strong effect suggests that the supramolecular periodic character of the helices somehow greatly enhances the spin ﬁltering, but more work is needed to understand the phenomenon. The high efﬁciency of the filtering action observed by Göhler et al. makes supramolecular chiral materials very

interesting for spintronics. The richness of supramolecular chiral organic synthesis (6) holds great promise for detailed studies and ﬁne-tuning of this new effect, which may one day ﬁnd application in spintronics devices. Periodic chiral structures created by other methods, like glancing angle deposition (7), may also prove to be efﬁcient spin ﬁlters. In any case, Göhler et al. have deﬁnitely given a new twist to spintronics. References

1. S. D. Bader, S. S. P. Parkin, Annu. Rev. Cond. Mat. Phys 1, 71 (2010). 2. Y. Lassailly, H.-J. Drouhin, A. van der Sluijs, G. Lampel, C. Marlière, Phys. Rev. B 50, 13054 (1994). 3. B. Göhler et al., Science 331, 894 (2011). 4. S. Mayer, J. Kessler, Phys. Rev. Lett. 74, 4803 (1995). 5. G. L. J. A. Rikken, J. Fölling, P. Wyder, Phys. Rev. Lett. 87, 236602 (2001). 6. Y. Furusho, E. Yashima, J. Polym. Sci. A Polym. Chem. 47, 5195 (2009). 7. K. Robbie, M. J. Brett, A. Lakhtakia, Nature 384, 616 (1996).

Downloaded from www.sciencemag.org on February 17, 2011

whether the spin direction is parallel or antiparallel to the current (see the ﬁgure). The everyday frustration of putting your left hand in your right-hand glove immediately illustrates that the interaction between objects of opposite handedness is not the same as that between objects of the same handedness. It came therefore as no surprise that the transmission of spin-polarized electron beams through a vapor of chiral molecules was reported by Mayer et al. in 1995 to depend on the relative handedness of the spin-polarized electron current and the molecular chirality (4). This effect was, however, found to be quite small (~10−4), as predicted by theory. The mechanism behind this selectivity is the spin-orbit coupling; an electron moving through the electric ﬁeld of the atomic nucleus experiences effectively also a magnetic ﬁeld (a weak, relativistic effect), which acts on the electron spin. This coupling is stronger if the nucleus carries more charge, that is, it is stronger for heavier elements.

10.1126/science.1201663

PLANT SCIENCE

Unlocking the Door to Invasion

A single plant receptor recognizes related bacterial and fungal signals to initiate symbiosis.

Attila Kereszt1,2 and Eva Kondorosi1,3

CREDIT: S. SCHAARSCHMIDT AND B. HORVATH

T

o increase crop yields, farmers use inorganic fertilizers that provide plants with usable forms of nitrogen and phosphorus. The production and application of fertilizers, however, requires huge amounts of fossil energy and causes pollution. To reduce these problems and support the growing human population, sustainable agriculture will require new plant varieties capable of supplying themselves with these elements. On page 909 of this issue, Op den Camp et al. (1) take a step toward this goal by describing a cell surface receptor that enables a plant to establish symbiotic relationships with both the bacteria and fungi that help it to ﬁx nitrogen from the atmosphere and take up phosphorus. Most land plants, including the major crops, are able to establish endosymbiotic “partnerships” with arbuscular mycorrhizal (AM) fungi that enhance uptake of phosphate (2). More recently in evolutionary Institute for Plant Genomics, Human Biotechnology and Bioenergy, Bay Zoltan Foundation for Applied Research, Derkovits fasor 2, Szeged, Hungary. 2Karoly Robert College, Matrai ut 36, Gyöngyös, Hungary. 3Institut des Sciences du Végétal, Centre National de la Recherche Scientiﬁque, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. E-mail: [email protected]; [email protected]

terms, a small group of plants belonging to certain orders of the eurosid I group of eudicots gained the ability to develop novel symbiotic organs called nodules to accommodate nitrogen-ﬁxing (NF) bacteria. These include Gram-negative (rhizobia) and Gram-positive A

(Frankia) species. Most past studies of symbiotic nitrogen ﬁxation have focused on rhizobial symbiosis with commercially valuable and model legume plants such as soybean and alfalfa. Genetic analysis of mutant plants have revealed that the development of B

1

Root symbioses. (A) Mycorrhizal hyphae, arbuscules, and vesicules of AM fungi stained with ink. (B) Rhizobial root nodules.

www.sciencemag.org SCIENCE VOL 331 18 FEBRUARY 2011 Published by AAAS

865

PERSPECTIVES Mayer et al. (4) observed spin selectivity only if a heavy atom, in their case ytterbium, was incorporated in the chiral molecule. They did not observe any selectivity for molecules consisting only of light atoms, like carbon, oxygen, or hydrogen. Other experiments studying the transmission of electrons polarized by an external magnetic ﬁeld through chiral conductors also showed only very small spinﬁltering effects (5). The spin ﬁlter effect reported by Göhler et al. is almost four orders of magnitude stronger than the one observed by Mayer et al. As there are no heavy atoms in the DNA molecule, spin-orbit coupling alone cannot explain it. The fact that only well-ordered long DNA helices show a strong effect suggests that the supramolecular periodic character of the helices somehow greatly enhances the spin ﬁltering, but more work is needed to understand the phenomenon. The high efﬁciency of the filtering action observed by Göhler et al. makes supramolecular chiral materials very

interesting for spintronics. The richness of supramolecular chiral organic synthesis (6) holds great promise for detailed studies and ﬁne-tuning of this new effect, which may one day ﬁnd application in spintronics devices. Periodic chiral structures created by other methods, like glancing angle deposition (7), may also prove to be efﬁcient spin ﬁlters. In any case, Göhler et al. have deﬁnitely given a new twist to spintronics. References

1. S. D. Bader, S. S. P. Parkin, Annu. Rev. Cond. Mat. Phys 1, 71 (2010). 2. Y. Lassailly, H.-J. Drouhin, A. van der Sluijs, G. Lampel, C. Marlière, Phys. Rev. B 50, 13054 (1994). 3. B. Göhler et al., Science 331, 894 (2011). 4. S. Mayer, J. Kessler, Phys. Rev. Lett. 74, 4803 (1995). 5. G. L. J. A. Rikken, J. Fölling, P. Wyder, Phys. Rev. Lett. 87, 236602 (2001). 6. Y. Furusho, E. Yashima, J. Polym. Sci. A Polym. Chem. 47, 5195 (2009). 7. K. Robbie, M. J. Brett, A. Lakhtakia, Nature 384, 616 (1996).

Downloaded from www.sciencemag.org on February 17, 2011

whether the spin direction is parallel or antiparallel to the current (see the ﬁgure). The everyday frustration of putting your left hand in your right-hand glove immediately illustrates that the interaction between objects of opposite handedness is not the same as that between objects of the same handedness. It came therefore as no surprise that the transmission of spin-polarized electron beams through a vapor of chiral molecules was reported by Mayer et al. in 1995 to depend on the relative handedness of the spin-polarized electron current and the molecular chirality (4). This effect was, however, found to be quite small (~10−4), as predicted by theory. The mechanism behind this selectivity is the spin-orbit coupling; an electron moving through the electric ﬁeld of the atomic nucleus experiences effectively also a magnetic ﬁeld (a weak, relativistic effect), which acts on the electron spin. This coupling is stronger if the nucleus carries more charge, that is, it is stronger for heavier elements.

10.1126/science.1201663

PLANT SCIENCE

Unlocking the Door to Invasion

A single plant receptor recognizes related bacterial and fungal signals to initiate symbiosis.

Attila Kereszt1,2 and Eva Kondorosi1,3

CREDIT: S. SCHAARSCHMIDT AND B. HORVATH

T

o increase crop yields, farmers use inorganic fertilizers that provide plants with usable forms of nitrogen and phosphorus. The production and application of fertilizers, however, requires huge amounts of fossil energy and causes pollution. To reduce these problems and support the growing human population, sustainable agriculture will require new plant varieties capable of supplying themselves with these elements. On page 909 of this issue, Op den Camp et al. (1) take a step toward this goal by describing a cell surface receptor that enables a plant to establish symbiotic relationships with both the bacteria and fungi that help it to ﬁx nitrogen from the atmosphere and take up phosphorus. Most land plants, including the major crops, are able to establish endosymbiotic “partnerships” with arbuscular mycorrhizal (AM) fungi that enhance uptake of phosphate (2). More recently in evolutionary Institute for Plant Genomics, Human Biotechnology and Bioenergy, Bay Zoltan Foundation for Applied Research, Derkovits fasor 2, Szeged, Hungary. 2Karoly Robert College, Matrai ut 36, Gyöngyös, Hungary. 3Institut des Sciences du Végétal, Centre National de la Recherche Scientiﬁque, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. E-mail: [email protected]; [email protected]

terms, a small group of plants belonging to certain orders of the eurosid I group of eudicots gained the ability to develop novel symbiotic organs called nodules to accommodate nitrogen-ﬁxing (NF) bacteria. These include Gram-negative (rhizobia) and Gram-positive A

(Frankia) species. Most past studies of symbiotic nitrogen ﬁxation have focused on rhizobial symbiosis with commercially valuable and model legume plants such as soybean and alfalfa. Genetic analysis of mutant plants have revealed that the development of B

1

Root symbioses. (A) Mycorrhizal hyphae, arbuscules, and vesicules of AM fungi stained with ink. (B) Rhizobial root nodules.

www.sciencemag.org SCIENCE VOL 331 18 FEBRUARY 2011 Published by AAAS

865

both the AM and the NF symbioses are based on the shared activity of a set of plant genes, SYM genes (3–5). This indicates that bacteria hijacked the signal transduction pathway that fungi had used to gain entry into plant tissues and cells. Op den Camp et al. provide evidence that in Parasponia, the only nonlegume partner of rhizobia, a single receptor can recognize both the fungal and bacterial signals and induce the common SYM pathway to promote the intracellular accommodation of the partner microorganisms. Op den Camp et al. used Rhizobium strains, some of which were able to form nodules, and some of which were unable to form nodules, to prove that both Parasponia and legumes use lipochito-oligosaccharides called Nod factors to induce nodule development. They also showed that Nod factors act similarly in both symbioses via a common signaling cascade; in Parasponia, the introduction of a dominant active form of calcium/ calmodulin-dependent kinase (CCaMK), a key element of this pathway, resulted in spontaneous nodulation, as in legumes. Op den Camp et al. also provide insight into how bacterial Nod factor receptors (NFRs) evolved from receptors involved in plant-fungi partnerships. The most-studied legumes recognize rhizobia—or, more accurately, the bacterial Nod factors—via a pair of LysM-type receptor-like kinases, NFR1/ LYK3 and NFR5/NFP (6, 7). Because these NFRs are specific to bacterial symbiosis, investigators had hypothesized that they evolved either by duplication of the mycorrhiza-speciﬁc receptors, which then gained new functions, or by the recruitment of new

receptors that turned on the common signaling pathway. Op den Camp et al.’s analysis indicates that receptor duplication was not essential for plants to acquire the ability to form a symbiotic relationship with NF bacteria. Instead, the presence of a single NFR5like receptor in Parasponia, and its indispensable role in both symbioses, strongly suggests that rhizobia entered symbiotic interactions with plants through the same entrance used by mycorrhizal fungi. It also means that the molecular “keycard” that opens the door to plant partnerships for both bacteria and fungi—the bacterial Nod factor and mycorrhizal (Myc) factor—must be very similar. Indeed, Maillet et al. (8) recently described the Myc factors of AM fungi as lipochito-oligosaccharide molecules that are very similar to Nod factors. These results raise several questions: Why is the appearance of nitrogen-fixing nodules, especially rhizobial ones, restricted to a small fraction of mycorrhizal plants? How do plants discriminate between symbiotic fungi and bacteria? Was it necessary for host plants to distinguish between the microbes to create different niches? Studies of genes from related plants suggest that plant families establishing rhizobial or actinorhizal (Frankia) symbioses belong to the same large lineage. This raises the possibility that, during the evolution of ﬂowering plants, a predisposition for symbiotic nodule formation originated only once (9). Did this predisposition occur by changing the activity of one or more component(s) of the common symbiotic pathway, for example, by enabling it to provide different outputs?

Both bacteria and mycorrhizal fungi induce changes in intracellular calcium (Ca2+-) concentrations (termed calcium spiking). However, the frequency and duration of the oscillations, as well as the speed of Ca movement, are different in the two symbioses (10). Early elements of the common SYM pathway, such as the LysM-type receptors and another receptor protein, the symbiosis receptor kinase (SYMRK), are required for the induction of the calcium spiking, which is then deciphered by CCaMK. It will be interesting to compare calcium spiking upon rhizobial and fungal inoculations in species that possess dual-functioning receptors. There is not yet enough systematic data from different plant lineages to determine exactly how molecules like SYMRK and CCaMK contributed to the evolution of a predisposition to nodule formation. The real challenge is to ﬁnd out why lineages with predisposition for nodulation (for example, certain legumes) are unable to establish NF symbiosis. References

1. R. Op den Camp et al., Science 331, 909 (2011). 2. M. J. Harrison, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 361 (1999). 3. C. Kistner et al., Plant Cell 17, 2217 (2005). 4. K. Markmann et al., PLoS Biol. 6, e68 (2008). 5. C. Chen, M. Gao, J. Liu, H. Zhu, Plant Physiol. 145, 1619 (2007). 6. S. Radutoiu et al., Nature 425, 585 (2003). 7. H. Kouchi et al., Plant Cell Physiol. 51, 1381 (2010). 8. F. Maillet et al., Nature 469, 58 (2011). 9. D. E. Soltis et al., Proc. Natl. Acad. Sci. U.S.A. 92, 2647 (1995). 10. S. Kosuta et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9823 (2008). 10.1126/science.1202342

PHYSIOLOGY

Life on Low Flame in Hibernation Gerhard Heldmaier

D

o bears really hibernate? Their high body temperature during winter dormancy has raised some doubt about this behavior, as it is unlike the pronounced decreases observed in small mammals that enter this nonactive state. On page 906 of this issue, Tøien et al. (1) show that bears do indeed hibernate. Through continuous measurement of oxygen consumption, body temperature, and heart, muscle, and brain activities, the authors show that black bears disAnimal Physiology, Philipps Universitaet, Marburg, Hessen 35043, Germany. E-mail: [email protected]

866

play unusual patterns of metabolic and thermal regulation during hibernation as well as when they emerge from this resting state in the spring. Hibernation is a powerful behavior that reduces energy costs in mammals. However, in small mammals, it is frequently interrupted by arousals (2, 3), thereby reducing its effectiveness. Generally, after entrance into torpor, deep torpor is maintained for 1 or 2 weeks with body temperature close to the freezing point of body fluids, and is terminated by an arousal for about 1 day. During arousal, body temperature rises to a normal 36°C by

In hibernating black bears, changes in metabolic rate and core body temperature occur independently.

endogenous heat production. Collectively, the arousal episodes require about 80% of the entire energy cost of the animal during the hibernation season. The reasons for the repeated arousals are still a mystery, but they may allow for the repair of neuronal damage induced by prolonged hypometabolism and brain inactivity at low temperature (4, 5). Spontaneous hibernation behavior is difficult to observe in captive animals, so its study has mostly relied on ﬁeld studies of subjects in their natural habitat, or on animals kept in conditions similar to their natural environment. Studying large mammals

18 FEBRUARY 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on February 17, 2011

PERSPECTIVES

both the AM and the NF symbioses are based on the shared activity of a set of plant genes, SYM genes (3–5). This indicates that bacteria hijacked the signal transduction pathway that fungi had used to gain entry into plant tissues and cells. Op den Camp et al. provide evidence that in Parasponia, the only nonlegume partner of rhizobia, a single receptor can recognize both the fungal and bacterial signals and induce the common SYM pathway to promote the intracellular accommodation of the partner microorganisms. Op den Camp et al. used Rhizobium strains, some of which were able to form nodules, and some of which were unable to form nodules, to prove that both Parasponia and legumes use lipochito-oligosaccharides called Nod factors to induce nodule development. They also showed that Nod factors act similarly in both symbioses via a common signaling cascade; in Parasponia, the introduction of a dominant active form of calcium/ calmodulin-dependent kinase (CCaMK), a key element of this pathway, resulted in spontaneous nodulation, as in legumes. Op den Camp et al. also provide insight into how bacterial Nod factor receptors (NFRs) evolved from receptors involved in plant-fungi partnerships. The most-studied legumes recognize rhizobia—or, more accurately, the bacterial Nod factors—via a pair of LysM-type receptor-like kinases, NFR1/ LYK3 and NFR5/NFP (6, 7). Because these NFRs are specific to bacterial symbiosis, investigators had hypothesized that they evolved either by duplication of the mycorrhiza-speciﬁc receptors, which then gained new functions, or by the recruitment of new

receptors that turned on the common signaling pathway. Op den Camp et al.’s analysis indicates that receptor duplication was not essential for plants to acquire the ability to form a symbiotic relationship with NF bacteria. Instead, the presence of a single NFR5like receptor in Parasponia, and its indispensable role in both symbioses, strongly suggests that rhizobia entered symbiotic interactions with plants through the same entrance used by mycorrhizal fungi. It also means that the molecular “keycard” that opens the door to plant partnerships for both bacteria and fungi—the bacterial Nod factor and mycorrhizal (Myc) factor—must be very similar. Indeed, Maillet et al. (8) recently described the Myc factors of AM fungi as lipochito-oligosaccharide molecules that are very similar to Nod factors. These results raise several questions: Why is the appearance of nitrogen-fixing nodules, especially rhizobial ones, restricted to a small fraction of mycorrhizal plants? How do plants discriminate between symbiotic fungi and bacteria? Was it necessary for host plants to distinguish between the microbes to create different niches? Studies of genes from related plants suggest that plant families establishing rhizobial or actinorhizal (Frankia) symbioses belong to the same large lineage. This raises the possibility that, during the evolution of ﬂowering plants, a predisposition for symbiotic nodule formation originated only once (9). Did this predisposition occur by changing the activity of one or more component(s) of the common symbiotic pathway, for example, by enabling it to provide different outputs?

Both bacteria and mycorrhizal fungi induce changes in intracellular calcium (Ca2+-) concentrations (termed calcium spiking). However, the frequency and duration of the oscillations, as well as the speed of Ca movement, are different in the two symbioses (10). Early elements of the common SYM pathway, such as the LysM-type receptors and another receptor protein, the symbiosis receptor kinase (SYMRK), are required for the induction of the calcium spiking, which is then deciphered by CCaMK. It will be interesting to compare calcium spiking upon rhizobial and fungal inoculations in species that possess dual-functioning receptors. There is not yet enough systematic data from different plant lineages to determine exactly how molecules like SYMRK and CCaMK contributed to the evolution of a predisposition to nodule formation. The real challenge is to ﬁnd out why lineages with predisposition for nodulation (for example, certain legumes) are unable to establish NF symbiosis. References

1. R. Op den Camp et al., Science 331, 909 (2011). 2. M. J. Harrison, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 361 (1999). 3. C. Kistner et al., Plant Cell 17, 2217 (2005). 4. K. Markmann et al., PLoS Biol. 6, e68 (2008). 5. C. Chen, M. Gao, J. Liu, H. Zhu, Plant Physiol. 145, 1619 (2007). 6. S. Radutoiu et al., Nature 425, 585 (2003). 7. H. Kouchi et al., Plant Cell Physiol. 51, 1381 (2010). 8. F. Maillet et al., Nature 469, 58 (2011). 9. D. E. Soltis et al., Proc. Natl. Acad. Sci. U.S.A. 92, 2647 (1995). 10. S. Kosuta et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9823 (2008). 10.1126/science.1202342

PHYSIOLOGY

Life on Low Flame in Hibernation Gerhard Heldmaier

D

o bears really hibernate? Their high body temperature during winter dormancy has raised some doubt about this behavior, as it is unlike the pronounced decreases observed in small mammals that enter this nonactive state. On page 906 of this issue, Tøien et al. (1) show that bears do indeed hibernate. Through continuous measurement of oxygen consumption, body temperature, and heart, muscle, and brain activities, the authors show that black bears disAnimal Physiology, Philipps Universitaet, Marburg, Hessen 35043, Germany. E-mail: [email protected]

866

play unusual patterns of metabolic and thermal regulation during hibernation as well as when they emerge from this resting state in the spring. Hibernation is a powerful behavior that reduces energy costs in mammals. However, in small mammals, it is frequently interrupted by arousals (2, 3), thereby reducing its effectiveness. Generally, after entrance into torpor, deep torpor is maintained for 1 or 2 weeks with body temperature close to the freezing point of body fluids, and is terminated by an arousal for about 1 day. During arousal, body temperature rises to a normal 36°C by

In hibernating black bears, changes in metabolic rate and core body temperature occur independently.

endogenous heat production. Collectively, the arousal episodes require about 80% of the entire energy cost of the animal during the hibernation season. The reasons for the repeated arousals are still a mystery, but they may allow for the repair of neuronal damage induced by prolonged hypometabolism and brain inactivity at low temperature (4, 5). Spontaneous hibernation behavior is difficult to observe in captive animals, so its study has mostly relied on ﬁeld studies of subjects in their natural habitat, or on animals kept in conditions similar to their natural environment. Studying large mammals

18 FEBRUARY 2011 VOL 331 SCIENCE www.sciencemag.org Published by AAAS

Downloaded from www.sciencemag.org on February 17, 2011

PERSPECTIVES

PERSPECTIVES

CREDIT: COMSTOCK

(at least 10 kg) is particularly difficult because of the challenges of continuous and long-term monitoring. Tøien et al. observed ﬁve Alaskan black bears (Ursus americanus) (two females and three males ranging in body mass from 34.3 to 103.9 kg) that were kept in outdoor enclosures in a forest near Fairbanks, Alaska. The bears hibernated in isolated wooden nest boxes, which allowed continuous observation and measurement of oxygen consumption and body temperature as well as monitoring of physiological activities from implanted transmitters. The authors observed that during the hibernation period (November to March), the bears did not display repeated arousals, but instead showed multiday oscillations of body temperature between 30° and 36°C. Such a lack of periodic arousals during hibernation has so far only been observed in one small mammal [fat-tailed lemur (6)]. However, Tøien et al. found that the hibernating bears reduced their metabolic rate to 75% below basal metabolic rate (BMR). The observed minimum metabolic rate in hibernating bears (0.056 ml O2 g−1 hour−1) is within the range of those observed in small hibernating mammals (0.02 to 0.06 ml O2 g−1 hour−1) (2, 3). This implies that bears use the entire mammalian scope of metabolic inhibition in torpor and are true hibernators. This reduction of metabolic rate to 75% below BMR is substantially less prominent than that for small

mammals (98% below BMR). The difference is largely due to the allometric scaling of BMR, indicating that hibernation is more effective in small mammals below 1 kg body mass. Tøien et al. also observed that when the bears emerged from their dens in midApril, they had a normal body temperature of 36.6°C. Yet, they maintained a low metabolic rate that was 47% below their BMR, and it took several weeks for it to rise to that of the active season (2.76 ml O2 g−1 hour−1). It is generally assumed that BMR is a speciesspeciﬁc constant that is necessary to maintain the vital physiological functions of an endothermic mammal resting at thermoneutrality. The ﬁndings of Tøien et al. show that bears can maintain their vital functions with a metabolic rate that is reduced to nearly half of that normally required in an active state, indicating that BMR is not a constant but a physiologically controlled variable. Transition into the torpid state includes three processes. Thermoregulatory heat production (by shivering or nonshivering thermogenesis) is inhibited because thermoregulation is adjusted to a lower body temperature. Metabolic rate is depressed below the BMR at normothermic body temperature (active metabolic inhibition). This inhibition can be assisted by temperature effects on metabolic rate. Tøien et al. make the surprising ﬁnding that in hibernating bears, metabolic depres-

References

1. Ø. Tøien et al., Science 331, 906 (2011). 2. F. Geiser, T. Ruf, Physiol. Biochem. Zool. 68, 935 (1995). 3. G. Heldmaier, S. Ortmann, R. Elvert, Respir. Physiol. Neurobiol. 141, 317 (2004). 4. J. Ruediger et al., Synapse 61, 343 (2007). 5. J. T. Stieler et al., PLoS ONE 6, e14530 (2011). 6. K. H. Dausmann, J. Glos, G. Heldmaier, J. Comp. Physiol. B 179, 345 (2009). 7. C. L. Buck, B. M. Barnes, Am. J. Physiol. 279, R255 (2000). 8. G. Heldmaier, R. Elvert, in Life in the Cold: Evolution, Mechanisms, Adaptation and Application, B. M. Barnes, H. V. Carey, Eds. (Univ. of Alaska, Fairbanks, 2004), pp. 185–198. 9. F. van Breukelen, S. Martin, J. Comp. Physiol. B 172, 355 (2002). 10. H. V. Carey, M. T. Andrews, S. L. Martin, Physiol. Rev. 83, 1153 (2003). 11. J. F. Staples, J. C. L. Brown, J. Comp. Physiol. B 178, 811 (2008). 12. M. T. Andrews, Bioessays 29, 431 (2007). 13. P. Morin Jr., K. B. Storey, Int. J. Dev. Biol. 53, 433 (2009). 14. W. Arnold et al., Am. J. Physiol. 286, R174 (2004).

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Barely active bear? Black bears emerging from hibernation have a metabolic rate that is nearly half of that found in a normal active state.

sion is largely achieved by active metabolic inhibition, whereas temperature effects play only a minor role. In small mammals that hibernate, active inhibition and temperaturerelated metabolic depression, on average, may each be responsible for about 50% of total metabolic depression (3, 7, 8). The molecular mechanisms and biochemical pathways that underlie metabolic adjustment in torpor are still unclear. In general, torpor metabolism involves inhibition of processes that generate adenosine 5′-triphosphate such as glycolysis (metabolism is rerouted to lipid utilization instead) and mitochondrial respiration, as well as energy-consuming processes such as transcription, translation, and protein degradation (9–11). This ultimately impairs cell proliferation and differentiation. However, entrance into torpor also requires increased expression of hibernation-speciﬁc genes to support lipid metabolism, gluconeogenesis, cytoprotection, and other measures required to maintain cells (12, 13). In most mammalian orders, one or several species use torpid metabolic depression. The greatest numbers are found among marsupials, rodents, and bats, but also in small numbers in insectivores, primates, and elephant shrews; and it is likely that more such examples will be discovered in large mammals (14). Although long considered an adaptation to cold, hibernation is also found in tropical animals and desert species, and, as in bears, can occur without substantial drops in body temperature. Perhaps we will find that a hypometabolic state is the primary means by which most, if not all mammals, can reduce their energy expenditures for prolonged periods of time.

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Controlling the Flow of Suspensions

Adding a small amount of an immiscible liquid to a suspension can change it from a viscous ﬂuid to an elastic gel.

Hans-Jürgen Butt

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I

f you’ve painted a wall, you from forming a large interfacial area A B know that you want the paint with the bulk, nonpolar liquid (see to ﬂow smoothly onto the surthe ﬁgure, panel B). Here, the contact face but be viscous enough that angles are around or above 90°, and θ it doesn’t drip. Paint is a suspenthe ﬂuid would tend to form droplets sion—small, solid particles of pigon the surface. In contrast to the penment and polymer dispersed in a dular state, this is called the capillary Liquid b liquid—and manufacturers devote state (1, 3). Koos and Willenbacher much effort to controlling its ﬂow studied many different combinations behavior, or rheology. Suspenof immiscible liquids and solid parθ sion rheology is critical not only ticles and observed the same changes in coatings but also in many food in the rheological properties, which C and materials processing steps, and suggests that this effect could be seen it depends on two factors. First, the in many other suspensions. content and shape of the dispersed A closely related effect has been particles are important, but these used for more than a century to stabifactors usually cannot be varied lize emulsions (a mixture of one liqto optimize performance. The size uid dispersed in a second immiscible and shape of the particles are usuliquid, such as oil in water). A Pickerθ ally predetermined, and a high voling emulsion is stabilized by adding Liquid a ume ratio of particles to solvent is colloidal particles that move to the often required. The second factor oil-water interface (8, 9) and prevent θ <<90°, wetting θ ≥ 90°, nonwetting is the interaction between the pardrops of the secondary liquid from ticles, which affects viscosity. To Stabilizing dispersed phases. These schematic representations show how merging (see the ﬁgure, panel C). In achieve low viscosity yet deliver a suspensions and emulsions can be stabilized with a third added phase. (A) contrast to the capillary state in sushigh volume ratio of particles, the Dispersed particles forming a pendular state in a primary liquid a (con- pensions, the amount of secondary particles should repel each other. tinuous phase, gray). The particles are kept together by pendular menisci liquid in a Pickering emulsion is comof a second, immiscible liquid b (blue). This happens if the contact angle To achieve a high viscosity and cre- is low and the added liquid wets the particles. (B) Koos and Willenbacher parable to that of the primary liquid, ate an elastic material like a gel, report on a capillary state, in which drops of the secondary liquid form the and the drops are much larger than the particles need to aggregate via center of particle agglomerates. In this case, the contact angle is about 90° the particles. More recently, colloidoattractive forces. Forces between and the added liquid fails to wet the particles. (C) A similar effect is used somes (10, 11), which have potenthe particles are determined by to stabilize Pickering emulsions, where colloidal particles prevent droplets tial applications in drug delivery, and their surface properties and are tra- in an emulsion from merging into larger droplets. small clusters of a deﬁned number of ditionally adjusted by adding surparticles—also called colloidal molefactants. On page 897 of this issue, Koos and sion draws the particles together. These capil- cules (12, 13)—have been made by conﬁning Willenbacher (1) propose another option, the lary forces (4–6) are relatively strong, much particles to the interface of oil drops in water, addition of a small amount of a second liquid, stronger than the van der Waals forces. The or vice versa. immiscible with the primary liquid. particles aggregate (7) and form an elastic What determines whether a suspension Koos and Willenbacher first dispersed network (see the ﬁgure, panel A). enters the pendular state or the capillary state? hydrophilic glass beads in a primary liquid Capillary forces act between two particles The answer lies in Young’s equation, which (an organic solvent, diisononyl phthalate) if the liquid-liquid interface of the pendular relates the interfacial tensions to the contact and then added 1 weight percent of water as meniscus forms a low contact angle θ with angle: cos θ = (γSA – γSB)/γ AB. Here, γSA is the a secondary liquid. Upon stirring, the sus- the solid-liquid interface. On a ﬂat surface, interfacial tension of the solid particle with pension changed from a viscous ﬂuid to an the secondary liquid would spread and from a the primary liquid A, γSB is the interfacial tenelastic or gel-like material. Such a transi- ﬂat pancake. However, capillary forces should sion of the solid particle with the secondary tion from a viscous to an elastic state caused not be operating for high contact angles. liquid B, and γAB is the interfacial tension of by trace amounts of a secondary liquid had Therefore, Koos and Willenbacher were the liquid-liquid interface. If the secondary already been observed (2, 3) for the case in quite surprised to observe a similar transition liquid wets the particles better than the priwhich the added liquid wets the particles. A when repeating the experiment with hydro- mary liquid (γSA > γSB), the contact angle is pendular water meniscus forms around the phobized glass beads. Hydrophobized glass low and the pendular state should form. If the contact point between two particles. Once beads form a large contact angle with water secondary liquid wets the particle surface less such a meniscus has formed, interfacial ten- in nonpolar liquids. Koos and Willenbacher or equally well (γSA ≤ γSB), the contact angle is explained this surprising result in terms of ≈90° and the capillary state should form. a collective effect in which several particles The discovery of Koos and Willenbacher Polymer Physics, Max Planck Institute for Polymer Research Mainz, 55128 Germany. E-mail: [email protected] gather around a droplet of water and protect it will have considerable impact. Process engi-

PERSPECTIVES of granular materials (14, 15), the interaction energies between particles are much higher than the thermal energy that governs particle and solvent motions. Large energy barriers have to be overcome in a transition, so a system is likely not to be in thermodynamic equilibrium. This situation makes a systematic study more difﬁcult because the structure and properties of a suspension will depend on its history. References

1. E. Koos, N. Willenbacher, Science 331, 897 (2011). 2. H. R. Kruyt, F. G. van Selms, Recueil Des Travaux Chimiques Des Pays-Bas 62, 415 (1943). 3. S. van Kao, L. E. Nielsen, C. T. Hill, J. Colloid Interface Sci. 53, 367 (1975).

4. R. A. Fisher, J. Agric. Sci. 16, 492 (1926). 5. W. B. Haines, J. Agric. Sci. 17, 264 (1927). 6. H.-J. Butt, M. Kappl, Adv. Colloid Interface Sci. 146, 48 (2009). 7. A. E. J. Eggleton, I. E. Puddington, Can. J. Chem. 32, 86 (1954). 8. S. H. Pickering, J. Chem. Soc. 91, 2001 (1907). 9. R. Aveyard, B. P. Binks, J. H. Clint, Adv. Colloid Interface Sci. 100-102, 503 (2003). 10. O. D. Velev, K. Furusawa, K. Nagayama, Langmuir 12, 2374 (1996). 11. A. D. Dinsmore et al., Science 298, 1006 (2002). 12. V. N. Manoharan, M. T. Elsesser, D. J. Pine, Science 301, 483 (2003). 13. C. S. Wagner, B. Fischer, M. May, A. Wittemann, Colloid Polym. Sci. 288, 487 (2010). 14. S. Herminghaus, Adv. Phys. 54, 221 (2005). 15. N. Mitarai, F. Nori, Adv. Phys. 55, 1 (2006). 10.1126/science.1201543

Downloaded from www.sciencemag.org on February 17, 2011

neers now have an additional way of tuning the ﬂow properties of suspensions in a simple, environmentally friendly, and inexpensive manner. Rather than adding surfactants or polymers, a suitable secondary liquid can be added. The discovery is also a warning to avoid contaminating a suspension with an immiscible secondary liquid and risk a drastic increase in suspension viscosity (clogged pipes or jammed machinery). For basic research, their ﬁndings lead to several questions. What is the inﬂuence of added surfactants and polymers, considering their adsorption to all three interfaces? What is the influence of polydispersity, particle shape, or surface roughness? As in the case

NEUROSCIENCE

Creating Stable Memories

Epigenetic mechanisms are involved in “tagging” rat neurons active in long-term memory of food odors.

J. David Sweatt

S

ocial animals with a keen sense of smell, such as rats, can teach each other about the safety of new foods by a practice called social transmission of food preference (STFP). In this olfactory learning paradigm, a rat that has just eaten can familiarize another rat with the taste odor of a new food by allowing the naïve rat to sniff its breath. The naïve rat apparently infers that the novel food is safe to eat because another rat has eaten it, and exhibits greatly diminished fear of the new food at ﬁrst exposure. On page 924 of this issue, Lesburguéres et al. (1) describe their discovery of two fascinating aspects of this form of long-term memory: Department of Neurobiology and Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA. E-mail: [email protected]

Speciﬁc neurons or synapses in the cerebral cortex are speciﬁcally “tagged” and thereby allocated to participate in a memory for a particular food odor, and this process involves epigenetic molecular mechanisms. STFP is a form of associative memory that depends on a particular part of the brain, the hippocampus (HPC), and can last an animal’s lifetime (2–4). When being formed, the memory is downloaded from the HPC to the cortex via a process called systems consolidation, a term that describes an ongoing synaptic dialog between the HPC and the orbitofrontal cortex (OFC) that occurs over the course of about 1 week (5, 6). In their experimental paradigm, Lesburguéres et al. showed that STFP is dependent on the OFC at 30 days after the training experience (“remote” memory), but independent of the OFC at 24 hours (“recent”

A

memory). Thus, STFP is HPC-dependent for its acquisition, and the long-term storage of the olfactory information requires the OFC. Lesburguéres et al. investigated how the interplay of neural circuits in the HPC and OFC allows systems-based consolidation. In their experiments (see the ﬁgure), they found that the glutamate receptor antagonist CNQX blocks memory when infused into the OFC during the 2 weeks immediately after a rat is exposed to the novel taste. The observation of memory loss with blockade of OFC activity immediately after novel taste odor exposure raised a conundrum. At 30 days, taste odor memory is consolidated and stored in the OFC. Hence, during the ﬁrst week that the memory is being processed in the HPC, it does not yet reside in the OFC. So how can early inhibition of the OFC block its subse-

B

Tagging hypothesis OFC neurons

Cumin taste odor

CREDIT: ADAPTED BY P. HUEY/SCIENCE

0 1.

Neural activity blockade in OFC

Test memory 1

2

Weeks

3

Effect

3. OFC blockade

Hippocampus neurons

Blocks remote memory

OFC blockade

2.

4

OFC blockade

Blocks remote memory Also blocks remote memory

Taste test. (A) Infusing CNQX into the OFC blocks neuronal ﬁring and leads to loss of long-term memory consolidation, both when infused early (during the 2 weeks immediately after novel taste exposure) and late (in weeks 3 and 4). (B) After expo-

Tagged cell and synapse Allocated to taste 1 Allocated to taste 2

sure to a novel taste, a subset of OFC neurons are “tagged” for different taste memories, marking them for subsequent activity-dependent plasticity driven by the hippocampus. This tag might reside at the synaptic or at the whole-cell level (15).

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PERSPECTIVES of granular materials (14, 15), the interaction energies between particles are much higher than the thermal energy that governs particle and solvent motions. Large energy barriers have to be overcome in a transition, so a system is likely not to be in thermodynamic equilibrium. This situation makes a systematic study more difﬁcult because the structure and properties of a suspension will depend on its history. References

1. E. Koos, N. Willenbacher, Science 331, 897 (2011). 2. H. R. Kruyt, F. G. van Selms, Recueil Des Travaux Chimiques Des Pays-Bas 62, 415 (1943). 3. S. van Kao, L. E. Nielsen, C. T. Hill, J. Colloid Interface Sci. 53, 367 (1975).

4. R. A. Fisher, J. Agric. Sci. 16, 492 (1926). 5. W. B. Haines, J. Agric. Sci. 17, 264 (1927). 6. H.-J. Butt, M. Kappl, Adv. Colloid Interface Sci. 146, 48 (2009). 7. A. E. J. Eggleton, I. E. Puddington, Can. J. Chem. 32, 86 (1954). 8. S. H. Pickering, J. Chem. Soc. 91, 2001 (1907). 9. R. Aveyard, B. P. Binks, J. H. Clint, Adv. Colloid Interface Sci. 100-102, 503 (2003). 10. O. D. Velev, K. Furusawa, K. Nagayama, Langmuir 12, 2374 (1996). 11. A. D. Dinsmore et al., Science 298, 1006 (2002). 12. V. N. Manoharan, M. T. Elsesser, D. J. Pine, Science 301, 483 (2003). 13. C. S. Wagner, B. Fischer, M. May, A. Wittemann, Colloid Polym. Sci. 288, 487 (2010). 14. S. Herminghaus, Adv. Phys. 54, 221 (2005). 15. N. Mitarai, F. Nori, Adv. Phys. 55, 1 (2006). 10.1126/science.1201543

Downloaded from www.sciencemag.org on February 17, 2011

neers now have an additional way of tuning the ﬂow properties of suspensions in a simple, environmentally friendly, and inexpensive manner. Rather than adding surfactants or polymers, a suitable secondary liquid can be added. The discovery is also a warning to avoid contaminating a suspension with an immiscible secondary liquid and risk a drastic increase in suspension viscosity (clogged pipes or jammed machinery). For basic research, their ﬁndings lead to several questions. What is the inﬂuence of added surfactants and polymers, considering their adsorption to all three interfaces? What is the influence of polydispersity, particle shape, or surface roughness? As in the case

NEUROSCIENCE

Creating Stable Memories

Epigenetic mechanisms are involved in “tagging” rat neurons active in long-term memory of food odors.

J. David Sweatt

S

ocial animals with a keen sense of smell, such as rats, can teach each other about the safety of new foods by a practice called social transmission of food preference (STFP). In this olfactory learning paradigm, a rat that has just eaten can familiarize another rat with the taste odor of a new food by allowing the naïve rat to sniff its breath. The naïve rat apparently infers that the novel food is safe to eat because another rat has eaten it, and exhibits greatly diminished fear of the new food at ﬁrst exposure. On page 924 of this issue, Lesburguéres et al. (1) describe their discovery of two fascinating aspects of this form of long-term memory: Department of Neurobiology and Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA. E-mail: [email protected]

Speciﬁc neurons or synapses in the cerebral cortex are speciﬁcally “tagged” and thereby allocated to participate in a memory for a particular food odor, and this process involves epigenetic molecular mechanisms. STFP is a form of associative memory that depends on a particular part of the brain, the hippocampus (HPC), and can last an animal’s lifetime (2–4). When being formed, the memory is downloaded from the HPC to the cortex via a process called systems consolidation, a term that describes an ongoing synaptic dialog between the HPC and the orbitofrontal cortex (OFC) that occurs over the course of about 1 week (5, 6). In their experimental paradigm, Lesburguéres et al. showed that STFP is dependent on the OFC at 30 days after the training experience (“remote” memory), but independent of the OFC at 24 hours (“recent”

A

memory). Thus, STFP is HPC-dependent for its acquisition, and the long-term storage of the olfactory information requires the OFC. Lesburguéres et al. investigated how the interplay of neural circuits in the HPC and OFC allows systems-based consolidation. In their experiments (see the ﬁgure), they found that the glutamate receptor antagonist CNQX blocks memory when infused into the OFC during the 2 weeks immediately after a rat is exposed to the novel taste. The observation of memory loss with blockade of OFC activity immediately after novel taste odor exposure raised a conundrum. At 30 days, taste odor memory is consolidated and stored in the OFC. Hence, during the ﬁrst week that the memory is being processed in the HPC, it does not yet reside in the OFC. So how can early inhibition of the OFC block its subse-

B

Tagging hypothesis OFC neurons

Cumin taste odor

CREDIT: ADAPTED BY P. HUEY/SCIENCE

0 1.

Neural activity blockade in OFC

Test memory 1

2

Weeks

3

Effect

3. OFC blockade

Hippocampus neurons

Blocks remote memory

OFC blockade

2.

4

OFC blockade

Blocks remote memory Also blocks remote memory

Taste test. (A) Infusing CNQX into the OFC blocks neuronal ﬁring and leads to loss of long-term memory consolidation, both when infused early (during the 2 weeks immediately after novel taste exposure) and late (in weeks 3 and 4). (B) After expo-

Tagged cell and synapse Allocated to taste 1 Allocated to taste 2

sure to a novel taste, a subset of OFC neurons are “tagged” for different taste memories, marking them for subsequent activity-dependent plasticity driven by the hippocampus. This tag might reside at the synaptic or at the whole-cell level (15).

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PERSPECTIVES

870

as the memory behavior itself. Furthermore, increasing OFC histone acetylation with infusions of a drug early after memory acquisition increased the robustness of the memory. These ﬁndings show that increasing histone (or other protein) acetylation is likely a necessary component of the tagging process, and implicate epigenetic molecular mechanisms in generating the tags necessary for systems consolidation in the OFC. Lesburguéres et al. move the ﬁeld in a new direction with two conceptual advances— the idea of synaptic tagging in the cortex as a gateway for remote memory consolidation, and the idea of epigenetic mechanisms as contributors to the formation of the tag and resulting systems consolidation. Although synaptic tagging is a component of longterm potentiation (LTP), a cellular mechanism underlying memory, there is no reason to believe that the molecular processes studied by Lesburguéres et al. need to be the same as those operating in hippocampal LTP. They do, after all, occur on very different time scales—minutes versus weeks. An interesting implication of the work by Lesburguéres et al. is that presumably cell-wide transcriptional changes driven by

epigenetic marks are interacting with synapse-speciﬁc mechanisms as part of generating and perpetuating the appropriate cortical tags (15). How does this happen when one set of mechanisms is operating at the level of the entire genome, while the other is synapse-speciﬁc? References

1. E. Lesburguéres et al., Science 331, 924 (2010). 2. M. Bunsey, H. Eichenbaum, Hippocampus 5, 546 (1995). 3. R. E. Clark, N. J. Broadbent, S. M. Zola, L. R. Squire, J. Neurosci. 22, 4663 (2002). 4. P. Alvarez, P. A. Lipton, R. Melrose, H. Eichenbaum, Learn. Mem. 8, 79 (2001). 5. R. S. Ross, H. Eichenbaum, J. Neurosci. 26, 4852 (2006). 6. B. J. Wiltgen, R. A. Brown, L. E. Talton, A. J. Silva, Neuron 44, 101 (2004). 7. U. Frey, R. G. M. Morris, Nature 385, 533 (1997). 8. U. Frey, R. G. M. Morris, Trends Neurosci. 21, 181 (1998). 9. R. L. Redondo, R. G. M. Morris, Nat. Rev. Neurosci. 12, 17 (2011). 10. M. W. Swank, J. D. Sweatt, J. Neurosci. 21, 3383 (2001). 11. J. M. Levenson et al., J. Biol. Chem. 279, 40545 (2004). 12. W. B. Chwang, J. S. Arthur, A. Schumacher, J. D. Sweatt, J. Neurosci. 27, 12732 (2007). 13. T. L. Roth, J. D. Sweatt, Curr. Opin. Neurobiol. 19, 336 (2009). 14. E. Borrelli, E. J. Nestler, C. D. Allis, P. Sassone-Corsi, Neuron 60, 961 (2008). 15. J. J. Day, J. D. Sweatt, Nat. Neurosci. 13, 1319 (2010). 10.1126/science.1202283

MOLECULAR BIOLOGY

Hitting a Tiny Target in the Dark R. Scott Hawley A region in mammalian sex chromosomes has distinct properties that ensure proper segregation during gamete development.

T

he success of sexual reproduction in mammals depends on segregation of the male sex chromosomes, X and Y, during the cell division process that produces gametes (meiosis). This requires that in male germ cells, the X and Y chromosomes reliably pair and exchange genetic material (1). This process, known as recombination or crossing-over, serves to physically interlock homologous chromosomes and ensure their proper segregation during the ﬁrst meiotic cell division. Failure of X-Y recombination has dire consequences—either germ cell death or X-Y missegregation (1, 2). On page 916 (3) of this issue, Kauppi et al. identify multiple mechanisms that ensure efficient Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA, and Department of Molecular and Integrative Physiology, Kansas University Medical Center, Kansas City, KS 66160, USA. E-mail: rsh@ stowers.org

X-Y pairing and recombination during meiosis in the mouse. The processes that underlie these recombination events are distinct from those governing crossing-over on the other non–sex-determining chromosomes (autosomes) and play important roles in integrating the Y chromosome into a successful meiotic program. In many organisms (including mammals), both the pairing of homologous chromosomes and the initiation of recombination depend on the programmed introduction of double-strand breaks in the DNA by the SPO11 protein (4, 5). Pairing of the X and Y chromosomes is mediated by a region of homology located near the ends of these chromosomes called the pseudoautosomal region (PAR) (6) (see the ﬁgure). Kauppi et al. show that X-Y pairing is greatly delayed relative to autosomes. Using antibodies that recognize the RAD51 and DMC1 pro-

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quent ability to stabilize the memory? What happens early in the OFC, during and immediately after the initial novel taste odor exposure, that prepares the OFC to be the recipient of the memory trace 2 weeks later? To answer this question, Lesburguéres et al. posited a tagging process (7–9). Upon the ﬁrst experience of a novel taste, a subset of neurons in the HPC and OFC are tagged and allocated to a speciﬁc memory, and thereafter are coordinately driven by the HPC-OFC systems consolidation circuitry (see the ﬁgure). They tested this idea in three principal experiments. First, they showed that inactivating the OFC immediately before a rat was exposed to a novel taste odor (cumin) blocked 30-day remote memory, but left 7-day immediate memory intact (see the ﬁgure). This result shows that circuit activity in the OFC during the novel taste experience is necessary for subsequent development of the capacity to store the memory. This strongly suggests the existence of an activity-dependent process in the OFC that prepares at least a subset of neurons in the OFC for subsequent, delayed reception of plasticity signals originating in the HPC. These signals drive the instantiation of a memory trace in the OFC. The inference is also that only a subset of OFC neurons are enabled (i.e., tagged for subsequent use), because the OFC has a capacity for memorizing at least several hundred different taste odors. To more directly test this inference, the investigators trained an individual animal with two different taste odors (cumin and cocoa). They allowed the animal to experience the ﬁrst (cocoa) normally, but inactivated the OFC 1 week later, before that animal experienced the second taste odor (cumin). Thus, the animal needed to develop two independent memory traces for two different taste odors, both residing in the OFC. Remarkably, only the memory for the second taste odor was diminished, whereas the ﬁrst remained intact after OFC inactivation. This result is consistent with early memory-speciﬁc synaptic or neuronal tagging in the OFC. In a final experiment, Lesburguéres et al. tested the role of epigenetic molecular mechanisms in tagging and stabilizing memory-associated neural circuits in the OFC (10–15). They found that formation of novel taste odor memory is associated with acetylation of a histone protein involved in chromatin structure (H3); this is considered an epigenetic “mark” in the OFC. Blocking signaling that involves extracellular signal– regulated kinases (ERKs) and mitogen- and stress-activated protein kinases (MSKs) prevented this increase in H3 acetylation, as well

PERSPECTIVES

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as the memory behavior itself. Furthermore, increasing OFC histone acetylation with infusions of a drug early after memory acquisition increased the robustness of the memory. These ﬁndings show that increasing histone (or other protein) acetylation is likely a necessary component of the tagging process, and implicate epigenetic molecular mechanisms in generating the tags necessary for systems consolidation in the OFC. Lesburguéres et al. move the ﬁeld in a new direction with two conceptual advances— the idea of synaptic tagging in the cortex as a gateway for remote memory consolidation, and the idea of epigenetic mechanisms as contributors to the formation of the tag and resulting systems consolidation. Although synaptic tagging is a component of longterm potentiation (LTP), a cellular mechanism underlying memory, there is no reason to believe that the molecular processes studied by Lesburguéres et al. need to be the same as those operating in hippocampal LTP. They do, after all, occur on very different time scales—minutes versus weeks. An interesting implication of the work by Lesburguéres et al. is that presumably cell-wide transcriptional changes driven by

epigenetic marks are interacting with synapse-speciﬁc mechanisms as part of generating and perpetuating the appropriate cortical tags (15). How does this happen when one set of mechanisms is operating at the level of the entire genome, while the other is synapse-speciﬁc? References

1. E. Lesburguéres et al., Science 331, 924 (2010). 2. M. Bunsey, H. Eichenbaum, Hippocampus 5, 546 (1995). 3. R. E. Clark, N. J. Broadbent, S. M. Zola, L. R. Squire, J. Neurosci. 22, 4663 (2002). 4. P. Alvarez, P. A. Lipton, R. Melrose, H. Eichenbaum, Learn. Mem. 8, 79 (2001). 5. R. S. Ross, H. Eichenbaum, J. Neurosci. 26, 4852 (2006). 6. B. J. Wiltgen, R. A. Brown, L. E. Talton, A. J. Silva, Neuron 44, 101 (2004). 7. U. Frey, R. G. M. Morris, Nature 385, 533 (1997). 8. U. Frey, R. G. M. Morris, Trends Neurosci. 21, 181 (1998). 9. R. L. Redondo, R. G. M. Morris, Nat. Rev. Neurosci. 12, 17 (2011). 10. M. W. Swank, J. D. Sweatt, J. Neurosci. 21, 3383 (2001). 11. J. M. Levenson et al., J. Biol. Chem. 279, 40545 (2004). 12. W. B. Chwang, J. S. Arthur, A. Schumacher, J. D. Sweatt, J. Neurosci. 27, 12732 (2007). 13. T. L. Roth, J. D. Sweatt, Curr. Opin. Neurobiol. 19, 336 (2009). 14. E. Borrelli, E. J. Nestler, C. D. Allis, P. Sassone-Corsi, Neuron 60, 961 (2008). 15. J. J. Day, J. D. Sweatt, Nat. Neurosci. 13, 1319 (2010). 10.1126/science.1202283

MOLECULAR BIOLOGY

Hitting a Tiny Target in the Dark R. Scott Hawley A region in mammalian sex chromosomes has distinct properties that ensure proper segregation during gamete development.

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he success of sexual reproduction in mammals depends on segregation of the male sex chromosomes, X and Y, during the cell division process that produces gametes (meiosis). This requires that in male germ cells, the X and Y chromosomes reliably pair and exchange genetic material (1). This process, known as recombination or crossing-over, serves to physically interlock homologous chromosomes and ensure their proper segregation during the ﬁrst meiotic cell division. Failure of X-Y recombination has dire consequences—either germ cell death or X-Y missegregation (1, 2). On page 916 (3) of this issue, Kauppi et al. identify multiple mechanisms that ensure efficient Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA, and Department of Molecular and Integrative Physiology, Kansas University Medical Center, Kansas City, KS 66160, USA. E-mail: rsh@ stowers.org

X-Y pairing and recombination during meiosis in the mouse. The processes that underlie these recombination events are distinct from those governing crossing-over on the other non–sex-determining chromosomes (autosomes) and play important roles in integrating the Y chromosome into a successful meiotic program. In many organisms (including mammals), both the pairing of homologous chromosomes and the initiation of recombination depend on the programmed introduction of double-strand breaks in the DNA by the SPO11 protein (4, 5). Pairing of the X and Y chromosomes is mediated by a region of homology located near the ends of these chromosomes called the pseudoautosomal region (PAR) (6) (see the ﬁgure). Kauppi et al. show that X-Y pairing is greatly delayed relative to autosomes. Using antibodies that recognize the RAD51 and DMC1 pro-

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quent ability to stabilize the memory? What happens early in the OFC, during and immediately after the initial novel taste odor exposure, that prepares the OFC to be the recipient of the memory trace 2 weeks later? To answer this question, Lesburguéres et al. posited a tagging process (7–9). Upon the ﬁrst experience of a novel taste, a subset of neurons in the HPC and OFC are tagged and allocated to a speciﬁc memory, and thereafter are coordinately driven by the HPC-OFC systems consolidation circuitry (see the ﬁgure). They tested this idea in three principal experiments. First, they showed that inactivating the OFC immediately before a rat was exposed to a novel taste odor (cumin) blocked 30-day remote memory, but left 7-day immediate memory intact (see the ﬁgure). This result shows that circuit activity in the OFC during the novel taste experience is necessary for subsequent development of the capacity to store the memory. This strongly suggests the existence of an activity-dependent process in the OFC that prepares at least a subset of neurons in the OFC for subsequent, delayed reception of plasticity signals originating in the HPC. These signals drive the instantiation of a memory trace in the OFC. The inference is also that only a subset of OFC neurons are enabled (i.e., tagged for subsequent use), because the OFC has a capacity for memorizing at least several hundred different taste odors. To more directly test this inference, the investigators trained an individual animal with two different taste odors (cumin and cocoa). They allowed the animal to experience the ﬁrst (cocoa) normally, but inactivated the OFC 1 week later, before that animal experienced the second taste odor (cumin). Thus, the animal needed to develop two independent memory traces for two different taste odors, both residing in the OFC. Remarkably, only the memory for the second taste odor was diminished, whereas the ﬁrst remained intact after OFC inactivation. This result is consistent with early memory-speciﬁc synaptic or neuronal tagging in the OFC. In a final experiment, Lesburguéres et al. tested the role of epigenetic molecular mechanisms in tagging and stabilizing memory-associated neural circuits in the OFC (10–15). They found that formation of novel taste odor memory is associated with acetylation of a histone protein involved in chromatin structure (H3); this is considered an epigenetic “mark” in the OFC. Blocking signaling that involves extracellular signal– regulated kinases (ERKs) and mitogen- and stress-activated protein kinases (MSKs) prevented this increase in H3 acetylation, as well

PERSPECTIVES

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teins, which mark the ends of double-strand breaks, Kauppi et al. also show that double-strand breaks have a delayed appearance within the PAR regions of the X and Y chromosomes. They note that most doublestrand breaks in the PAR occur after the abundance of such breaks in the autosomes starts to decline. Perhaps the late occurrence of most breaks in the PAR reﬂects an alternative mechanism for inducing recombination to ensure that those germ cells that did not receive an early PAR double-strand break do eventually get one. The sex body, a chromatin domain that encompasses the X and Y chromosomes, forms contemporaneously with the delayed PAR breaks (1), which suggests that it facilitates X-Y pairing. Such a requirement for the sex body to help ensure this pairing may explain the authors’ observation that those breaks arising in the PAR at earlier times during meiosis cannot facilitate pairing immediately. Thus, X-Y pairing and recombination initiation occur within a narrow temporal window, unlike the rest of the genome. The male meiotic cell must also ensure that the rate of double-strand break formation within the PAR is higher than the genome average by a factor of 10 to 20. The frequency of recombination within a region

along a chromosome does not always correlate with the total length of DNA contained in that region, but it is correlated with the length of the structures known as chromosome axes that organize the chromosomes into a series of loops (7). The length of this chromosome axis varies inversely with the size of chromatin loops attached to the axis. DNA arranged into smaller loops along a longer axis may increase the frequency of double-strand breaks (8). Kauppi et al. show that relative to DNA content, the PAR axes are substantially longer (by the necessary factor of 10) than are the axes of two autosomal regions examined, with concomitantly shorter chromatin loops. This provides a structure in the sex chromosomes that increases the probability of double-strand break formation by SPO11. The odds for getting a double-strand break into the PARs may also be facilitated by a specific isoform of SPO11. In mice and humans, there are two major isoforms of SPO11 that are produced by alternative splicing, SPO11α and SPO11β. SPO11β appears earlier during male meiosis, when most double-strand breaks are being created throughout the genome, whereas SPO11α is produced later (5). In female mice, where the two fully homologous X chromosomes do not require PAR-based recombination, Kauppi et

References

1. P. S. Burgoyne, S. K. Mahadevaiah, J. M. Turner, Nat. Rev. Genet. 10, 207 (2009). 2. N. S. Thomas, T. J. Hassold, Hum. Reprod. Update 9, 309 (2003). 3. L. Kauppi et al., Science 331, 916 (2011) 4. F. Baudat, K. Manova, J. P. Yuen, M. Jasin, S. Keeney, Mol. Cell 6, 989 (2000). 5. P. J. Romanienko, R. D. Camerini-Otero, Mol. Cell 6, 975 (2000). 6. F. Rouyer et al., Nature 319, 291 (1986). 7. L. Froenicke, L. K. Anderson, J. Wienberg, T. Ashley, Am. J. Hum. Genet. 71, 1353 (2002). 8. N. Kleckner, A. Storlazzi, D. Zickler, Trends Genet. 19, 623 (2003). 9. J. V. Henzel et al., Genetics, 10.1534/genetics. 110.124958 (2011).

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Controlling recombination. In mammals, the process of genetic recombination between a pair of homologous chromosomes (autosomes) occurs along the length of the chromosomes (top). However, for the sex chromosomes, recombination is restricted to the PAR regions shared by the X and Y chromosomes (bottom). Shown are scenarios for human chromosomes.

al. demonstrate that SPO11β is sufﬁcient for near normal levels of recombination. But in males, the sole expression of SPO11β supports only autosomal pairing and recombination—the X and Y PARs fail to experience double-strand breaks or pair in about 70% of meiotic nuclei. The low level of X-Y pairing observed following the sole expression of SPO11β is likely the consequence of those few early double-strand breaks within the PAR that can be induced by SPO11β. Perhaps the most interesting facet of this story is the multiple layers of control that mediate the extraordinarily high levels of recombination that occur within the PAR in male meiosis. It was thought (and is often still taught) that the frequency of meiotic recombination between two points on a chromosome is proportional to the physical distance between them. But in reality, the distribution of recombination throughout the genome is under tight genetic control, such that recombination events occur rarely in regions such as heterochromatin (highly repetitive and highly condensed DNA) or very close to telomeres (the ends of chromosomes) but at high rates in some autosomal regions and in the PAR. The studies by Kauppi et al. thus provide crucial insights into the mechanisms that control exchange at the very small target of the PAR. One possible insight as to how such mechanisms might evolve comes from studies in the nematode Caenorhabditis elegans, in which a pair of structurally dissimilar chromosomes was generated by a chromosome fusion event (9). This fusion created a pair of heteromorphic “sex chromosomes” analogous to the mammalian X and Y chromosomes. Despite sharing an extensive domain of homology, most of the recombination that occurred between these two chromosomes was restricted to a limited region of the distal end of the chromosome pair—a situation not so different from what is observed in mammalian PAR.

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ESSAY GENOME-SEQUENCING ANNIVERSARY

A Celebration of the Genome, Part III

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his is the third in our series of vignettes celebrating the 10th anniversary of the sequencing of the human genome. You will ﬁnd vastly different perceptions of what the genome represents and its impact.

Sheila Jasanoff Pforzheimer Professor of Science and Technology Studies, John F. Kennedy School of Government, Harvard University, Cambridge, MA, USA A decade is not nearly enough time to measure the impact of a scientiﬁc revolution. We should not presume to judge how human lives will change as a result of the publication of the human genome sequence in Science in February 2001. It is clear, though, that simply weighing past predictions against present realities misses the mark. It is too soon to tell whether cures for genetic disease were oversold or fears of producing a genetic underclass were much overblown. What matters is that we found a powerful new way to represent human identity, and the moral implications of that re-representation are just beginning to unfold. Like the Constitution of the United States, the human genome turned out to be a sparse document, containing fewer genes than expected. This means that, as with the Constitution, the genome’s meanings will evolve over time, as scientists, lawmakers, and publics make sense of the ﬁxed elements of the sequence in relation to the variables and unknowns in the surrounding environment. Some outlines of new thinking are already emerging, as importantly in law and ethics as in biomedicine. Unsettled questions about who can patent genes, how to regulate unconsented uses of genetic information, when to allow familial searching of data banks, who owns immortal cell lines, and what constitutes impermissible mixing of human and nonhuman genetic material all point to deeper uncertainty about the moral status of the human genome. These questions are contentious, but in disrupting our taken-for-

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granted assumptions, they force us to rethink the nature and consequences of being human. In retrospect, this point leaps out as most signiﬁcant: 2001 marked the issuance of a founding document, a biological living constitution that remakes how we, as individuals and species, interpret our obligations to each other and to other entities on Earth.

What Does a “Normal” Human Genome Look Like? Maynard V. Olson Professor Emeritus of Medicine and Genome Sciences, University of Washington, Seattle, WA, USA During the past 10 years, we have had our ﬁrst glimpse of why individuals differ so much from one another. This is not the right place to revisit the nature-nurture wars or to reﬂect on the extent to which our impression of high phenotypic variability in humans is a narcissistic illusion. For the moment, let us just acknowledge the obvious: In any small group of people, we see a lot of variation in traits we care about— and not just those that affect health. Geneticists shy away from studying these traits—and for good reason. They prefer not to revisit a subject whose premature investigation accounted for the worst moments of their young science. Nonetheless, questions about the genetic basis of variability among generally healthy humans will not go away. Public fascination alone will keep it alive, as will the realization that genetics is unlikely to revolutionize medicine until we develop a better understanding of normal phenotypic variation. So, what have our ﬁrst glimpses of variation in the genomes of generally healthy people taught us? First, balancing selection, the evolutionary process that favors genetic diversiﬁcation rather than the ﬁxation of a single “best” variant, appears to play a minor role outside the immune system. Local adaptation, which accounts for variation in traits such as pigmentation, dietary specialization, and susceptibility to particular pathogens is also a second-tier player. What is on the top tier? Increasingly, the answer appears to be mutations that are “deleterious” by biochemical or standard evolutionary criteria. These mutations, as has long been appreciated, overwhelmingly make up the most abundant form of nonneutral variation in all genomes. A model for human genetic individuality is emerging in which there actually is a “wild-type” human genome—one in which most genes exist in an evolutionarily optimized form. There just are no “wild-type” humans: We each fall short of this Platonic ideal in our own distinctive ways.

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A Living Constitution

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–Barbara R. Jasny and Laura M. Zahn

ESSAY Shinya Yamanaka Director, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan Induced pluripotent stem (iPS) cells have great potential for human therapies. Although the ﬁrst such cells were generated by introducing four transcription factors—Oct4, Sox2, Klf4, and c-Myc—into mouse somatic cells, we and our research benefited from the atmosphere generated in the community by the human genome project. We started in 2000 with the goal of generating embryonic-like stem cells from somatic cells. During the ﬁrst phase of the research, we decided to identify genes that are expressed specifically in embryonic stem cells. The most effective weapon for us to ﬁnd such genes—veritable needles in the haystack of transcribed genes—was an expressed sequence tag database, a mouse cDNA library developed and made public by RIKEN. Construction of this database was inspired by the U.S.-led international project to decipher the human genome. Mice are usually used as a model organism for humans. As Japan lagged behind in genomic science in the mid-1990s, RIKEN launched the project of sequencing all RNAs that are expressed as genes and built the mouse cDNA library. The human genome sequence publications contributed to the development of numerous genetic databases and fundamental technologies, such as microarray analysis. The human genome project has brought tremendous changes to research methods today and has contributed to building the foundation of medical and life sciences.

Human Genome Sequencing: Celebrating 10 years Lihadh Al-Gazali Professor, Clinical Genetics and Paediatrics, Department of Pediatrics, United Arab Emirates University, Al-Ain, United Arab Emirates The sequencing of the human genome has had a signiﬁcant impact on pediatric practice in the Arab region (the 23 states of the Arab league). It has facilitated research into the identiﬁcation of genes of single-gene disorders, particularly the autosomal recessive type, which are a major cause of infant mortality, morbidity, and handicap in Arab countries. Of the 955 genetic diseases described in Arabs, 60% (586) are caused by recessive genes.

The information obtained from the human genome, coupled with the high level of consanguinity (25 to 60%) and large family sizes in this region, has provided the perfect opportunity to identify the genes of recessive disorders via homozygozity mapping and DNA sequencing. The CTGA database, curated by the Center for Arab Genomic Studies, indicates that more than 60 recessive genes were identiﬁed, mostly during the past 10 years, in families from this region. Undoubtedly, these studies contributed to genome annotation and to unraveling the extent of genome variability. In addition, molecular characterization of well-described phenotypes in large families from this region will also, it is hoped, reveal genes that underlie complex and heterogeneous conditions. Despite recent progress, many recessive disorders in the Arab region are still not studied. At present, 36% of the genetic disorders reported in this region are only known as clinical observations. We expect that the molecular causes of the vast majority will be resolved within the next few years. This will result in a high-value knowledge base and will have direct implications on genetic counseling, prenatal diagnosis, and patient management. However, translating human genome research into clinical practice requires the development of comprehensive and effective programs by health authorities. Although progress has been made in this respect in some Arab countries—such as the mandatory premarital screening for thalassemia and sickle cell anemia program, prenatal screening, and the diagnosis of congenital anomalies—the limited options available for carrier couples remain a challenge in making these programs effective in the prevention of genetic diseases.

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Of Mice and Humans

Gene Patents: The Shadow of Uncertainty

Robert Cook-Deegan Director, Institute for Genome Sciences and Policy, Duke University, Durham, NC, USA. Twenty-two years ago, the Congressional Ofﬁce of Technology Assessment released a report about policy options for federal support in scaling up genome research. It contained the misleading statement, “genome projects raise no new questions of patent or copyright law.” I wrote that sentence and have spent the past decade studying how wrong it was. The general parameters of intellectual property law do indeed cover all technologies, not just genomics. Patent law applies to new technologies in distinctive ways, however, and using and interpreting it differs markedly among ﬁelds. Genomics has served up more than its share of controversy. The year 2010 was tumultuous in the world of gene patents. In February, the Secretary’s Advisory Committee on Genetics, Health, and Society approved a report that recommended exemptions from patent infringement liability for research and for diagnostic use. On 29 March, Judge Robert Sweet of the federal district court for southern New York turned gene patents on their heads by ruling that contested claims over BRCA1 and BRCA2 genes were invalid. Most of the ruling argued that DNA molecules cannot be patented because they are embodiments of information

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John S. Mattick Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia For me, the most important outcome of the human genome project has been to expose the fallacy that most genetic information is expressed as proteins. The biggest shock was that the number of conventional genes, despite some lineage-speciﬁc expansions and occasional innovations, does not scale with developmental and cognitive complexity: Humans and other vertebrates have only about 20,000 protein-coding genes, a similar number with functions largely similar to those in nematode worms that have just 1000 somatic cells. There have been several aftershocks: In contrast to protein-coding genes, the extent of noncoding intronic and intergenic sequences increases markedly with complexity; only 1.5% of the human genome encodes proteins. Subsequent tremors include the ﬁndings that the vast majority of the mammalian genome is dynamically transcribed in a developmental stage- and cell type– speciﬁc manner to produce huge numbers of noncoding RNAs and that the extent of the genome imputed to be functional by this measure and oth874

Famine in the Presence of the Genomic Data Feast Eileen Hoal Associate Professor, Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa Current graduate students cannot conceive of the world before the human genome was available for their daily practical tasks. In Africa, the shortage of resources (in terms of scientists, equipment, and operating funds) for data acquisition has made the publicly available data particularly valuable. However, in a sense, the sheer volume of accessible data is a drawback. We have been made acutely aware of our need for computational tools and expertise, exacerbated by the brain drain through emigration of those who acquire these scarce skills. For complex analyses such as admixture mapping or copy-number variation, we often now have the data spread out in a tantalizing buffet, but are left feeling as though we are looking into the dining room window, our noses are pressed to the glass. The solution, to avoid simply exporting data or samples, is North-South collaboration and the formation of consortia to maximize the promise of the information still locked in the rich variety of African genomes. In a wholly South African effort, publicly available data allowed us to dissect the structure of a uniquely African population. Further application of this knowledge required us to foster collaborations with more experienced laboratories and researchers, which are already showing results. Human genome analysis has the potential to act as an accelerating enabler, helping us to understand how an individual’s genome may affect susceptibility to infectious diseases such as tuberculosis. Approaches such as genome-wide association studies have been disappointing in this disease, and a search for rare variants—or simply understanding variation in the older, more diverse African populations—may hold some answers. Ultimately, local initiatives need to grow local expertise and to retain the necessary talent.

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The Genomic Foundation Is Shifting

ers, such as chromatin modiﬁcations, is far greater than that suggested by analyses of sequence conservation based on circular assumptions about the nonfunctionality of transposon-derived sequences. These observations suggest that we need to reassess the underlying genetic orthodoxy, which is deeply ingrained and has been given superﬁcial reprieve by uncritically accepted assumptions about the nature and power of combinatorial control. As Nobel laureate Barbara McClintock wrote in 1950: “Are we letting a philosophy of the [protein-coding] gene control [our] reasoning? What, then, is the philosophy of the gene? Is it a valid philosophy?” … There is an alternative: Human complexity has been built on a massive expansion of genomic regulatory sequences, most of which are transacted by RNAs that use generic protein infrastructure and control the epigenetic mechanisms underpinning embryogenesis and brain function. I see the human genome not simply as providing detail, but more importantly, as the beginning of a conceptual enlightenment in biology.

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and products of nature, and another section argued that the methods claims did not pass muster. That ruling is being appealed, and the ﬁnal resolution may not be for several more years. In December, the Court of Appeals for the Federal Circuit reafﬁrmed the patentability of a diagnostic method. The stakes are high, because present uncertainty over the existence and extent of patent rights casts a shadow over those making decisions about clinical and other applications of gene patents. Promising new methods for full-genome analysis might or might not face patent infringement liability. Some assert that patent rights have hindered research, although evidence of such harm is not compelling, but the evidence that patent rights have fostered diagnostic innovation is even weaker. Many laboratories have gotten genetic tests to market without patent rights. Those with exclusive rights have rarely, if ever, been ﬁrst to market. Companies that became sole U.S. providers did so by clearing the market of competitors. This is not unique to gene patents, but it creates intense controversy. Patent rights could, however, prove crucial to product development if payers demand evidence of clinical utility or if the U.S. Food and Drug Administration begins to regulate genetic tests, because developing such tests would suddenly become more costly and time-consuming, and a patent incentive could help induce private investment. In 2011, we may ﬁnally begin to develop case law on how patent law will apply to DNA technologies beyond protein therapeutics. The debate over establishing a human genome project included disagreement over patent policy; that disagreement continues, but the effects of uncertainty are moving from equivocal and probably weak effects on research to actual impact on private investment decisions that will affect what genomic technologies get developed in the real world. Reducing the uncertainty surrounding diagnostic uses of gene patents is important.

Synthetic Clonal Reproduction Through Seeds Mohan P. A. Marimuthu,1* Sylvie Jolivet,2* Maruthachalam Ravi,3* Lucie Pereira,2 Jayeshkumar N. Davda,1 Laurence Cromer,2 Lili Wang,3 Fabien Nogué,2 Simon W. L. Chan,3† Imran Siddiqi,1† Raphaël Mercier2† ngineering cloning through seeds (apomixis) in food crops would revolutionize agriculture by fixing hybrid vigor and allowing the perpetuation of any elite heterozygous genotype (1). The first component of apomixis, the formation of diploid clonal gametes, can be introduced in a sexual plant, with Arabidopsis mutants (MiMe and dyad ) (2, 3). However these clonal gametes participate in normal fertilization, giving rise to progeny with more chromosomes than their parent. Clonal gametes would be turned into clonal seeds if fertilized by a strain whose chromosomes are eliminated from the resultant progeny (Fig. 1A). Genome elimination can be induced by manipulating the centromere-specific histone CENH3 (4). By co-expressing green fluorescent protein (GFP)–CENH3 and GFP-tailswap variants in the cenh3-1 mutant, we made a line with increased fertility that causes genome elimination when crossed to a parent with diploid gametes (5) [supporting online material (SOM) text]. We named this line GEM for Genome Elimination induced by a Mix of CENH3 variants. When GEM was crossed to a tetraploid wild type (which has diploid gametes) or to osd1, a diploid mutant that produces a very high proportion of diploid male and female gametes (2),

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it induced conversion of diploid gametes into uniparental diploid progeny in addition to giving triploid and aneuploid progeny (table S1). However, uniparental diploid plants from osd1 crosses are not clones, because the diploid gametes produced by osd1 are recombined (2, 5) (SOM text and fig. S2). We then crossed dyad and MiMe plants as female to the GEM line and screened for clones in the F1 generation, which comprised diploids, triploids, and aneuploids (table S2). Because the genetic background of the parents differed, we could trace the origin of the chromosomes. MiMe × GEM gave an average of 14 viable (i.e., able to germinate) seeds per fruit; 34% (53/155) were diploid (table S2). Among these diploid plants, 98% (52/53) had only maternal chromosomes, lacking any paternal contribution for the eight diagnostic loci tested (Fig. 1B). Furthermore, these maternal diploids (named diploid eliminants) retained the heterozygosity of the mother plant at all tested loci (Fig. 1B). Likewise, dyad × GEM resulted in 0.9 viable seeds per fruit, 13% (29/220) of which were diploid eliminants retaining full maternal heterozygosity (table S2 and Fig. 1C). Although not fully penetrant, this demonstrates clonal propagation through seed in a manner akin to the outcome of apomixis.

Because MiMe also produces male diploid clonal gametes (2), we tested for clonal male inheritance by crossing GEM as the female to a MiMe male. Although seed viability was lower in this cross, likely because the Col-0 strain is sensitive to paternal genome excess (6), 42% of progeny were diploids that lacked maternal contribution and had the heterozygosity of the male parent (fig. S3A and table S2). Thus, these plants are clones of their MiMe father, mimicking male apomixis (7). We crossed a maternal MiMe clone to GEM for a second generation and obtained 14 seeds per fruit. In this population, 24% (19/79) of progeny were diploid and genetically identical to their mother and grandmother (fig. S3B and table S2), demonstrating clonal propagation through seed for more than one generation. Our experiments show that clonal reproduction can be engineered in a sexual plant by manipulating two to four conserved genes controlling meiosis and chromosome segregation. The system we have described still relies on crossing, whereas to achieve the full benefits of apomixis the plant needs to be self-propagating. However, this proof of principle suggests a new strategy for development of apomixis in food crops. References and Notes 1. C. Spillane, M. D. Curtis, U. Grossniklaus, Nat. Biotechnol. 22, 687 (2004). 2. I. d’Erfurth et al., PLoS Biol. 7, e1000124 (2009). 3. M. Ravi, M. P. Marimuthu, I. Siddiqi, Nature 451, 1121 (2008). 4. M. Ravi, S. W. Chan, Nature 464, 615 (2010). 5. Materials and methods are available as supporting material on Science Online. 6. B. P. Dilkes et al., PLoS Biol. 6, e308 (2008). 7. C. Pichot, M. El Maâtaoui, S. Raddi, P. Raddi, Nature 412, 39 (2001). 8. We thank D. Singh for fluorescent in situ hybridization analysis; E. Gugan and N. Shukla for technical assistance; and L. Comai, O. Loudet, J. P. Vielle-Calzada, W. Lucas, and V. Sundaresan for comments on the manuscript. This research was funded by Agence Nationale de la Recherche (ANR-09-EBIO-011 to R.M.), INRA GAP Department (R.M.), NSF (IOS-1026094 to S.W.L.C.), the Basil O’Connor Starter Scholar Program from the March of Dimes (S.W.L.C.), a targeted allocation grant from CSIR (I.S.), and a Centre of Excellence grant from Department of Biotechnology (I.S.). Two provisional patent applications based on the work have been filed jointly by INRA and UC Davis (USA 61/418,792) and jointly by CSIR and UC Davis (India 619/DEL/2010).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/876/DC1 Materials and Methods SOM Text Figs. S1 to S5 Tables S1 to S3 References 28 October 2010; accepted 14 January 2011 10.1126/science.1199682

Fig. 1. (A) In natural apomixis, clonal seeds develop without fertilization. To induce clonal seed from sexual plants, we fertilized clonal gametes with a parent whose chromosomes are modified to be eliminated after fertilization. (B and C) Parents and diploid progeny were genotyped for polymorphic loci (table S3). Each row represents one plant, and each column is a locus.

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1 Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research (CSIR), Uppal Road, Hyderabad 500007, India. 2Institut Jean-Pierre Bourgin, UMR1318, Institut National de la Recherche Agronomique (INRA), Route de Saint Cyr, 78026 Versailles, France. 3Department of Plant Biology, University of California (UC) Davis, 1 Shields Avenue, Davis, CA 95616, USA.

*These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected] (R.M.); srchan@ucdavis. edu (S.W.L.C.); [email protected] (I.S.)

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Michel Bouchon,1* Hayrullah Karabulut,2 Mustafa Aktar,2 Serdar Özalaybey,3 Jean Schmittbuhl,4 Marie-Paule Bouin5 Laboratory and theoretical studies suggest that earthquakes are preceded by a phase of developing slip instability in which the fault slips slowly before accelerating to dynamic rupture. We report here that one of the best-recorded large earthquakes to date, the 1999 moment magnitude (Mw) 7.6 Izmit (Turkey) earthquake, was preceded by a seismic signal of long duration that originated from the hypocenter. The signal consisted of a succession of repetitive seismic bursts, accelerating with time, and increased low-frequency seismic noise. These observations show that the earthquake was preceded for 44 minutes by a phase of slow slip occurring at the base of the brittle crust. This slip accelerated slowly initially, and then rapidly accelerated in the 2 minutes preceding the earthquake. he existence or observation of a detectable nucleation phase before earthquakes is a long-standing goal with implications for earthquake prediction and risk assessment. Although it is well established that some earthquakes are preceded by foreshocks, nothing so far distinguishes these foreshocks from regular earthquake occurrences, so there is no objective way to identify these events as foreshocks until they are followed by a larger earthquake (1). On the other hand, the presence of a short-duration (a few seconds or less) nucleation phase on some earthquake records, which would indicate the growth of slip instability on the fault before the earthquake, has been strongly debated (2). Laboratory (3–6) and theoretical (7–14) models of earthquake nucleation predict that slip instability should occur before earthquakes, but whether that instability is large enough to be detected in real Earth conditions and of long enough duration to be helpful is unknown (15). In this context, we examined the seismic signal preceding the 1999 Izmit earthquake, the largest well-recorded strikeslip earthquake to date, to characterize any existing nucleation phase. The earthquake. The Izmit earthquake occurred at 0 hours, 1 min, 38 s (00h01mn38s; universal time) on 17 August 1999 and, in ~25 s, broke the North Anatolian fault, a major plate boundary, over a length of 150 km across northwestern Turkey (16–19). During the earthquake, the two plates bounding the fault moved horizontally

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past each other by ~3 m. This sudden motion of the plates occurred over the brittle part of the crust, which in this region extends from Earth’s surface to a depth of ~17 km (20). Below, rocks are more ductile because of the increase in temperature, and the two plates move continuously past each other by ~2.5 cm per year (21). What happens in the transition zone between the brittle and the ductile parts of the crust—possibly including the occurrence of nonvolcanic tremor— may influence the nucleation of the large earthquakes but is largely unknown. Estimates of the depth of the hypocenter vary slightly (16, 20), but are all comprised in the range 15 T 2 km, which places the zone of nucleation near the base of the brittle crust. The earthquake was preceded by foreshocks (20, 22), the largest one being reported by two local seismic stations (20, 22). The station closest to the epicenter (located 14 km away) reported the occurrence of seven other foreshocks (20). This station (UCG; fig. S1) was equipped with a shortperiod (1 Hz, L4C) seismometer with a vertical component, coupled with a digital recorder (23). This instrument recorded the vertical velocity of the ground at a sampling rate of 100 Hz. Its x 10

Fig. 1. Record of the vertical ground motion 20 min before the earthquake. This recording is part of the second triggered window. The numbers indicate the foreshocks and refer to their chronological order from the start of the sequence.

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1 1 Centre National de la Recherche Scientifique and Université Joseph Fourier, Grenoble, ISTerre, BP 53, 38041 Grenoble, France. 2Kandilli Observatory and Earthquake Research Institute, Bogaziçi University, 81220 Cengelköy, Istanbul, Turkey. 3TÜBITAK, Marmara Research Center, Earth and Marine Sciences Institute, 41470 Gebze, Kocaeli, Turkey. 4Centre National de la Recherche Scientifique and Université de Strasbourg, Ecole et Observatoire des Sciences de la Terre, 5 rue Descartes, 67084 Strasbourg, France. 5 Centre National de la Recherche Scientifique and Institut de Physique du Globe de Paris, Observatoire Volcanologique et Sismologique de Guadeloupe, 97113 Gourbeyre, Guadeloupe, France.

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Extended Nucleation of the 1999 Mw 7.6 Izmit Earthquake

records shed some light on the mechanical processes taking place in the hypocentral area of a large earthquake before the earthquake occurs. Nucleation signal. Station UCG was located on limestone, a hard rock which does not substantially attenuate or affect seismic waves and thus provides good recording conditions. It was operated in triggered mode like many seismic stations, meaning that it recorded only when a preset threshold of ground-motion amplitude was exceeded (24). In all, the ground motion was recorded in six triggered windows during the 45 min preceding the earthquake, providing almost 10 min (563 s) of records. In one of these recordings, 20 min before the earthquake, five shocks are visible (Fig. 1). Each one begins with a P-wave sequence followed 2.4 s later by higher-amplitude S waves. Some of the shocks occur less than 5 s apart. A total of 18 events, ranging in magnitude from 0.3 to 2.7 (table S1), can be visually identified in the six triggered windows (Fig. 2). Two characteristics of these shocks stand out: Their S-minus-P travel times are the same, within the digitization precision (0.01 s), and their waveforms are surprisingly similar, in spite of the wide range of magnitude they cover. Several other stations, located in various azimuths from the epicenter, also recorded the largest foreshock. At the closest station other than UCG (fig. S1), the P-wave arrival is clear so that the time difference between the largest foreshock and the main shock can be measured accurately. This time difference (105.22 s) is exactly the same, within the digitization precision (0.01 s), as the one measured at UCG, which shows that the travel times of the P-wave from the foreshock and from the main shock hypocenter to the stations are the same and implies that the source of the foreshocks is the hypocenter. Furthermore, at all of the stations where the largest foreshock was recorded, P-wave polarity is the same as the main shock, indicating that the slip mechanism of the foreshocks is the same as the main shock. Because all of the events share nearly the same waveform, the waveform of one of them

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occurrence of at least six shocks in the 40-s-long recording leading to the earthquake (Fig. 3A). Less than 1 s before the earthquake, the process accelerated again, and the instability increased dramatically: One shock occurred 0.14 s before the earthquake, followed by another shock 0.07 s later, finally followed by the earthquake itself (Fig. 3B). In spite of the short time span between the first two

shocks, the P-wave pulse of the first one can be clearly observed. Its shape and width are the same as the ones of the other events (Fig. 3B). A comparison of their amplitude shows that its magnitude is ~2.0. The shock that immediately follows has a broader P pulse and a higher amplitude than any of the previous shocks. The earthquake occurs 0.07 s later. More events or a

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may be used as a template to try to detect the presence of smaller shocks for which the signal is within the noise level. This is performed by cross-correlating the template with the records (24). As a result, many more events than the ones visually recognized can be detected (Fig. 3A), bringing the total number of identified events to about 40. Interevent separation. To confirm that the events originate from the same or nearly the same place, we compared the first two events. Visually, their waveforms (Fig. 4A) are nearly the same. Cross-correlating them (Fig. 4B) yields a difference between the S-minus-P travel times of the two events of ~0.0006 s. This value, which is at the limit of the resolution of the measurement, implies a separation distance of ~5 m. Although this separation between the two events is the one seen from the station and is thus the projection of the real separation on the travel path of the waves, its value is surprisingly small compared with the size of the sources (25). We extended this analysis by cross-correlating the P and S waves of all possible pairs of events (136, as the P-wave of foreshock 8, which is outside the triggered window, was not recorded) and calculating the difference in S-minus-P time for each pair. As the position and length of the windows selected for the correlation may affect the results (because of the presence of noise and the asymmetry of the pulse shapes), we considered 200 different windows for each event pair. The results show that any one of the events differs in S-minus-P travel time by less than 0.0024 s from the majority of the other events, regardless of the correlation windows that we considered. This implies that any one shock is located within 20 m or less from the majority of the other events. Although this separation is the one seen from the station, its small value, compared with the likely source size of the largest shocks (25), shows that all of the events originate from an area of the fault that is no larger than the size of the largest events. Slip acceleration. Laboratory and theoretical studies suggest that earthquakes are preceded by a phase of developing slip instability in which the fault begins to slip slowly before accelerating to dynamic rupture (3–14). A correlation between the onset of slip acceleration and the emission of tremorlike signals has also been observed in friction experiments (26). If we consider only the largest events of the sequence, for which the detection should be complete because they are above the triggering threshold, we note that the fourth-largest event occurs 43 min before the earthquake, the third-largest one takes place 20 min before, the second-largest one 12 min before, and the largest one 1 min 45 s before the earthquake (Fig. 2 and table S1). The magnitudes of the fourth, third, and second events are relatively close, which suggests a slowly accelerating process in the first 42 min of the sequence, followed by a rapid acceleration 2 min before the earthquake. This rapid acceleration is also suggested by the

RESEARCH ARTICLES events with the same waveform (Figs. 2 and 3). Events occurred at 40-s (in the first triggered window), ~10-s (on average), ~5-s, and then at tenths-of-a-second intervals. The comparison of the spectra of the shocks (fig. S2) confirms the similarity observed in the

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Fig. 5. (A to C) Comparison of the S-wave ground-velocity spectra of some events. Numbers refer to the chronological order of the foreshocks in Fig. 2. All of the spectra are corrected for the instrument response and normalized independently. Spectral amplitudes below 5 Hz are affected by the low-frequency background seismic noise, which scales differently from event to event. The peak recorded amplitude of each event is given in parenthesis and is expressed in micrometers per second. The two largest shocks are shown in (C). www.sciencemag.org

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waveforms. Most of the shocks have nearly the same spectral shape and corner frequencies (the frequency beyond which the spectral amplitude decays). This similitude is especially clear for some of the events (Fig. 5). In particular, the two strongest shocks have nearly identical spectral shapes (Fig. 5C). Considering these two shocks, two possible reasons may cause this similitude: (i) Their spectral corner frequencies are both higher than the maximum frequency displayed (35 Hz), which means that the two shocks appear as point sources over the frequency range considered and that their source size is not resolvable with the data. (ii) Alternatively, the two events may have nearly the same corner frequencies, because spectral amplitude drops off rapidly beyond an event corner frequency. The first hypothesis would imply an extremely high stress drop on the order of 800 MPa for the largest shock [based on the approach in (27)]. Such a high value lies well outside the range of values observed for tectonic earthquakes (28, 29). Thus, it appears likely that the two events have similar corner frequencies. As corner frequency is inversely proportional to source dimension, it implies that their source size is nearly the same. This is surprising because the shocks have very different amplitudes (table S1). An estimate of the source extent of these two shocks can be obtained by modeling their source as a patch of shear slip located at the hypocenter and matching the calculated spectral shape with the one observed. This yields a patch size of ~300 m (fig. S3). As the calculation does not account for the anelastic attenuation of the waves, which is not precisely known and might shift the spectral peak to lower frequency, this measurement should be considered as an upper limit of the source size. Regardless of its precise value, however, the similitude of the spectral shapes of the two events well beyond their spectral peak seems to require that their source size is nearly the same. The small separation distance of the two events, seen from the station and estimated from their waveform crosscorrelation (~9 m), further supports the notion that the same patch is the source of the two events. The amplitude of the two shocks differs by a factor of 10, which implies that their stress drops (the release of stress on the source patch) also differ by a factor of 10. If the stress drop had been similar for the two shocks, their amplitude ratio would have implied a little more than a factor of 2 difference in their corner frequencies (seismic moment, which is the physical measure of an earthquake strength, scales as the cube of the source dimension). This is clearly not what we have observed (Fig. 5C). The source dimension inferred (fig. S3) implies that the slip on the patch is, on average, a little less than 1 cm (0.8 cm) for the largest shock and a little less than 1 mm (0.8 mm) for the other one. These values lead to a stress drop of ~2.6 MPa for the largest shock, a value typical for earthquakes, and ~0.3 MPa for the second event, a lower value than commonly observed. The similarity of the spectra of the other shocks (figs. S2 and S4) is intriguing but is more

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cascade of events may have occurred in the short time span leading to the earthquake, but the data resolution cannot distinguish anything else other than these two immediate foreshocks. Repeating events? The seismic signal before the earthquake shows a repetitive pattern of

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difficult to interpret. Either the relatively small magnitude of these events results in corner frequencies too high to be resolvable by the data, or all of these shocks have nearly the same source size, just slightly smaller than that of the two largest shocks. To make sure that this spectral similitude is not due to a local site effect at the station (such as a possible seismic resonance of the geological structure) or to anelastic attenuation along the travel path of the waves, we also present the spectra of some aftershocks, occurring a few days later and located at a comparable distance from the station (fig. S5). The wide range of spectral shapes that we observed suggests that the corner frequency of the shocks is not the result of a site or an attenuation effect, but is more likely a source effect. If this is the case, it would indicate that the pre-Izmit events have nearly the same source size. A consequence of this would be that the small events have extremely low stress drops, possibly comparable to the stress drops of the lowfrequency earthquakes (30, 31) that make up nonvolcanic tremor (32, 33). Regardless of this possibility, the near duplicate spectrum of some of the events (Fig. 5, A and B) suggests that at least some repeating events occur, as is the case with the two strongest shocks. Fault creep. As earthquakes are known to release stress on the part of the fault on which they occur, the presence of repeating events at fewminute (events 13 and 16, Fig. 5C) or possibly at few-second (events 5 and 6, Fig. 5B) intervals is intriguing. The repeating rupture of the same fault patch seems to require a reloading of stress on the patch between events. A simple mechanism that can provide this reloading is creep on the area surrounding the patch. Repeating events have been observed elsewhere, particularly on the San Andreas fault in California (34–38), and creep in the area surrounding the source is thought to be the cause. Compared with the present events, they usually have recurrent times of months or years and similar magnitudes. A possible reason for the magnitude difference observed here is that the interevent time of the pre-Izmit shocks is extremely short, possibly forcing the response of the patch. In other words, loading may be too fast to produce a steady response. Another possibility is that the

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patch responds not only to the loading but also to the loading rate, which may be highly irregular. Another intriguing feature of the records is the change in the background low-frequency seismic noise that takes place after the first foreshock (Fig. 6). Coincidentally with the first foreshock, the low-frequency ground motion increases. Then, whereas the amplitude of the first foreshock decays rapidly after the S-wave arrival (bottom trace in Fig. 6), the low-frequency motion stays at a higher level than before the foreshock (top trace in Fig. 6). From then on, this low-frequency signal, which is also visible on the unfiltered records, was continuously present on all the recordings until the onset of the earthquake (fig. S6). Its spectrum shows that its frequency is below 2 Hz (fig. S7). Although it is not possible with one station to determine its origin, this lowfrequency noise, whose beginning coincides with the occurrence of the first foreshock, may be the seismic signature of the fault creep that was then probably occurring around the hypocentral area. Outlook. These observations show that this particular earthquake was preceded by a phase of slow slip occurring at the base of the brittle crust. The relatively long duration of the nucleation and the observation that it emits a characteristic signal are encouraging for possible early warning systems, but it remains to be seen whether this behavior is applicable to other large earthquakes. Some other well-recorded earthquakes, such as the 1999 Chi-Chi (Taiwan) or the 2004 Parkfield (California) earthquakes, do not show evidence for similar foreshocks or nucleation events. The next steps include reexamining the near-fault seismic records of other large, well-recorded earthquakes for similar signals. Continued seismic monitoring networks will also be necessary to understand if such extended nucleation events apply beyond this example.

Supporting Online Material

References and Notes 1. C. H. Scholz, The Mechanics of Earthquakes and Faulting (Cambridge Univ. Press, Cambridge, 1990). 2. W. L. Ellsworth, G. C. Beroza, Science 268, 851 (1995). 3. J. H. Dieterich, J. Geophys. Res. 83, 3940 (1978). 4. J. H. Dieterich, Tectonophysics 211, 115 (1992). 5. M. Ohnaka, J. Phys. Earth 41, 45 (1993). 6. M. Ohnaka, L. F. Shen, J. Geophys. Res. 104, 817 (1999).

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7. S. Das, C. H. Scholz, J. Geophys. Res. 86, 6039 (1981). 8. J. R. Rice, J. Geophys. Res. 98, 9885 (1993). 9. B. Shibazaki, M. Matsu’ura, Geophys. J. Int. 132, 14 (1998). 10. M. Campillo, P. Favreau, I. R. Ionescu, C. Voisin, J. Geophys. Res. 106, 16307 (2001). 11. M. Cocco, A. Bizzarri, Geophys. Res. Lett. 29, 1516 (2002). 12. P. Favreau, M. Campillo, I. R. Ionescu, J. Geophys. Res. 107, 2147 (2002). 13. N. Lapusta, J. R. Rice, J. Geophys. Res. 108, 2205 (2003). 14. J. P. Ampuero, A. M. Rubin, J. Geophys. Res. 113, B01302 (2008). 15. R. E. Abercrombie, D. C. Agnew, F. K. Wyatt, Bull. Seismol. Soc. Am. 85, 1873 (1995). 16. M. N. Toksöz, R. E. Reilinger, C. G. Doll, A. A. Barka, N. Yalcin, Seismol. Res. Lett. 70, 669 (1999). 17. A. Barka et al., Bull. Seismol. Soc. Am. 92, 43 (2002). 18. M. Bouchon et al., Bull. Seismol. Soc. Am. 92, 256 (2002). 19. Z. Çakir et al., Geophys. J. Int. 155, 93 (2003). 20. S. Özalaybey et al., Bull. Seismol. Soc. Am. 92, 376 (2002). 21. R. Reilinger et al., J. Geophys. Res. 111, B05411 (2006). 22. O. Polat et al., Bull. Seismol. Soc. Am. 92, 361 (2002). 23. M. Aktar, F. Biçmen, Cah. Cent. Eur. Géodyn. Sismol. 1, 11 (1989). 24. Materials and methods are available as supporting material on Science Online. 25. Assuming a stress drop of 3 MPa for the events, which is close to the average of the values commonly observed, and a circular source would lead to source dimension (diameter) of ~66 and 108 m for the first two foreshocks, respectively, and 290 m for the largest one. 26. D. Zigone, C. Voisin, E. Larose, F. Renard, M. Campillo, Geophys. Res. Lett. 38, L01315 (2011). 27. R. Madariaga, Bull. Seismol. Soc. Am. 66, 639 (1976). 28. R. E. Abercrombie, J. Geophys. Res. 100, 24015 (1995). 29. K. Imanishi, W. L. Ellsworth, S. G. Prejean, Geophys. Res. Lett. 31, L12S09 (2004). 30. S. Ide, G. C. Beroza, D. R. Shelly, T. Uchide, Nature 447, 76 (2007). 31. G. C. Beroza, S. Ide, Science 324, 1025 (2009). 32. D. R. Shelly, G. C. Beroza, S. Ide, S. Nakamula, Nature 442, 188 (2006). 33. D. R. Shelly, G. C. Beroza, S. Ide, Nature 446, 305 (2007). 34. J. E. Vidale, W. L. Ellsworth, A. Cole, C. Marone, Nature 368, 624 (1994). 35. C. Marone, J. E. Vidale, W. L. Ellsworth, Geophys. Res. Lett. 22, 3095 (1995). 36. D. P. Schaff, G. Beroza, B. E. Shaw, Geophys. Res. Lett. 25, 4549 (1998). 37. R. M. Nadeau, T. V. McEvilly, Science 285, 718 (1999). 38. O. Lengliné, D. Marsan, J. Geophys. Res. 114, B10303 (2009). 39. We thank the TÜBITAK Marmara Research Center Earth and Marine Sciences Institute, specifically M. Ergin, C. Tapirdamaz, F. Biçmen, and A. Yörük, who participated in the operation of the seismic station and in the processing of the data. This study benefited from discussions with M. Campillo, O. Konca, D. Marsan, L. Géli, O. Lenginé, N. Toksöz, F. Renard, C. Voisin, P. Bernard, G. Poupinet, A. Paul, D. Zigone, V. Durand, A. M. Boullier, P. Henri, P. Y. Bard, O. Coutant, and H. Perfettini.

www.sciencemag.org/cgi/content/full/331/6019/877/DC1 Materials and Methods Figs. S1 to S7 Table S1 References 3 September 2010; accepted 13 January 2011 10.1126/science.1197341

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Sandro F. Ataide,1 Nikolaus Schmitz,1* Kuang Shen,2* Ailong Ke,3 Shu-ou Shan,2 Jennifer A. Doudna,4† Nenad Ban1† Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)–dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon. he signal recognition particle (SRP) is a ubiquitous ribonucleoprotein complex that cotranslationally delivers membrane and secretory proteins to the plasma membrane in prokaryotes and to the endoplasmic reticulum in eukaryotes (1, 2). The SRP targeting process starts with recognition of a hydrophobic signal sequence on the ribosome-nascent chain complex (RNC or cargo) by the SRP to yield an RNC:SRP complex. Subsequently, the RNC:SRP complex associates on the membrane with the SRP receptor (SR). The binding between SRP and SR induces conformational changes of yet-unknown nature that allow the cargo to be transferred to the protein-conducting channel (translocon). The cycle can be resumed after guanine triphosphate (GTP) hydrolysis that drives dissociation of the SRP:SR complex (1–3). Both the SRP and SR include components that are structurally and functionally conserved across the different domains of life (1, 2). In Escherichia coli, SRP is composed of two universally conserved components, the Ffh protein (SRP54 in eukaryotes) and the 4.5S SRP RNA. Ffh contains a methionine-rich (M) domain responsible for highaffinity RNA binding and recognition of signal sequences (4), a helical N domain responsible for interactions with the ribosome, and a Ras-like guanine triphosphatase (GTPase) (G) domain. The E. coli SR is a single protein, FtsY, that consists of an N and a G domain that are structurally similar to those found in Ffh (5, 6) and an additional

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1 Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zurich (ETH Zurich), 8093 Zurich, Switzerland. 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA. 3 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA. 4Howard Hughes Medical Institute, Department of Molecular and Cell Biology, Department of Chemistry, University of California Berkeley; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

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

A domain responsible for interactions with the membrane and the translocon (7). When bound to GTP, SRP and SR form a stable complex through extensive interactions between their NG domains (5, 6). At the heterodimer interface, a composite active site is formed in which SRP and SR act as reciprocal activating proteins for one another. GTPase activation within the SRP:SR complex is achieved by a set of conformational changes in both proteins that are distinct from those required for their initial complex assembly (8). Mutations that block GTPase activation severely disrupt protein targeting and translocation (8), suggesting that conformational changes leading to GTPase activation play an essential role in the unloading of cargo (5, 9, 10). The RNA moiety in the SRP system is essential for cell viability in vivo (4, 11) and for protein targeting and translocation in vitro (12, 13). The E. coli 4.5S RNA has two characterized biochemical functions: acceleration of the interaction between Ffh and FtsY by increasing their complex assembly and disassembly rates and stimulation of GTPase activity once a stable SRP:SR complex is formed (13–16). Additionally, the SRP RNA has been described to act as a platform for conformational changes in Ffh and FtsY after recognition of the signal sequence by the M domain (15, 17, 18). Despite extensive prior studies of cotranslational protein targeting, several fundamental questions remain unanswered: How does the SRP RNA stimulate GTP hydrolysis of the SRP:SR complex, why is this GTPase activation essential for protein targeting, and how is cargo transfer to the translocon coupled to GTP hydrolysis by Ffh and FtsY? To address these questions, we solved the three-dimensional structure of the SRP:SR complex with the nonhydrolyzable GTP analog b-g methylene-guanosine 5′-triphosphate (GMPPCP). The SRP:SR complex structure. The prokaryotic SRP:SR complexes were crystallized in the

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pre-GTP hydrolysis state (Fig. 1, A and B) (19, 20). Stable complexes were assembled by using E. coli Ffh1-432, full-length 4.5S RNA from E. coli or Deinococcus radiodurans, and E. coli FtsY196-497 in the presence of GMPPCP and the nonionic detergent C12E8, which is proposed to mimic a signal peptide (21). Crystals of the homologous E. coli SRP:SR complex and the heterologous complex containing D. radiodurans 4.5S RNA were isomorphous, but the latter diffracted x-rays to higher resolution. After extensive screening, a single crystal of the heterologous complex was identified that diffracted x-rays better than others and permitted recording of a complete data set to 3.9 Å resolution (table S1). These data were used to produce an atomic-resolution model of the complex. The structure was solved by molecular replacement using high-resolution structures of isolated parts of the assembly, including the NG dimer (5) and the 4.5S RNA domain IV in complex with the M domain (4). Iterative rounds of rebuilding and refinement produced excellent electron density maps at 3.9 Å resolution, which allowed unambiguous tracing of the molecules and placement of side chains and bases for wellordered parts of the structure (Fig. 1C and fig. S1). The missing parts in previous structures, specifically the distal portion of the SRP RNA and the connective linker between the NG and M domains of Ffh, could be unambiguously identified in the calculated electron density maps (omit map for the linker helix shown in Fig. 1D). The two molecules in the crystallographic asymmetric unit form a head-to-tail dimer with the N terminus of the symmetry-related Ffh molecule interacting with a groove on the M domain of the opposing molecule defined by portions of the finger loop (excluding residues 354 to 368) (22) (fig. S2). Electron density maps for the homologous complex calculated at 7.0 Å resolution, using the coordinates of the heterologous complex for phasing, revealed that their global structures are nearly identical (table S1, Fig. 1A, and fig. S3). The structure shows an unexpected domain arrangement of the Ffh and FtsY proteins relative to the SRP RNA. Whereas the M domain binds near the tetraloop region (also known as helix 8 or domain IV) as observed previously (4), the NG heterodimer contacts the opposite end of the 4.5S RNA (also named helix 5) (23). The linker connecting the Ffh NG and M domains forms a well-defined, 30-amino-acids-long helix (Fig. 1, A and D). In previous studies, the linker region was either too flexible to be assigned (22) or was modeled as an a helix and a loop structure (24). There has been no clear evidence for a structural role of the linker helix in previous studies. However, mutations of conserved residues in the linker region abolish the ability of the SRP RNA to stimulate SRP:SR complex formation as well as GTP hydrolysis, indicating that the linker is intimately involved in the function of the SRP RNA (17). In the structure reported here, the linker acts as a spacer placing the activated NG domains of the Ffh and FtsY at the distal end of the 4.5S

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The Crystal Structure of the Signal Recognition Particle in Complex with Its Receptor

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RNA. This conformation of the SRP:SR complex had not been detected previously (15, 25), possibly because this configuration of the SRP:SR complex is a transient state that was stabilized by intermolecular interactions in the crystal. Nevertheless, the functional importance of this conformational state is strongly supported by the following analyses. New function of the distal end region of the 4.5S RNA. In addition to the structural data presented here, another clue to the importance of the distal RNA region is its sequence conservation (Fig. 2A and fig S4). The sequence motif GUGCCG (bases 83 to 88 in E. coli) can be found in the helix 5 region of most known 4.5S RNAs (Fig. 2A) and is also conserved in the longer prokaryotic 6S SRP RNA (23). The secondary structure prediction for this region of the SRP RNA always features a bulged or unpaired base (Fig. 2A). The conservation of this region is similar to that of the SRP RNA tetraloop and the region recognized by the M domain (Fig. 2, A and B, and fig. S4).

In our structures, C83 (C86 in E. coli, fig. S5) is positioned close to both GMPPCP molecules and interacts directly with both Ffh and FtsY proteins at regions that regulate the GTPase activity (Fig. 2C and fig. S5) (26–28). Two residues from Ffh can form H bonds with C83, Lys278 interacts with the phosphate group of C83 and Glu277 interacts with the N4 of C83 and the 2′O from GMPPCP. In contrast, the interaction of C83 with FtsY is hydrophobic: Residues Leu198 and Phe137 stack with the base; the latter resides in the insertion box domain (IBD) motif previously described as essential for GTP binding and hydrolysis (5, 10, 29). Identical interactions have been described in the Ffh:FtsY NG domains crystal structure [Protein Data Bank (PDB) code 2CNW] containing guanine diphosphate (GDP):AlF4, in which a peripheral nucleotide [guanine monophosphate (GMP)] was found to bind in the same position as C83 (28) (fig. S6). The superposition of our NG heterodimer structure with previously determined structures containing GMPPCP (PDB

Fig. 1. Structure of SRP in complex with SR. (A) Top view of the SRP:SR complex. Ffh is colored blue, 4.5S RNA in gray, and FtsY (SR) is shown in green. The atoms of the two GMPPCP molecules are displayed as red spheres. (B) Side view of the SRP:SR complex rotated 70° relative to the view in (A). N denotes the N domain, G denotes the G domain, M denotes the M domain, L is the flexible linker, and F denotes the finger loop. (C) Visualization of the 2Fobs – Fcalc (where Fobs and Fcalc are observed and calculated structure factors, respectively) electron density con-

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code 1OKK), b-g-imido-guanosine 5′-triphosphate (GMPPNP) (PDB code 1RJ9), and GDP:AlF4 (PDB code 2CNW) reveals no large differences in the GTPase center within the resolution limits of our structure (5, 6, 26, 27). The NG heterodimer exhibits an extensive interaction interface with the 4.5S RNA, burying 890 Å2 (Fig. 3A). FtsY is responsible for the majority of these contacts, whereas Ffh interaction with the distal region of the RNA is limited to two bases, C82 and C83. However, the M domain of Ffh is also responsible for the interaction with domain IVof the 4.5S RNA. Taken together, the total surface area buried between Ffh and the 4.5S RNA is 780 Å2, which is similar to the extent of interaction between FtsY and the distal portion of the 4.5S RNA (Fig. 3B). These observations explain why FtsY will bind the distal portion of the 4.5S RNA as part of the GTP-activated twin complex but not in its free state (Fig. 2C and 3B). First, the contact to the RNA is mediated by both FtsY and Ffh. Second, the local concentra-

toured at 1s and the stick representation of the SRP:SR complex (41). (D) Side view of the SRP:SR complex with the contour of a 2Fobs – Fcalc unbiased omit map calculated for linker region residues 300 to 330 of Ffh. The linker is displayed as a tube together with the difference density Fobs – Fcalc electron density contoured at 3s, shown as green mesh. The 2Fobs – Fcalc electron density for the entire complex is contoured at 1s and displayed as gray mesh. The cover radius used had a cutoff of 2.6 Å for the 2Fobs – Fcalc and Fobs – Fcalc. SCIENCE

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RESEARCH ARTICLES

RESEARCH ARTICLES fact that, at subsaturating protein concentrations, the observed GTPase reaction rate is limited by the assembly of the complex. Recent studies that more rigorously dissected the complex assembly versus GTP hydrolysis steps established that the tetraloop is essential for accelerating SRP:FtsY complex assembly, whereas it does not substantially affect GTPase activation once the GTPase complex is formed (12, 32, 33). To define the RNA site(s) responsible for stimulating GTP hy-

Fig. 2. Interaction of the Ffh-SR NG domain with a conserved flipped base at 5′,3′ distal end of the 4.5S RNA. (A) Secondary structure of the D. radiodurans 4.5S RNA with conserved residues indicated in red (sequence alignment displayed in fig. S4). Base pairings are indicated as straight lines and noncanonical interactions are indicated as pound symbols; bulged residues are unpaired. (B) Overall view of the interaction of Ffh and FtsY (displayed as ribbons and colored as in Fig. 1A) with the 4.5S RNA (represented as a contoured surface colored according to the conservation indicated in fig. S4). GMPPCP molecules are shown as red spheres. (C) Closeup view of the interaction of the conserved flipped C83 with the interface of Ffh and FtsY. 4.5S RNA is displayed as sticks colored in gray with C83 colored in orange. Ffh and FtsY residues that interact directly with C83 are displayed as sticks and colored as in (B). GMPPCP residues are represented as sticks colored with white carbons, red oxygens, blue nitrogens, and orange phosphates.

Fig. 3. Major contact area of the Ffh:FtsY NG domain with 5′,3′ region of the 4.5S RNA. (A) Surface representation of the SRP:SR complex in the 5′,3′ region of the 4.5S RNA. (B) Surface representation of the separated Ffh and FtsY (rotate each to one direction) from the 4.5S RNA [maintained in the same orientation as in (A)]. The interface between Ffh and FtsY is displayed in white in both proteins with GMPPCP displayed as red spheres. The contact area of each protein to the RNA is indicated in gray. The FtsY contact area in the RNA is indicated in green and the Ffh contact area in blue; C83 is indicated in blue but contacted by both proteins. www.sciencemag.org

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drolysis, we systematically truncated the E. coli 4.5S RNA from the distal end (Fig. 4A and fig. S7, A and B) and tested the effects of these mutations on the stimulated GTPase reaction between SRP and FtsY. The heterologous complex involving D. radiodurans 4.5S RNAwas not used for biochemical experiments because it showed substantially lower GTPase activity. This could be either due to small differences in side chain positions at the interface of the RNA and activated NG domains, beyond the resolution limit of our current data, or due to the possibility that the experimental conditions have been optimized for measuring the activity of the E. coli complex. Truncations of up to 10 base pairs from the distal end did not substantially affect the GTPase rate (kcat) of the SRP:FtsY complex at saturating protein concentrations (Fig. 4, B and C, and fig. S7B). However, truncation of an additional five base pairs (92mer → 82mer) reduced kcat over eightfold to values approaching that observed in the absence of RNA (Fig. 4, B and C, and fig. S7B). Therefore, the base-paired region C97–C101:G10–G14 at the distal end plays an important role in the SRP RNA-mediated stimulation of GTPase hydrolysis. Consistent with these findings, this region provides a major site for docking of the SRP:SR NG domain complex at the RNA distal end (Fig. 3, A and B). In contrast, the values of kcat/Km (where Km is the Michaelis constant) in the stimulated GTPase reaction were largely unaffected unless the SRP RNA was truncated to less than 56 nucleotides (Fig. 4B and fig. S7B). Because the kcat/Km value in this reaction rate is limited by SRP:FtsY complex formation (19), this result strongly argues that the distal end accelerates the actual GTP catalysis step but not the initial SRP:FtsY complex assembly. To provide independent evidence for this possibility, we directly measured the complex assembly rate constants by using acrylodan conjugated at Ffh-C235, a probe that changes fluorescence upon formation of a GTP-dependent SRP:FtsY complex (19). SRP:FtsY complex assembly rates were affected no more than twofold for RNA truncations up to the 56mer; a substantial defect was detected only with the 43mer (Fig. 4D and fig. S7C). Therefore, the distal end of the SRP RNA specifically stimulates GTPase activation in the SRP:FtsY complex without affecting the initial assembly of the complex. Together with previous work (12, 32, 33), these results demonstrate that the SRP RNA contains two separate motifs that regulate distinct stages of the SRP:FtsY interaction: The tetraloop end accelerates the initial assembly of the complex, whereas the distal end facilitates GTPase activation at late stages. To test the role of the extruded base C86 in GTPase activation, we either removed C86 (DC86) or mutated it to A, G, or U. Deletion of C86 or its mutation to G reduced the GTPase rate constant by half, whereas the C86A and C86U mutations reduced GTPase activity by factors of 7 to 10, to rates approaching that in the absence of the SRP RNA (Fig. 4E and fig. S7D). These data

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tion of the tethered homodimeric NG domains is very high because they are connected via the linker region to the M domain, which is tightly bound to the SRP RNA [dissociation constant (KD) of 5 pM] (30, 31). The SRP RNA distal end specifically stimulates GTPase activation. Although earlier work implicated the tetraloop region of the SRP RNA in GTP hydrolysis (13, 15), interpretation of the observed GTPase activity is complicated by the

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support a role of the extruded base in modulating GTPase activity and explain the conservation of the identity of this base. Modeling the conformation of the SRP:SR on the ribosome. The data presented here, together with previous structural and biochemical studies, show that the SRP complex can exist in distinct conformational states depending on the orientation of the linker between the NG and M domains of Ffh. We propose that these conformational states represent different stages of the SRP-mediated protein targeting pathway. Indeed, the large rearrangement of the NG domains from their initial position in the vicinity of the 4.5S RNA tetraloop as observed in electron microscopy (EM) and biochemical studies (15, 18, 34, 35) to the opposite end of this RNA suggests a plausible mechanism of transferring the RNC from the SRP to the translocon. In order to better understand the implications of the observed conformational changes in the targeting process, we modeled our structure onto the ribosome by using available cryogenic EM (cryo-EM) data (34) (Fig. 5A). The SRP bound to the RNC in the absence of the SR is in a conformation in which its NG domain is next to the M domain with the linker region wrapping around the M domain (Fig. 5B) (24, 34, 36). We propose that the structure described here represents the cargo-release state of the protein targeting cycle. Repositioning the NG domains of Ffh and FtsY to the distal region of 4.5S RNA exposes ribosomal proteins L23 and L29, which constitutes the main translocon binding site. Therefore, this conformational change simultaneously exposes the signal sequence-binding cleft of the M domain and liberates the translocon binding region on the ribosome (36–38). Because the SRP cycle is evolutionarily conserved, we speculate that many of the molecular features that govern the prokaryotic SRP cycle will also occur in eukaryotes. Upon comparison of the molecular model in Fig. 5 with the cryoEM density of the eukaryotic RNC:SRP:SR complex arrested with the GTP analog GMPPNP, certain similarities can be observed (37). Most noticeable, in the eukaryotic complex the density for the NG domains of the SRP54 and SR proteins (homologous to Ffh) is absent from the tetraloop end of the 4.5S RNA, whereas additional density appears in the distal end of the SRP RNA as observed in our complex (fig. S8). It is therefore possible that this density originates from the activated twin NG domains rather than from SR alone, as suggested (37). The SRP cycle revised. On the basis of these data, a mechanism can be envisioned that governs the handoff of substrates to the translocon and the role of GTP in this process. The initial interaction of free SRP with the RNC involves binding of the N domain of Ffh to the ribosomal proteins L23 and L29 (Fig. 6A) as well as interaction of the M domain of Ffh with the signal sequence, possibly aided by the linker helix that wraps around the M domain in the RNC-bound

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Fig. 4. The SRP RNA distal end specifically stimulates GTP hydrolysis in the SRP:SR complex. (A) E. coli SRP RNA systematically truncated from the distal end that were used in this study (see fig. S7A for the sequence of the truncated RNA constructs). The FtsY contact area in the RNA is indicated in green and the Ffh contact area in blue; C86 is indicated in blue but contacted by both proteins. (B) Stimulated GTPase activity between SRP and FtsY with the SRP RNA mutants in (A). The data were fit to the Michaelis-Menten equation, and the kinetic constants kcat and kcat/Km are summarized in fig. S7B. wt, wild type. (C) A substantial defect in the GTPase rate of the SRP:FtsY complex was observed upon truncation of the SRP RNA from the 92mer to 82mer. Rate constants were from (B) and normalized to that of the wild-type SRP RNA. Error bars indicate SDs from two independent experiments. (D) Truncation of the RNA distal end did not substantially affect SRP-FtsY complex formation rates (kon) until the C loop is truncated (43mer). Values of kon were derived from fig. S7B. (E) Mutation of the conserved base C86 (C83 in D. radiodurans 4.5S RNA) impairs the ability of SRP RNA to stimulate GTPase hydrolysis of the SRP:FtsY complex. Rate constants were relative to that of the wild-type SRP RNA and were derived from the data in fig. S7D. Error bars indicate SDs from three independent experiments.

Fig. 5. Conformation of the SRP and SRP:SR structure on the ribosome showing the large rearrangement between cargo-binding and cargo-release modes. (A) The structure of the SRP:SR is superimposed on the SRP:RNC bound structure from cryo-EM reconstruction (34), with Ffh in the cryoEM structure omitted. The signal peptide from the SRP:RNC structure was maintained (red surface) as a reference for the exit tunnel. The RNC is displayed as a white surface with the protein L23 highlighted in yellow. SRP:SR are presented as spheres, with 4.5S RNA colored with dark gray for phosphate and ribose and light gray for bases; Ffh is blue and FtsY is green. (B) The cargo-binding conformation of SRP in the SRP:RNC model structure from cryo-EM is indicated as spheres with Ffh colored in purple.

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RESEARCH ARTICLES RNA because the reduced GTPase rate would provide a longer time frame for the less-efficient signal sequence transfer to occur. The results presented here, together with previous biochemical evidence (13–18, 21), define the SRP RNA as a bifunctional molecule acting as a binding platform for the initial receptor interactions on one end of the 142 Å long molecule and for the activated GTPase domains of the Ffh:FtsY complex on the other end. Such conformational change provides a mechanism for the temporal and spatial exchange of large factors that have to access the signal sequence as it emerges from the ribosomal exit tunnel. References and Notes

conformation of the SRP (Fig. 6B) (34, 36). In this RNC preorganized state, the Ffh G domain is positioned in the vicinity of the tetraloop end of the 4.5S RNA and primed for interaction with the SR (Fig. 6B) (15, 16, 34–36). Receptor binding is initially facilitated by the RNA tetraloop (13, 14, 32, 39), which stabilizes an early conformation of the heterodimeric NG complex through interaction with the SR (Fig. 6C) (5, 10, 16). Subsequent structural rearrangements in the GTPase complex may reduce the affinity of the RNA tetraloop for the SR as well as that of the Ffh N domain for the ribosome (Fig. 6C) (5, 6, 37). Consequently, the activated NG heterodimer detaches from the ribosome exit site, but stays tethered to the SRP RNA via the tightly bound M domain and relocates to the alternative binding site at the distal end of the 4.5S RNA hairpin (Fig. 6D). Because the A domain of FtsYassociates with the translocon (7, 40), repositioning of the NG domain heterodimer will carry the translocon toward its ribosome binding site (L23), which is now exposed (Fig. 6D). This repositioning could also stabilize

the linker domain in the extended helical conformation away from the M domain, thus exposing the signal sequence-binding cleft for peptide release to the translocon. Concurrently, the interaction of the activated NG heterodimer with the distal portion of the 4.5S RNA will stimulate GTPase activity in both Ffh and FtsY, leading to their dissociation and completing the targeting cycle (Fig. 6D). The conformation of the SRP in complex with its receptor observed here also explains many previous observations, including the density features observed in the cryo-EM reconstruction of the eukaryotic RNC:SRP:SR complex (37) and the observation that mutations that disrupt the conformational changes leading to GTPase activation block late stages of protein targeting (8). Previous studies also indicated that overexpression of a truncated 4.5S RNA (domain IV) is capable of sustaining cell viability in a 4.5S depleted strain (4). According to the model described here, signal sequence transfer would be possible even in the absence of the distal region of the SRP

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Fig. 6. Model of the SRP targeting cycle. Schematic depiction of the sequence of conformational changes involved in the SRP cycle (viewed in the membrane plane). (A) SRP recognizes the RNC and M domain interacts with the signal peptide (finger loop is indicated in pink). (B) The N domain interacts with L23, and the linker region folds on top of the signal peptide covering/shielding the M domain. (C) SRP bound cargo (RNC) is transferred to the membrane vicinity via SRP interaction with SR. (D) GTP-dependent rearrangements in the SRP:SR complex enables detachment of the Ffh N domain from L23 and the RNA tetraloop, and the NG domain complex relocates to the 5′,3′ end of the 4.5S RNA sandwiching the C83 (orange arrowhead) at the interface of the two G domains. This repositioning simultaneously exposes the signal sequence bound to the M domain and the L23 region of the ribosome for interactions with the translocon (Sec YEG), which is associated with the A domain of FtsY (black tail). In the final step, signal sequence is transferred from the M domain to the translocon, and the distal region of the 4.5S RNA promotes GTP hydrolysis and subsequent Ffh and FtsY dissociation.

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37. M. Halic et al., Science 312, 745 (2006). 38. K. Mitra et al., Nature 438, 318 (2005). 39. J. R. Jagath, M. V. Rodnina, W. Wintermeyer, J. Mol. Biol. 295, 745 (2000). 40. B. Weiche et al., J. Mol. Biol. 377, 761 (2008). 41. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; G, Gly; Q, Gln; and R, Arg. 42. We thank K. Zhou for excellent technical assistance and help with crystal preparation during the early stages of the project. Initial crystallographic analysis was performed at beamline 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory; we acknowledge C. Ralston for outstanding technical assistance at the ALS. Crystallographic data were

collected at the beamline X06SA at the Swiss Light Source (SLS). We thank A. Brunger for the prerelease version of CNS and for helpful comments on the refinement, C. Schulze-Briese and T. Tomizaki for their outstanding support at the SLS, T. Maier and S. Klinge for critical discussion and reading of the manuscript, and T. Maier and M. Leibundgut for help and assistance with data collection and solving the structure. S.F.A. was funded initially by the Howard Hughes Medical Institute and currently by an ETH postdoctoral fellowship, N.S. is funded by Boehringer Ingelheim Fonds, and K.S. is funded by NIH grant GM078024 to S.S. This work was supported in part by the Howard Hughes Medical Institute (J.A.D.) and by the Swiss National Science Foundation

(SNSF) and the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF. Atomic coordinates and structure factors for the SRP:SR crystal structure have been deposited with the Protein Data Bank under accession code 2xxa.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/881/DC1 Materials and Methods Figs. S1 to S8 Tables S1 and S2 References 13 August 2010; accepted 18 January 2011 10.1126/science.1196473

Metallic and Insulating Oxide Interfaces Controlled by Electronic Correlations H. W. Jang,1 D. A. Felker,2 C. W. Bark,1 Y. Wang,3 M. K. Niranjan,3 C. T. Nelson,4 Y. Zhang,4,5 D. Su,6 C. M. Folkman,1 S. H. Baek,1 S. Lee,1 K. Janicka,3 Y. Zhu,6 X. Q. Pan,4 D. D. Fong,7 E. Y. Tsymbal,3 M. S. Rzchowski,2 C. B. Eom1* The formation of two-dimensional electron gases (2DEGs) at complex oxide interfaces is directly influenced by the oxide electronic properties. We investigated how local electron correlations control the 2DEG by inserting a single atomic layer of a rare-earth oxide (RO) [(R is lanthanum (La), praseodymium (Pr), neodymium (Nd), samarium (Sm), or yttrium (Y)] into an epitaxial strontium titanate oxide (SrTiO3) matrix using pulsed-laser deposition with atomic layer control. We find that structures with La, Pr, and Nd ions result in conducting 2DEGs at the inserted layer, whereas the structures with Sm or Y ions are insulating. Our local spectroscopic and theoretical results indicate that the interfacial conductivity is dependent on electronic correlations that decay spatially into the SrTiO3 matrix. Such correlation effects can lead to new functionalities in designed heterostructures. dvanced deposition techniques enable the growth of epitaxial heterostructures with atomically controlled interfaces such as multilayers (1), superlattices (2–4), and ultrathin films (5, 6). In these artificial structures, the interfaces play a prominent role in determining the functionalities of the structures and their applications (7). A recent example is the discovery of two-dimensional electron gases (2DEGs) at the interface between complex insulating oxides (8) such as LaAlO3/SrTiO3 (9, 10), LaTiO3/SrTiO3 (2), and LaVO3/SrTiO3 (11) heterostructures, in

A

which the 2DEG is confined near the LaO/TiO2 interface. Magnetic and superconducting ground states of the 2DEG have been identified (12–14),

and applications to field-effect transistors and tunnel junctions have been demonstrated (15–17). Theoretical work on LaTiO3/SrTiO3 superlattices (18) suggests that for a several-unit-cellthick LaTiO3 layer, the LaTiO3/SrTiO3 interface region is metallic; however, nonmetallic behavior dominates in the LaTiO3 region away from the interface, resulting from strong electron correlations similar to those found in bulk LaTiO3. In other bulk rare-earth titanates, the effect of electron correlations depends critically on the rareearth ion (19). We used the unique electronic character of oxide interfaces, and atomic level control of their structure and composition, to deliberately manipulate the 2DEG electronic properties. We studied the effect of strong electron correlations on an oxide 2DEG by inserting a single atomic layer of RO (R is La, Pr, Nd, Sm, or Y) into an epitaxial SrTiO3 matrix using pulsed-laser deposition with atomic layer control. The RO layer donates electrons to the conduction band of SrTiO3. These electrons remain near the inserted RO layer due to Coulomb attraction. We find that the transport properties of these electrons range from metallic to insulating, depending critically Fig. 1. (A) Schematic representation of a SrTiO3 / 1-ML RO/SrTiO3 heterostructure. The atomic structure near the interface is enlarged. The +1 valent RO layer donates electrons to neighboring TiO2 planes, leading to the larger electron density ne near the interface. (B) Typical RHEED oscillations for the growth of 1-ML LaO and 10-uc SrTiO3 layers in sequence on a TiO2terminated SrTiO3 substrate. (C) AFM image of a 10-uc SrTiO3/1-ML LaO/SrTiO3 heterostructure showing an atomically smooth surface.

1 Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison, WI 53706, USA. 2Department of Physics, University of Wisconsin–Madison, Madison, WI 53706, USA. 3Department of Physics and Astronomy, Nebraska Center for Materials and Nanoscience, University of Nebraska– Lincoln, Lincoln, NE 68588, USA. 4Department of Materials Science and Engineering, University of Michigan–Ann Arbor, Ann Arbor, MI 48109, USA. 5National Laboratory of Solid State Microstructures and Department of Materials Science and Engineering, Nanjing University, Nanjing 210093, P.R. China. 6 Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY 11973, USA. 7Materials Science Division, Argonne National Laboratory, Argonne, IL 60439, USA.

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

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37. M. Halic et al., Science 312, 745 (2006). 38. K. Mitra et al., Nature 438, 318 (2005). 39. J. R. Jagath, M. V. Rodnina, W. Wintermeyer, J. Mol. Biol. 295, 745 (2000). 40. B. Weiche et al., J. Mol. Biol. 377, 761 (2008). 41. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; G, Gly; Q, Gln; and R, Arg. 42. We thank K. Zhou for excellent technical assistance and help with crystal preparation during the early stages of the project. Initial crystallographic analysis was performed at beamline 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory; we acknowledge C. Ralston for outstanding technical assistance at the ALS. Crystallographic data were

collected at the beamline X06SA at the Swiss Light Source (SLS). We thank A. Brunger for the prerelease version of CNS and for helpful comments on the refinement, C. Schulze-Briese and T. Tomizaki for their outstanding support at the SLS, T. Maier and S. Klinge for critical discussion and reading of the manuscript, and T. Maier and M. Leibundgut for help and assistance with data collection and solving the structure. S.F.A. was funded initially by the Howard Hughes Medical Institute and currently by an ETH postdoctoral fellowship, N.S. is funded by Boehringer Ingelheim Fonds, and K.S. is funded by NIH grant GM078024 to S.S. This work was supported in part by the Howard Hughes Medical Institute (J.A.D.) and by the Swiss National Science Foundation

(SNSF) and the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF. Atomic coordinates and structure factors for the SRP:SR crystal structure have been deposited with the Protein Data Bank under accession code 2xxa.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/881/DC1 Materials and Methods Figs. S1 to S8 Tables S1 and S2 References 13 August 2010; accepted 18 January 2011 10.1126/science.1196473

Metallic and Insulating Oxide Interfaces Controlled by Electronic Correlations H. W. Jang,1 D. A. Felker,2 C. W. Bark,1 Y. Wang,3 M. K. Niranjan,3 C. T. Nelson,4 Y. Zhang,4,5 D. Su,6 C. M. Folkman,1 S. H. Baek,1 S. Lee,1 K. Janicka,3 Y. Zhu,6 X. Q. Pan,4 D. D. Fong,7 E. Y. Tsymbal,3 M. S. Rzchowski,2 C. B. Eom1* The formation of two-dimensional electron gases (2DEGs) at complex oxide interfaces is directly influenced by the oxide electronic properties. We investigated how local electron correlations control the 2DEG by inserting a single atomic layer of a rare-earth oxide (RO) [(R is lanthanum (La), praseodymium (Pr), neodymium (Nd), samarium (Sm), or yttrium (Y)] into an epitaxial strontium titanate oxide (SrTiO3) matrix using pulsed-laser deposition with atomic layer control. We find that structures with La, Pr, and Nd ions result in conducting 2DEGs at the inserted layer, whereas the structures with Sm or Y ions are insulating. Our local spectroscopic and theoretical results indicate that the interfacial conductivity is dependent on electronic correlations that decay spatially into the SrTiO3 matrix. Such correlation effects can lead to new functionalities in designed heterostructures. dvanced deposition techniques enable the growth of epitaxial heterostructures with atomically controlled interfaces such as multilayers (1), superlattices (2–4), and ultrathin films (5, 6). In these artificial structures, the interfaces play a prominent role in determining the functionalities of the structures and their applications (7). A recent example is the discovery of two-dimensional electron gases (2DEGs) at the interface between complex insulating oxides (8) such as LaAlO3/SrTiO3 (9, 10), LaTiO3/SrTiO3 (2), and LaVO3/SrTiO3 (11) heterostructures, in

A

which the 2DEG is confined near the LaO/TiO2 interface. Magnetic and superconducting ground states of the 2DEG have been identified (12–14),

and applications to field-effect transistors and tunnel junctions have been demonstrated (15–17). Theoretical work on LaTiO3/SrTiO3 superlattices (18) suggests that for a several-unit-cellthick LaTiO3 layer, the LaTiO3/SrTiO3 interface region is metallic; however, nonmetallic behavior dominates in the LaTiO3 region away from the interface, resulting from strong electron correlations similar to those found in bulk LaTiO3. In other bulk rare-earth titanates, the effect of electron correlations depends critically on the rareearth ion (19). We used the unique electronic character of oxide interfaces, and atomic level control of their structure and composition, to deliberately manipulate the 2DEG electronic properties. We studied the effect of strong electron correlations on an oxide 2DEG by inserting a single atomic layer of RO (R is La, Pr, Nd, Sm, or Y) into an epitaxial SrTiO3 matrix using pulsed-laser deposition with atomic layer control. The RO layer donates electrons to the conduction band of SrTiO3. These electrons remain near the inserted RO layer due to Coulomb attraction. We find that the transport properties of these electrons range from metallic to insulating, depending critically Fig. 1. (A) Schematic representation of a SrTiO3 / 1-ML RO/SrTiO3 heterostructure. The atomic structure near the interface is enlarged. The +1 valent RO layer donates electrons to neighboring TiO2 planes, leading to the larger electron density ne near the interface. (B) Typical RHEED oscillations for the growth of 1-ML LaO and 10-uc SrTiO3 layers in sequence on a TiO2terminated SrTiO3 substrate. (C) AFM image of a 10-uc SrTiO3/1-ML LaO/SrTiO3 heterostructure showing an atomically smooth surface.

1 Department of Materials Science and Engineering, University of Wisconsin–Madison, Madison, WI 53706, USA. 2Department of Physics, University of Wisconsin–Madison, Madison, WI 53706, USA. 3Department of Physics and Astronomy, Nebraska Center for Materials and Nanoscience, University of Nebraska– Lincoln, Lincoln, NE 68588, USA. 4Department of Materials Science and Engineering, University of Michigan–Ann Arbor, Ann Arbor, MI 48109, USA. 5National Laboratory of Solid State Microstructures and Department of Materials Science and Engineering, Nanjing University, Nanjing 210093, P.R. China. 6 Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY 11973, USA. 7Materials Science Division, Argonne National Laboratory, Argonne, IL 60439, USA.

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

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REPORTS on a TiO2-terminated SrTiO3 substrate, followed by deposition of a SrTiO3 overlayer of varying thickness (20). A thick SrTiO3 overlayer approximates a single RO monolayer embedded in an infinite SrTiO3 matrix. Thicknesses of inserted 1-monolayer (ML)–thick RO and 1-unit-cell (uc)– thick RTiO3 layers were accurately controlled by monitoring in situ reflection high-energy elec-

Fig. 2. Dependence of sheet carrier concentration ns on the R ion in SrTiO3/1-ML RO/SrTiO3 heterostructures and the SrTiO3 overlayer thickness d. Sheet carrier concentration is plotted as a function of (A) the thickness of the SrTiO3 overlayer and (B) the RO doping layer for SrTiO3/1-ML RO/SrTiO3 heterostructures. SmO-based and YO-based heterostructures never become conducting, even with very thick SrTiO3 overlayers.

Fig. 3. STEM and EELS analysis. (A) High-angle annular dark field (HAADF) image of a 10-uc SrTiO3/1-ML LaO film grown on SrTiO3. The rectangular box represents the region of EELS line scans. (B) EELS spectra of T-L2,3 and O-K edges obtained from 2D line scans across the interface shown in (A). The spacing along the line scan between consecutive EELS spectra is 2.8 Å. The spectra at the LaO layer are highlighted by thicker lines. For the spectra for Ti L2 and L3 edges, peak broadening and less pronounced peak splitting at the interface are clearly observed. (C) HAADF images of 10-uc SrTiO3/1-ML LaO/SrTiO3 and 10-uc SrTiO3/1-ML SmO/SrTiO3 heterostructures. Both samples show no obvious defects or dislocations, indicating coherent interfaces. (D) Selected area Ti-L2,3 EELS spectra obtained at the interfaces for 10-uc SrTiO3/1-ML LaO/SrTiO3 and 10-uc SrTiO3/1-ML SmO/SrTiO3 heterostructures. The arrow is a guide for comparison. www.sciencemag.org

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tron diffraction (RHEED) intensity oscillations. Typical RHEED oscillations for the growth of a 10-uc SrTiO3/1-ML LaO heterostructure on a SrTiO3 substrate are shown in Fig. 1B. The atomic force microscopy (AFM) image of the surface of a complete heterostructure (Fig. 1C) shows the steps and terraces of the original substrate surface, indicating high-quality growth. Microstructure and electrical properties of both SrTiO3/1-ML RO/SrTiO3 and SrTiO3/1-uc RTiO3/SrTiO3 heterostructures were almost identical (20). Here, we focus on the SrTiO3/RO/SrTiO3 heterostructures. We first characterized the dependence of electrical properties on growth conditions, using the LaO-based heterostructure, and established the growth conditions of oxygen pressure (PO2 ¼ 10−3 mbar) and temperature (Tgrowth = 550°C) as optimal (20). These growth conditions were used to fabricate SrTiO3 heterostructures with single inserted atomic layers of LaO, PrO, NdO, SmO, and YO. Fig. 2A shows the mobile sheet carrier concentration ns for the five different RO layers as a function of the SrTiO3 overlayer thickness. It is seen that LaO-, PrO-, and NdO-based heterostructures become conducting above the critical thickness of SrTiO3 of three or four unit cells. However, SmO- and YO-based heterostructures are insulating, even with a SrTiO3 overlayer thickness of 100 uc. This is summarized in Fig. 2B, which shows the mobile sheet carrier concentration at fixed overlayer thickness as the rare-earth ion progresses from La to Y. The nominal room-temperature concentration of mobile carriers in crossover NdO-based heterostructures decreases dramatically at lower temperatures (fig. S3D), in contrast to the relatively temperatureindependent behavior of the conducting LaObased and PrO-based heterostructures. This trend is analogous to that in bulk RTiO3, where the effects of electron correlations increase as R is varied from La to Y (21). The mobilities of all conducting heterostructures are roughly independent of the rare-earth ion, showing a crossover from temperature-dependent phonon scattering at high temperature to a temperature-independent value at low temperatures. Our transport measurements are sensitive to mobile carriers near the interface. We also investigated charge transfer from the RO layer to nearby Ti states with electron energy-loss spectroscopy (EELS), sensitive to both mobile and nonmobile carriers (2, 10). For a conducting LaObased heterostructure, the spatial dependence of EELS spectra of Ti-L2,3 and O-K edges is shown in Fig. 3B. The EELS spectra are spatially separated by 0.28 nm, in a line scan across the interface of a 10-uc SrTiO3/1-ML LaO/SrTiO3 heterostructure (Fig. 3A). Four clear peaks in the Ti L2,3 edge become broader at the interface, with peak separations less pronounced. We attribute this broadening to the presence of a Ti3+ component. Compared with previous reports (2, 10), the relatively small modulation of the EELS signal at the interface may be related to the low ns determined from the Hall effect. Our depth profil-

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on the rare-earth ion, and that this dependence arises from strong electronic correlations. We grew epitaxial SrTiO3 heterostructures containing a symmetric TiO2/RO/TiO2 interface (Fig. 1A), resulting in RTiO3-like structure at the interface. Using pulsed-laser deposition, the heterostructures were fabricated by depositing either a RO monolayer or a RTiO3 unit cell

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ing of the Ti3+ to Ti4+ ratios indicates that the carriers are confined to within ~1 nm of the interface (fig. S6), in good agreement with recent theoretical calculations (22). Fig. 3, C and D, show scanning transmission electron microscope (STEM) images and selected area Ti-L2,3 EELS spectra, at TiO2 planes adjacent to the interface, for LaO (conducting) and SmO (insulating) heterostructures. For both heterostructures, the STEM images and the Ti-L2,3 spectra at the interface look very similar. In particular, the very similar peak splittings at ~462 eV in the Ti L2 edges suggest that the electron transfer from the RO layer to the neighboring TiO2 planes is the same for both LaO- and SmO-based heterostructures. Our transport measurements indicate that these electrons produce a conducting 2DEG in LaO heterostructures but are not mobile in SmO heterostructures. TiO6 octahedra rotations in bulk RTiO3 determine the width of the Ti-3d band of t2g symmetry, and hence the electronic properties, through a change in the Mott-Hubbard gap (23). SrTiO3, however, has no TiO6 octahedral rotations at room temperature. We investigated octahedral rotations in our SrTiO3/RO/SrTiO3 heterostructures, with synchrotron x-ray experiments at the Advanced Photon Source. We observed strong superlattice reflections (figs. S7 and S8) resulting from unitcell doubling TiO6 octahedra rotations, in good agreement with the density functional calculations discussed below. The octahedral rotations are well ordered in the interfacial plane, with typical rocking widths giving an in-plane domain size > 60 nm. The breadths of the half-order peaks in the out-of-plane direction are consistent with octahedral rotations at the RTiO3 layer rapidly decaying into the SrTiO3 matrix. These decaying octahedra rotations lead to a spatial gradient in the electronic structure, influencing the conduction. In addition, epitaxial strain in the interfacial RTiO3 layer also affects the interface conductiv-

ity. LaTiO3, PrTiO3, and NdTiO3 layers at the interface are strained under biaxial compression, but SmTiO3 and YTiO3 layers are under biaxial tension (table S1) (21). Compressive strain has been shown to induce conducting behavior in LaTiO3 thin films (24), attributed to an increased Ti t2g bandwidth and a weakened crystal field. This has been predicted theoretically to reduce the effect of electron correlations and to support metallic behavior (25). The tensile strain in the SmTiO3 and YTiO3 layers embedded in SrTiO3 appears to enforce the effect of strong correlations and favor insulating behavior. To understand the combined effects of charge transfer, spatially varying octahedral rotations, biaxial strain, and rare-earth electronic structure, we have performed density functional theory (DFT) calculations, including a Hubbard U term accounting for the on-site Coulomb interaction (20). The values of U that provide a realistic description of the electronic and atomic structure of bulk YTiO3 and LaTiO3 compounds (26) were used. The atomic positions were fully relaxed, under the constraint that the in-plane lattice constant be equal to the calculated lattice constant of bulk SrTiO3. The density of electronic states, and the corresponding atomic coordinates, calculated for periodic superlattices, are shown in Fig. 4A (3.5-uc SrTiO3/1-ML LaO) and in Fig. 4B (3.5-uc SrTiO3/1-ML YO). For the LaObased heterostructure, the Fermi energy lies in the region of nonzero density of states, consistent with the previous calculations (27, 28), whereas for the YO heterostructure the Fermi energy lies between the split-off lower Hubbard band and the higher energy density of states. This indicates that the LaO-based interface is metallic, whereas the YO-based interface is insulating, supporting our experimental observations. Our calculations predict that the ground state of the SrTiO3/LaO heterostructure is not chargeordered, whereas the SrTiO3/YO heterostructure

Fig. 4. Energy-dependent density of states and structural relaxation of 3.5-uc SrTiO3/1-ML LaO (A and C) periodic superlattice and 3.5-uc SrTiO3/1-ML YO periodic superlattice (B and D) obtained from DFT calculations. Positive density of states is for spin up and negative is for spin down. The dashed line indicates the position of the Fermi level. The results indicate conducting behavior for the 3.5-uc SrTiO3/1-ML LaO periodic superlattice and insulating behavior for the 3.5-uc SrTiO3/1-ML YO periodic superlattice.

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is unstable with respect to charge disproportionation and has a charge-ordered ground state similar to that found in (29). Octahedra rotations are clearly visible in the relaxed structures shown in Fig. 4, C and D, consistent with our synchrotron measurements. The electron donated by the RO embedded in the SrTiO3 matrix is localized to the nearby TiO2 layers. Filling of the Ti-3d band in these layers close to n = 0.5, and enhanced electroncorrelation effects due to 2D confinement, will strongly influence the interfacial conductivity. It is well known that the effect of Ti-3d band filling on electronic, magnetic, and transport properties of bulk RTiO3 Mott-Hubbard insulators depends critically on the rare-earth ion (23). It appears that for the relatively weakly correlated LaO-based heterostructure, several percent of hole doping is sufficient to cause a metalinsulator transition. In contrast, for the YO-based heterostructures with larger U, lower bandwidth W, and larger strain and structural distortions, the insulating phase persists. The number of electrons transferred in each case is the same, but stronger correlation effects in the YO heterostructure seem to be responsible for the insulating behavior. Our experimental and theoretical investigations suggest that these correlations arise from an interplay of strain, spatially varying rotational distortions, and rare-earth ion effects on the band structure. Indications of electron correlations have also been recently reported in LaIO3/SrTiO3 heterostructures (30). Strong correlations in 2DEGs at oxide interfaces have been shown to result from electronic properties of different RO inserted layers, as well as the structural and electronic modification of nearby layers. Quantitatively exploring the underlying physics of the experimental data presented here is complex and challenging, because strong correlations combined with atomic-scale structural and chemical variations severely limit the effectiveness of theoretical calculations. The details cannot be fully captured within the DFT+U calculations used here, and more advanced approaches— based on dynamical mean-field theory (31), for example—are likely necessary to capture the spatial variations. The work presented here is important in elucidating correlation effects in systems with atomic-scale perturbations (32) and external perturbation-induced changes in oxide 2DEG systems (8, 15–17). The ability to design and grow heterostructures with atomic-scale variations, and the demonstrated strong dependence of correlated 2DEGs on these variations, open new directions for oxide 2DEG heterostructures. References and Notes 1. H. Yamada et al., Science 305, 646 (2004). 2. A. Ohtomo, D. A. Muller, J. L. Grazul, H. Y. Hwang, Nature 419, 378 (2002). 3. M. P. Warusawithana, E. V. Colla, J. N. Eckstein, M. B. Weissman, Phys. Rev. Lett. 90, 036802 (2003). 4. E. Bousquet et al., Nature 452, 732 (2008). 5. M. P. Warusawithana et al., Science 324, 367 (2009).

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REPORTS 23. M. Imada, A. Fujimori, Y. Tokura, Rev. Mod. Phys. 70, 1039 (1998). 24. F. J. Wong et al., Phys. Rev. B 81, 161101 (2010). 25. H. Ishida, A. Liebsch, Phys. Rev. B 77, 115350 (2008). 26. S. Okatov, A. Poteryaev, A. Lichtenstein, Europhys. Lett. 70, 499 (2005). 27. Z. S. Popovic, S. Satpathy, Phys. Rev. Lett. 94, 176805 (2005). 28. S. Okamoto, A. J. Millis, N. A. Spaldin, Phys. Rev. Lett. 97, 056802 (2006). 29. R. Pentcheva, W. E. Pickett, Phys. Rev. Lett. 99, 016802 (2007). 30. M. Breitschaft et al., Phys. Rev. B 81, 153414 (2010). 31. G. Kotliar et al., Rev. Mod. Phys. 78, 865 (2006). 32. Q. Si, M. J. Rozenberg, G. Kotliar, A. E. Ruckenstein, Phys. Rev. Lett. 72, 2761 (1994). 33. We thank D. G. Schlom and D. A. Muller for fruitful discussions. This work was supported by the National Science Foundation under grant DMR-0906443 and a David and Lucile Packard Fellowship (C.B.E.). The research at University of Nebraska was supported by the Materials Research Science and Engineering Center (NSF grant DMR-0820521), the Nanoelectronics Research Initiative of the Semiconductor Research Corporation, the National Science Foundation (grant

Time-Reversed Lasing and Interferometric Control of Absorption Wenjie Wan, Yidong Chong, Li Ge, Heeso Noh, A. Douglas Stone, Hui Cao* In the time-reversed counterpart to laser emission, incident coherent optical fields are perfectly absorbed within a resonator that contains a loss medium instead of a gain medium. The incident fields and frequency must coincide with those of the corresponding laser with gain. We demonstrated this effect for two counterpropagating incident fields in a silicon cavity, showing that absorption can be enhanced by two orders of magnitude, the maximum predicted by theory for our experimental setup. In addition, we showed that absorption can be reduced substantially by varying the relative phase of the incident fields. The device, termed a “coherent perfect absorber,” functions as an absorptive interferometer, with potential practical applications in integrated optics. ime-reversal symmetry is a fundamental symmetry of classical electromagnetic theory and of nonrelativistic quantum mechanics. It implies that if a particular physical process is allowed, then there also exists a “timereversed process” that is related to the original process by reversing momenta and the direction of certain fields (typically external magnetic fields and internal spins). These symmetry operations are equivalent to changing the sign of the time variable in the dynamical equations, and for steadystate situations they correspond to interchanging incoming and outgoing fields. The power of time-reversal symmetry is that it enables exact predictions of the relationship between two processes of arbitrary complexity. A familiar example is spin echo in nuclear magnetic resonance (NMR) (1): A set of precessing spins in a magnetic field fall out of phase because of

T

Department of Applied Physics, Post Office Box 208284, Yale University, New Haven, CT 06520, USA. *To whom correspondence should be addressed. E-mail: [email protected]

slightly different local field environments, quenching the NMR signal. The signal can be restored by imposing an inversion pulse at time T, which has the effect of running the phase of each spin

EPS-1010674), and the Nebraska Research Initiative. Work at the University of Michigan was supported by the U.S. Department of Energy (DOE) under grant DE-FG02-07ER46416. We thank the National Center for Electron Microscopy at Lawrence Berkeley National Laboratory for their support under DOE grant DE-AC02-05CH11231 for user facilities. Work at Argonne and use of the Advanced Photon Source were supported by the DOE Office of Science, Office of Basic Energy Sciences, under contract DE-AC0206CH11357. Work at Brookhaven National Laboratory was sponsored by DOE/BES/MSE and the Center for Functional Nanomaterials under contract DE-AC0298CH10886. J. Karapetrova’s assistance at beamline 33-BM of the Advanced Photon Source is gratefully acknowledged.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/886/DC1 Materials and Methods Figs. S1 to S8 Table S1 References

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6. V. Garcia et al., Nature 460, 81 (2009). 7. H. Kroemer, Surf. Sci. 132, 543 (1983). 8. J. Mannhart, D. G. Schlom, Science 327, 1607 (2010). 9. A. Ohtomo, H. Y. Hwang, Nature 427, 423 (2004). 10. N. Nakagawa, H. Y. Hwang, D. A. Muller, Nat. Mater. 5, 204 (2006). 11. Y. Hotta, T. Susaki, H. Y. Hwang, Phys. Rev. Lett. 99, 236805 (2007). 12. A. Brinkman et al., Nat. Mater. 6, 493 (2007). 13. N. Reyren et al., Science 317, 1196 (2007). 14. Y. Kozuka et al., Nature 462, 487 (2009). 15. S. Thiel, G. Hammerl, A. Schmehl, C. W. Schneider, J. Mannhart, Science 313, 1942 (2006). 16. C. Cen et al., Nat. Mater. 7, 298 (2008). 17. C. Cen, S. Thiel, J. Mannhart, J. Levy, Science 323, 1026 (2009). 18. S. Okamoto, A. J. Millis, Nature 428, 630 (2004). 19. Y. Okimoto, T. Katsufuji, Y. Okada, T. Arima, Y. Tokura, Phys. Rev. B 51, 9581 (1995). 20. Materials and methods are available as supporting material on Science Online. 21. H. D. Zhou, J. B. Goodenough, J. Phys. Condens. Matter 17, 7395 (2005). 22. K. Janicka, J. P. Velev, E. Y. Tsymbal, Phys. Rev. Lett. 102, 106803 (2009).

7 October 2010; accepted 19 January 2011 10.1126/science.1198781

backward in time, so that after 2T they are back in phase, no matter how complicated their local field environment. Time-reversal symmetry is the origin of the well-known weak localization effect (2) in the resistance of metals, the coherent backscattering peak in the reflection from multiple scattering media (3–5), and the elastic enhancement factor familiar in nuclear scattering (6). Effects due to direct generation of time-reversed waves via special “mirrors” have been extensively studied for sound waves (7–9) and microwave radiation (10). Recently, several of the authors (11) explored theoretically an exact time-reversal symmetry property of optical systems: the time-reversed analog of laser emission. In the lasing process, a cavity with gain produces outgoing optical fields with a definite frequency and phase relationship, without being illuminated by coherent incoming fields at that frequency. The laser is coupled to an energy source (the pump) that inverts the electron population of the gain medium, causing the onset

Fig. 1. A laser beam from a tunable (800 to 1000 nm) continuous-wave Ti:sapphire source enters a beam splitter (designated 1). The two split beams are directed normally onto opposite sides of a silicon wafer of thickness ~110 mm, using a MachZehnder geometry. A phase delay in one of the beam paths controls the relative phase of the two beams. An additional attenuator ensures that the input beams have equal intensities, compensating for imbalances in the beam splitters and other imperfections. The output beams are rerouted, via beam splitters (designated 2, 3, and 4), into a spectrometer. The inset is a schematic of the CPA mechanism. The incident beams from left and right multiply scatter within the wafer with just the right amplitude and phase so that the total transmitted and reflected beams destructively interfere on both sides, leading to perfect absorption.

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REPORTS 23. M. Imada, A. Fujimori, Y. Tokura, Rev. Mod. Phys. 70, 1039 (1998). 24. F. J. Wong et al., Phys. Rev. B 81, 161101 (2010). 25. H. Ishida, A. Liebsch, Phys. Rev. B 77, 115350 (2008). 26. S. Okatov, A. Poteryaev, A. Lichtenstein, Europhys. Lett. 70, 499 (2005). 27. Z. S. Popovic, S. Satpathy, Phys. Rev. Lett. 94, 176805 (2005). 28. S. Okamoto, A. J. Millis, N. A. Spaldin, Phys. Rev. Lett. 97, 056802 (2006). 29. R. Pentcheva, W. E. Pickett, Phys. Rev. Lett. 99, 016802 (2007). 30. M. Breitschaft et al., Phys. Rev. B 81, 153414 (2010). 31. G. Kotliar et al., Rev. Mod. Phys. 78, 865 (2006). 32. Q. Si, M. J. Rozenberg, G. Kotliar, A. E. Ruckenstein, Phys. Rev. Lett. 72, 2761 (1994). 33. We thank D. G. Schlom and D. A. Muller for fruitful discussions. This work was supported by the National Science Foundation under grant DMR-0906443 and a David and Lucile Packard Fellowship (C.B.E.). The research at University of Nebraska was supported by the Materials Research Science and Engineering Center (NSF grant DMR-0820521), the Nanoelectronics Research Initiative of the Semiconductor Research Corporation, the National Science Foundation (grant

Time-Reversed Lasing and Interferometric Control of Absorption Wenjie Wan, Yidong Chong, Li Ge, Heeso Noh, A. Douglas Stone, Hui Cao* In the time-reversed counterpart to laser emission, incident coherent optical fields are perfectly absorbed within a resonator that contains a loss medium instead of a gain medium. The incident fields and frequency must coincide with those of the corresponding laser with gain. We demonstrated this effect for two counterpropagating incident fields in a silicon cavity, showing that absorption can be enhanced by two orders of magnitude, the maximum predicted by theory for our experimental setup. In addition, we showed that absorption can be reduced substantially by varying the relative phase of the incident fields. The device, termed a “coherent perfect absorber,” functions as an absorptive interferometer, with potential practical applications in integrated optics. ime-reversal symmetry is a fundamental symmetry of classical electromagnetic theory and of nonrelativistic quantum mechanics. It implies that if a particular physical process is allowed, then there also exists a “timereversed process” that is related to the original process by reversing momenta and the direction of certain fields (typically external magnetic fields and internal spins). These symmetry operations are equivalent to changing the sign of the time variable in the dynamical equations, and for steadystate situations they correspond to interchanging incoming and outgoing fields. The power of time-reversal symmetry is that it enables exact predictions of the relationship between two processes of arbitrary complexity. A familiar example is spin echo in nuclear magnetic resonance (NMR) (1): A set of precessing spins in a magnetic field fall out of phase because of

T

Department of Applied Physics, Post Office Box 208284, Yale University, New Haven, CT 06520, USA. *To whom correspondence should be addressed. E-mail: [email protected]

slightly different local field environments, quenching the NMR signal. The signal can be restored by imposing an inversion pulse at time T, which has the effect of running the phase of each spin

EPS-1010674), and the Nebraska Research Initiative. Work at the University of Michigan was supported by the U.S. Department of Energy (DOE) under grant DE-FG02-07ER46416. We thank the National Center for Electron Microscopy at Lawrence Berkeley National Laboratory for their support under DOE grant DE-AC02-05CH11231 for user facilities. Work at Argonne and use of the Advanced Photon Source were supported by the DOE Office of Science, Office of Basic Energy Sciences, under contract DE-AC0206CH11357. Work at Brookhaven National Laboratory was sponsored by DOE/BES/MSE and the Center for Functional Nanomaterials under contract DE-AC0298CH10886. J. Karapetrova’s assistance at beamline 33-BM of the Advanced Photon Source is gratefully acknowledged.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/886/DC1 Materials and Methods Figs. S1 to S8 Table S1 References

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6. V. Garcia et al., Nature 460, 81 (2009). 7. H. Kroemer, Surf. Sci. 132, 543 (1983). 8. J. Mannhart, D. G. Schlom, Science 327, 1607 (2010). 9. A. Ohtomo, H. Y. Hwang, Nature 427, 423 (2004). 10. N. Nakagawa, H. Y. Hwang, D. A. Muller, Nat. Mater. 5, 204 (2006). 11. Y. Hotta, T. Susaki, H. Y. Hwang, Phys. Rev. Lett. 99, 236805 (2007). 12. A. Brinkman et al., Nat. Mater. 6, 493 (2007). 13. N. Reyren et al., Science 317, 1196 (2007). 14. Y. Kozuka et al., Nature 462, 487 (2009). 15. S. Thiel, G. Hammerl, A. Schmehl, C. W. Schneider, J. Mannhart, Science 313, 1942 (2006). 16. C. Cen et al., Nat. Mater. 7, 298 (2008). 17. C. Cen, S. Thiel, J. Mannhart, J. Levy, Science 323, 1026 (2009). 18. S. Okamoto, A. J. Millis, Nature 428, 630 (2004). 19. Y. Okimoto, T. Katsufuji, Y. Okada, T. Arima, Y. Tokura, Phys. Rev. B 51, 9581 (1995). 20. Materials and methods are available as supporting material on Science Online. 21. H. D. Zhou, J. B. Goodenough, J. Phys. Condens. Matter 17, 7395 (2005). 22. K. Janicka, J. P. Velev, E. Y. Tsymbal, Phys. Rev. Lett. 102, 106803 (2009).

7 October 2010; accepted 19 January 2011 10.1126/science.1198781

backward in time, so that after 2T they are back in phase, no matter how complicated their local field environment. Time-reversal symmetry is the origin of the well-known weak localization effect (2) in the resistance of metals, the coherent backscattering peak in the reflection from multiple scattering media (3–5), and the elastic enhancement factor familiar in nuclear scattering (6). Effects due to direct generation of time-reversed waves via special “mirrors” have been extensively studied for sound waves (7–9) and microwave radiation (10). Recently, several of the authors (11) explored theoretically an exact time-reversal symmetry property of optical systems: the time-reversed analog of laser emission. In the lasing process, a cavity with gain produces outgoing optical fields with a definite frequency and phase relationship, without being illuminated by coherent incoming fields at that frequency. The laser is coupled to an energy source (the pump) that inverts the electron population of the gain medium, causing the onset

Fig. 1. A laser beam from a tunable (800 to 1000 nm) continuous-wave Ti:sapphire source enters a beam splitter (designated 1). The two split beams are directed normally onto opposite sides of a silicon wafer of thickness ~110 mm, using a MachZehnder geometry. A phase delay in one of the beam paths controls the relative phase of the two beams. An additional attenuator ensures that the input beams have equal intensities, compensating for imbalances in the beam splitters and other imperfections. The output beams are rerouted, via beam splitters (designated 2, 3, and 4), into a spectrometer. The inset is a schematic of the CPA mechanism. The incident beams from left and right multiply scatter within the wafer with just the right amplitude and phase so that the total transmitted and reflected beams destructively interfere on both sides, leading to perfect absorption.

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A

B

C

D

E

F

G

Fig. 2. Phase modulation of beam absorption. (A) Theoretical plot of normalized total output intensities as a function of wavelength l for parity-odd (blue) and parity-even (red) scattering eigenmodes. The dashed black line is the result for incoherent input beams. (B to D) Theoretical output intensities at three representative values of l as the relative phase of the input beams is varied, of coherent radiation at a threshold value of the pump. Above threshold the laser is a nonlinear device, but at threshold for the first lasing mode, the laser is described by the linear Maxwell equations with complex (amplifying) refractive index. Because of the properties of these equations under time reversal (11), it follows that the same cavity, with the gain medium replaced by an equivalent absorbing medium, will perfectly absorb the same frequency of light, if it is illuminated with incoming waves with the same field pattern. Additional analysis showed that if the cavity is illuminated with coherent field patterns not corresponding to the time-reversed emission pattern, it is possible to decrease the absorption well below the value for incoherent illumination. Such a device, related to a laser by time reversal, was termed a “coherent perfect absorber” (CPA) (11). The properties of CPAs point to a new method for controlling absorption through coherent illumination. Here we demonstrate both the strong enhancement and reduction of absorption in a simple realization of the CPA: a silicon wafer functioning as solid Fabry-Perot etalon. We now give a more precise statement of the CPA theorem. For simplicity, consider the scalar wave equation [see (12) for the vector generalization]: ½∇2 þ n2 ðrÞk 2 fk ðrÞ ¼ 0

890

A

B

Fig. 3. (A) Modulation depth—the ratio of maximum to minimum output intensity obtained by varying the relative input phase, M = max(Iout)/min(Iout)—as a function of wavelength. The wavelength spacing of adjacent M-peaks is ~1.27 nm, closely matching the free spectral range of the Si etalon. Between these maxima, M goes nearly to unity, corresponding to the “phase-insensitive points” where the two S-matrix eigenvalues have the same magnitude. (B) Ratio of these maximum and minimum values to the value 2(R + T), obtained when the two input beams do not interfere coherently, demonstrating both enhancement and suppression of cavity absorption by interference. Squares (A) and triangles (B) are experimental data [in (B), upright triangles denote reduced absorption, whereas inverted triangles denote enhanced absorption]; solid curves are theory, including resolution effects (12). which relates incoming and outgoing channel states whose weights are represented by complex vectors a, b, obeying S½nðrÞk ⋅ a ¼ b

ð1Þ

where k = w/c, w is the frequency, c is the speed of light, fk(r) is the electric field, and n = n1 + in2 is the refractive index (n2 < 0 for gain and n2 > 0 for absorption). Outside of the cavity, n is assumed to be real and constant. Steady-state solutions of these equations are described by the electromagnetic scattering matrix (S-matrix) (11),

showing intensities emitted to the right (magenta) and left (green) sides of the slab, and the total intensity (black). Values of l corresponding to (B) to (D) are marked by vertical lines in (A); (B) is the CPA resonance. (E to G) Experimental results at values of l approximately corresponding to (B) to (D). Solid lines are fits to the data, not theory curves; results are normalized to max(Iout) of the fit.

ð2Þ

The S-matrix is unitary if and only if n2 = 0. In general it satisfies the property that, under time reversal, S½n ðrÞk ⋅ b ¼ a

ð3Þ

Equations 2 and 3 imply that every scattering solution of the amplifying problem, with n = n1 −

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in2 (n2 > 0) and outgoing amplitudes b, is accompanied by a solution to the absorbing problem with n = n1 + in2 and incoming amplitudes b*. Now consider a laser at threshold: There exists a specific solution, described by a vector of nonzero outgoing amplitudes b (determined up to an overall scale factor), for infinitesimal incoming amplitudes (a → 0). Thus, the S-matrix has an eigenvalue that tends to infinity. By the timereversal property (Eq. 3), a lossy cavity with n1 = n1, −n2 → +n2 must possess a solution corresponding to the time-reversed lasing mode, for which the incident field (b*) is completely absorbed (a* → 0); the associated S-matrix

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REPORTS cause of the finite width of the free-space beams, our results indicate that this is not the main source of deviation from the ideal behavior (12). The output beams are collected into a high-resolution spectrometer, and the intensities in each individual output beam, as well as the total output intensity, are measured (12). In this geometry, the physical origin of the CPA effect is clear (11). As illustrated in the inset of Fig. 1, the multiply scattered transmission from the left beam interferes destructively with the multiply scattered reflection from the right beam at the right interface, and vice versa at the left face. At the precise CPA condition, this leads to an ideal interference “trap” for the two beams, so that eventually the radiation is entirely dissipated by the interband absorption processes in the silicon. Counterintuitively, increasing the single-pass absorption would actually reduce the net absorption by disturbing the ideal balance of absorption and interference at the operating wavelength. For a given cavity Q-value, only a certain narrow range of absorption coefficients will yield strong CPA resonances. The reflection symmetry of our cavity implies that CPA resonances arise when the reflectance (R) and transmittance (T ) are equal, which occurs as l varies through the band gap and strong absorption sets in. We use this condition to determine the operating wavelength range of 990 nm <~ l <~ 1010 nm for our system (12); fine-tuning l within this interval yields strong CPA resonances. The key quantity measured in the experiment is the total intensity of the scattered radiation (reflectance plus transmittance from both sides). This is determined theoretically by the eigenvectors of the 2 × 2 S-matrix, which satisfy S(k) · ai = siai for i = 1, 2. Because of the cavity’s reflection symmetry, pﬃﬃ pﬃﬃ the eigenvectors take the form ai ¼ ½ I ; I expðfi Þ, where I is the incident intensity of the balanced beams and fi = 0, p. The total scattered intensity for each eigenvector is 2|si|2I. Figure 2A shows a theoretical plot of S-matrix eigenvalue intensities, |s|2, assuming n = 3.6 + 0.0008i and slab thickness a = 115.79 mm (12). Multiple CPA resonances exist, occurring alternatively for even and odd eigenvectors. If we work at a wavelength corresponding to a CPA resonance, such as the central minimum shown in Fig. 2A, then, upon varying the relative phase f from 0 to p (keeping the two beam intensities constant and equal), the system goes from enhanced scattering (red curve) to nearly zero scattering (blue curve). Intermediate values of f do not correspond to a single S-matrix eigenstate, so the scattered intensity interpolates between the extremal values. The black curve in Fig. 2A shows the expected scattered intensity for incident beams neglecting their interference, 2(R + T)I. At the CPA resonance, it lies roughly a factor of 2 below the scattered intensity of the even (red) mode, demonstrating substantial coherent reduction of absorption for this mode. This is due to constructive interference in this mode

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for escape from the cavity, reducing its total absorption; other, more complicated structures allow even larger contrast between the CPA mode and the “scattered” mode (11). There also exist “phase-insensitive” points between each pair of CPA resonances, seen in Fig. 2A where the red and blue curves cross. Here, the scattered intensity is completely independent of the relative phase of the two input beams and equal to the value for incoherent illumination. The experimental data (Fig. 2, E to G) are in good agreement with the theoretical predictions (Fig. 2, B to D). The theoretical plots show the normalized total output intensity at three representative values of the wavelength, indicated by the corresponding labels (b to d) in Fig. 2A. They show, respectively, an odd-parity CPA resonance (Fig. 2B); an intermediate wavelength, with a smaller total intensity variation with f but still a factor of ~2.5 in the scattering of the evenand odd-parity beams (Fig. 2C); and a phaseinsensitive point (Fig. 2D). Also shown, along with the total output intensity, are the intensities as measured on the left and right. Generally, these two intensities have maximum attenuation at different values of f. However at the wavelength corresponding to the CPA resonance (Fig. 2B), their minima coincide at a single f (0 or p), producing an absorption contrast of several orders of magnitude. At the phase-insensitive point (Fig. 2D), the left and right outputs are precisely out of phase; varying f leaves the total output unchanged but switches the dominant output between left and right. A convenient figure of merit for how close the experiment comes to the exact CPA condition is the “modulation depth” M(l) = max(Iout)/ min(Iout), the ratio of the maximum to minimum total output intensity as we vary f. The observed values as a function of l are shown in Fig. 3A. For a device satisfying the exact CPA condition, min(Iout) → 0 and so M → ∞. This does not occur in the present device because we slightly miss the CPA condition by tuning only one parameter, l, leading to a maximum modulation depth of ~104 to 105. However, the limiting factors in the experiment are the temporal and spatial coherence of the laser, reducing M(l) to ~102. The finite laser linewidth (0.18 nm) “smears out” the CPA resonances, which are optimized for a monochromatic input. This effect can be partially compensated by filtering the output through a spectrometer [resolution ≈ 0.05 nm (12)]. This finite resolution of the spectrometer can be incorporated into the analysis, and the resulting theoretical curve, shown in Fig. 3A, agrees well with the experimental data. The dual role of interference in both enhancing and suppressing absorption can be seen more clearly in Fig. 3B, which compares the maximum and minimum output intensities to 2(R + T), the expected output intensity for two incoherent input beams. At the CPA resonant wavelength, the minimum output intensity is less than 1% of the input, while the incoherent

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eigenvector has eigenvalue zero. This lossy cavity is the CPA, the time-reversed counterpart to the original laser. Furthermore, if the CPA cavity is accessible by more than one asymptotic channel, it typically possesses other eigenvectors with nonzero eigenvalues, so there are many other incident radiation patterns, at the same frequency, that are not fully absorbed. Early laser studies (13, 14) had briefly noted the possibility of the time-reversed process of lasing, but detailed theory and investigation of practical realizations began with (11). The simplest realization, a single-channel CPA, consists of an asymmetric cavity with a perfectly reflecting mirror at one end and coupled at the other end to a single input channel. When the absorption of the cavity is tuned to an optimal value, the reflection from the “back mirror” of the cavity destructively interferes with the reflection from the front face, and the incident radiation is perfectly absorbed. Several investigators have discovered the coherent absorption effect for this case, without making use of the analogy to a laser at threshold. III-V semiconductor devices that are essentially equivalent to the singlechannel CPA have been widely developed over the past two decades as modulators (15–18) (“asymmetric Fabry-Perot” modulators) and detectors (19, 20) (“resonant cavity-enhanced photodetectors”). In addition, a closely related device, used as an optical switch or filter, is the “critically coupled resonator” (21–23), which typically has a ring geometry and is equivalent to two decoupled single-channel CPAs. Our two-channel CPA is qualitatively different from the single-channel case because it requires two coherent input beams, and perfect absorption is only achieved when these beams have the correct relative phase and amplitude. Thus, it is not only sensitive to frequency but to the amplitude and phase of the input light and can function as an absorptive interferometer, potentially useful as a modulator, detector, or phasecontrolled optical switch. Reaching the precise CPA condition of perfect absorption requires tuning two parameters (e.g., n1 and n2 or n2 and l); by analogy to the laser, the CPA must have the correct absorption to reach “threshold” and also must satisfy an appropriate interference condition in the cavity. However, simply tuning l near the band gap of a semiconductor (11) can bring the system very close to the CPA condition, increasing the absorption by many orders of magnitude. The simplest two-channel CPA has a uniform complex refractive index n approaching one of the values needed for the CPA condition, connected to a single propagating mode on the left and another on the right. In our implementation (Fig. 1), two collimated counterpropagating freespace laser beams are directed onto opposite surfaces of a Si wafer, which functions as a low-Q Fabry-Perot etalon based on Fresnel reflection at the surfaces (Q ≈ 840). Although these illumination conditions are not truly single-channel be-

891

illumination gives ~35% output. When the phase is adjusted to maximize the scattering, the output reaches ~70%. Although we have demonstrated coherent reduction of absorption in our experiment, this effect should be distinguished from the phenomenon of electromagnetically induced transparency, in which absorption is suppressed by coherently driving the absorbing medium itself (24), instead of by enhancing escape from the cavity by constructive interference, as in our system. Because this optical effect is easily realized in silicon, coherent perfect absorbers may enable novel functionalities in silicon integrated photonic circuits of the type envisioned for nextgeneration optical communications and computing applications (25) as well as for coherent laser spectroscopy. The simplest versions of the device immediately would serve as compact on-chip interferometers, which absorb or scatter the input beams instead of steering them. Although our current CPA operates near the silicon band edge, it should be possible to fabricate devices in which an additional parameter tunes the absorption coefficient independently of l (e.g., by free carrier injection or by optical pumping), allowing one to fix the operating wavelength by design. Direct–band gap semiconductors also are suitable materials for CPAs, assuming that fluorescent emission can be tolerated or avoided in a specific application. Recent theoretical work has proposed a fascinating extension of the CPA concept, suitable for direct–band gap materials: Systems with balanced gain and

loss can function simultaneously as a CPA and as a laser (i.e., as an interferometric amplifierattenuator) (26, 27). The CPA effect is not immediately applicable to photovoltaic or stealth technology because it is a narrow-band effect requiring coherent inputs. More generally, the exact time-reversal symmetry property that relates laser emission to coherent perfect absorption implies that an arbitrarily complicated scattering system can be made to perfectly absorb at discrete frequencies if its imaginary refractive index can be tuned continuously over a reasonable range of values, and if appropriate coherent incident beams can be imposed. Progress in these areas would open up interesting new avenues for future research and applications. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

E. L. Hahn, Phys. Rev. 80, 580 (1950). G. Bergmann, Phys. Rep. 107, 1 (1984). Y. Kuga, A. Ishimaru, J. Opt. Soc. Am. A 1, 831 (1984). M. P. Van Albada, A. Lagendijk, Phys. Rev. Lett. 55, 2692 (1985). P. E. Wolf, G. Maret, Phys. Rev. Lett. 55, 2696 (1985). C. H. Lewenkopf, H. A. Weidenmuller, Ann. Phys. 212, 53 (1991). M. Fink et al., Rep. Prog. Phys. 63, 1933 (2000). J. de Rosny, M. Fink, Phys. Rev. Lett. 89, 124301 (2002). De Rosny and Fink (8) demonstrated a process of perfect absorption of time-reversed acoustic waves, but this requires a dynamic sink that is driven externally with appropriate amplitude and phase. This process is not related to time-reversed lasing. G. Lerosey, J. de Rosny, A. Tourin, M. Fink, Science 315, 1120 (2007).

Quantum Reflection of He2 Several Nanometers Above a Grating Surface Bum Suk Zhao,* Gerard Meijer, Wieland Schöllkopf Quantum reflection allows an atom or molecule to be reflected from a solid before it reaches the region where it would encounter the repulsive potential of the surface. We observed nondestructive scattering of the helium dimer (He2), which has a binding energy of 10−7 electron volt, from a solid reflection grating. We scattered a beam containing the dimer as well as atomic helium and larger clusters, but could differentiate the dimer by its diffraction angle. Helium dimers are quantum reflected tens of nanometers above the surface, where the surface-induced forces are too weak to dissociate the fragile bond. neutral atom or molecule approaching a solid surface experiences an attractive force caused by the van der Waals atomsurface interaction potential, as sketched in Fig. 1A. In a classical picture, the particle accelerates toward the surface until it scatters back from the steep repulsive-potential branch. In quantummechanical scattering, a wave packet approaching the surface exhibits a nonvanishing reflection

A

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany. *To whom correspondence should be addressed. E-mail: [email protected]

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coefficient even when it is in the attractive part of the potential. Thus, despite the force acting toward the surface, there is some probability that the particle will reflect tens of nanometers or more above the surface, without ever colliding with the repulsive potential wall. The probability for this counterintuitive effect, termed quantum reflection, even approaches unity in the lowenergy limit of the incident particle [e.g., (1)]. Quantum reflection from a solid was first observed by Shimizu for ultracold metastable Ne (2) and He (3) atoms. Later, it was also observed with helium atom beams (4, 5). Here, we demonstrate that quantum reflection allows

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11. Y. D. Chong, L. Ge, H. Cao, A. D. Stone, Phys. Rev. Lett. 105, 053901 (2010). 12. See supporting material on Science Online. 13. A. E. Siegman, Phys. Rev. A 39, 1253 (1989). 14. W. A. Hamel, J. P. Woerdman, Phys. Rev. A 40, 2785 (1989). 15. R. H. Yan, R. J. Simes, L. A. Coldren, IEEE Photon. Technol. Lett. 1, 273 (1989). 16. K.-K. Law, R. H. Yan, J. L. Merz, L. A. Coldren, Appl. Phys. Lett. 56, 1886 (1990). 17. K.-K. Law, R. H. Yan, L. A. Coldren, J. L. Merz, Appl. Phys. Lett. 57, 1345 (1990). 18. J. F. Heffernan, M. H. Moloney, J. Hegarty, J. S. Roberts, M. Whitehead, Appl. Phys. Lett. 58, 2877 (1991). 19. K. Kishino et al., IEEE J. Quantum Electron. 27, 2025 (1991). 20. M. S. Ünlü, K. Kishino, H. J. Liaw, H. Morkoç, J. Appl. Phys. 71, 4049 (1992). 21. M. Cai, O. Painter, K. J. Vahala, Phys. Rev. Lett. 85, 74 (2000). 22. A. Yariv, IEEE Photon. Technol. Lett. 14, 483 (2002). 23. J. R. Tischler, M. S. Bradley, V. Bulović, Opt. Lett. 31, 2045 (2006). 24. S. E. Harris, Phys. Today 50, 36 (1997). 25. M. Lipson, J. Lightwave Technol. 23, 4222 (2005). 26. S. Longhi, Phys. Rev. A 82, 031801(R) (2010). 27. Y. D. Chong, L. Ge, A. D. Stone, http://arxiv4.library. cornell.edu/abs/1008.5156v1 (2010). 28. Supported by NSF grants DMR-0808937 and DMR0908437 and by seed funding from the Yale NSF-MRSEC (DMR-0520495). We thank E. Dufresne for use of his laser facility, and D. Miller and J. Bleuse for drawing our attention to the literature on asymmetric Fabry-Perot modulators and resonant cavity-enhanced photodetectors, respectively.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/889/DC1 Materials and Methods Figs. S1 and S2 22 November 2010; accepted 19 January 2011 10.1126/science.1200735

for nondestructive scattering of extremely fragile helium dimers from a ruled reflection grating. The van der Waals–bound dimer of two ground-state helium atoms, He2, is the most fragile ground-state molecule known (6, 7). The binding energy of 10−7 eV leads to an exceptionally large bond length (mean internuclear distance) of 5.2 nm (8). The helium dimer is a quantum system because the probability for He2 to be found in a classically forbidden state, where the internuclear separation is larger than the system’s classical outer turning point, is more than 80% (Fig. 1B). Moreover, because rotational or vibrational excitation leads to dissociation, there are no excited bound states. These distinctive features have made He2 an attractive model system for computational methods in quantum chemistry. Trying to observe nondestructive scattering of He2 from a solid surface appears to be an illconceived experiment. Even at grazing incidence, where the kinetic energy associated with the dimer’s momentum component perpendicular to the surface can be smaller than the binding energy, the dimer is likely to be torn apart by the forces exerted on both atoms in the surface– potential-well region sketched in Fig. 1A. The potential-well depth is typically ~5 meV, hence,

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illumination gives ~35% output. When the phase is adjusted to maximize the scattering, the output reaches ~70%. Although we have demonstrated coherent reduction of absorption in our experiment, this effect should be distinguished from the phenomenon of electromagnetically induced transparency, in which absorption is suppressed by coherently driving the absorbing medium itself (24), instead of by enhancing escape from the cavity by constructive interference, as in our system. Because this optical effect is easily realized in silicon, coherent perfect absorbers may enable novel functionalities in silicon integrated photonic circuits of the type envisioned for nextgeneration optical communications and computing applications (25) as well as for coherent laser spectroscopy. The simplest versions of the device immediately would serve as compact on-chip interferometers, which absorb or scatter the input beams instead of steering them. Although our current CPA operates near the silicon band edge, it should be possible to fabricate devices in which an additional parameter tunes the absorption coefficient independently of l (e.g., by free carrier injection or by optical pumping), allowing one to fix the operating wavelength by design. Direct–band gap semiconductors also are suitable materials for CPAs, assuming that fluorescent emission can be tolerated or avoided in a specific application. Recent theoretical work has proposed a fascinating extension of the CPA concept, suitable for direct–band gap materials: Systems with balanced gain and

loss can function simultaneously as a CPA and as a laser (i.e., as an interferometric amplifierattenuator) (26, 27). The CPA effect is not immediately applicable to photovoltaic or stealth technology because it is a narrow-band effect requiring coherent inputs. More generally, the exact time-reversal symmetry property that relates laser emission to coherent perfect absorption implies that an arbitrarily complicated scattering system can be made to perfectly absorb at discrete frequencies if its imaginary refractive index can be tuned continuously over a reasonable range of values, and if appropriate coherent incident beams can be imposed. Progress in these areas would open up interesting new avenues for future research and applications. References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

E. L. Hahn, Phys. Rev. 80, 580 (1950). G. Bergmann, Phys. Rep. 107, 1 (1984). Y. Kuga, A. Ishimaru, J. Opt. Soc. Am. A 1, 831 (1984). M. P. Van Albada, A. Lagendijk, Phys. Rev. Lett. 55, 2692 (1985). P. E. Wolf, G. Maret, Phys. Rev. Lett. 55, 2696 (1985). C. H. Lewenkopf, H. A. Weidenmuller, Ann. Phys. 212, 53 (1991). M. Fink et al., Rep. Prog. Phys. 63, 1933 (2000). J. de Rosny, M. Fink, Phys. Rev. Lett. 89, 124301 (2002). De Rosny and Fink (8) demonstrated a process of perfect absorption of time-reversed acoustic waves, but this requires a dynamic sink that is driven externally with appropriate amplitude and phase. This process is not related to time-reversed lasing. G. Lerosey, J. de Rosny, A. Tourin, M. Fink, Science 315, 1120 (2007).

Quantum Reflection of He2 Several Nanometers Above a Grating Surface Bum Suk Zhao,* Gerard Meijer, Wieland Schöllkopf Quantum reflection allows an atom or molecule to be reflected from a solid before it reaches the region where it would encounter the repulsive potential of the surface. We observed nondestructive scattering of the helium dimer (He2), which has a binding energy of 10−7 electron volt, from a solid reflection grating. We scattered a beam containing the dimer as well as atomic helium and larger clusters, but could differentiate the dimer by its diffraction angle. Helium dimers are quantum reflected tens of nanometers above the surface, where the surface-induced forces are too weak to dissociate the fragile bond. neutral atom or molecule approaching a solid surface experiences an attractive force caused by the van der Waals atomsurface interaction potential, as sketched in Fig. 1A. In a classical picture, the particle accelerates toward the surface until it scatters back from the steep repulsive-potential branch. In quantummechanical scattering, a wave packet approaching the surface exhibits a nonvanishing reflection

A

Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany. *To whom correspondence should be addressed. E-mail: [email protected]

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coefficient even when it is in the attractive part of the potential. Thus, despite the force acting toward the surface, there is some probability that the particle will reflect tens of nanometers or more above the surface, without ever colliding with the repulsive potential wall. The probability for this counterintuitive effect, termed quantum reflection, even approaches unity in the lowenergy limit of the incident particle [e.g., (1)]. Quantum reflection from a solid was first observed by Shimizu for ultracold metastable Ne (2) and He (3) atoms. Later, it was also observed with helium atom beams (4, 5). Here, we demonstrate that quantum reflection allows

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11. Y. D. Chong, L. Ge, H. Cao, A. D. Stone, Phys. Rev. Lett. 105, 053901 (2010). 12. See supporting material on Science Online. 13. A. E. Siegman, Phys. Rev. A 39, 1253 (1989). 14. W. A. Hamel, J. P. Woerdman, Phys. Rev. A 40, 2785 (1989). 15. R. H. Yan, R. J. Simes, L. A. Coldren, IEEE Photon. Technol. Lett. 1, 273 (1989). 16. K.-K. Law, R. H. Yan, J. L. Merz, L. A. Coldren, Appl. Phys. Lett. 56, 1886 (1990). 17. K.-K. Law, R. H. Yan, L. A. Coldren, J. L. Merz, Appl. Phys. Lett. 57, 1345 (1990). 18. J. F. Heffernan, M. H. Moloney, J. Hegarty, J. S. Roberts, M. Whitehead, Appl. Phys. Lett. 58, 2877 (1991). 19. K. Kishino et al., IEEE J. Quantum Electron. 27, 2025 (1991). 20. M. S. Ünlü, K. Kishino, H. J. Liaw, H. Morkoç, J. Appl. Phys. 71, 4049 (1992). 21. M. Cai, O. Painter, K. J. Vahala, Phys. Rev. Lett. 85, 74 (2000). 22. A. Yariv, IEEE Photon. Technol. Lett. 14, 483 (2002). 23. J. R. Tischler, M. S. Bradley, V. Bulović, Opt. Lett. 31, 2045 (2006). 24. S. E. Harris, Phys. Today 50, 36 (1997). 25. M. Lipson, J. Lightwave Technol. 23, 4222 (2005). 26. S. Longhi, Phys. Rev. A 82, 031801(R) (2010). 27. Y. D. Chong, L. Ge, A. D. Stone, http://arxiv4.library. cornell.edu/abs/1008.5156v1 (2010). 28. Supported by NSF grants DMR-0808937 and DMR0908437 and by seed funding from the Yale NSF-MRSEC (DMR-0520495). We thank E. Dufresne for use of his laser facility, and D. Miller and J. Bleuse for drawing our attention to the literature on asymmetric Fabry-Perot modulators and resonant cavity-enhanced photodetectors, respectively.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/889/DC1 Materials and Methods Figs. S1 and S2 22 November 2010; accepted 19 January 2011 10.1126/science.1200735

for nondestructive scattering of extremely fragile helium dimers from a ruled reflection grating. The van der Waals–bound dimer of two ground-state helium atoms, He2, is the most fragile ground-state molecule known (6, 7). The binding energy of 10−7 eV leads to an exceptionally large bond length (mean internuclear distance) of 5.2 nm (8). The helium dimer is a quantum system because the probability for He2 to be found in a classically forbidden state, where the internuclear separation is larger than the system’s classical outer turning point, is more than 80% (Fig. 1B). Moreover, because rotational or vibrational excitation leads to dissociation, there are no excited bound states. These distinctive features have made He2 an attractive model system for computational methods in quantum chemistry. Trying to observe nondestructive scattering of He2 from a solid surface appears to be an illconceived experiment. Even at grazing incidence, where the kinetic energy associated with the dimer’s momentum component perpendicular to the surface can be smaller than the binding energy, the dimer is likely to be torn apart by the forces exerted on both atoms in the surface– potential-well region sketched in Fig. 1A. The potential-well depth is typically ~5 meV, hence,

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15 = 52 Å

He-He potential [K]

He2-surface potent

200 100 0 -100

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1

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ght

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ce

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impact ionization mass spectrometer, set to a mass of 4 atomic mass units (amu). At this ionmass channel, not only atoms but also clusters can be detected, because the latter can fragment upon electron impact, thereby releasing a He+ ion. From its diffraction angle, the small peak between the intense specular and first-order diffraction peak of the monomer is identified as the first-order diffraction peak of the dimer. Another measurement for identical conditions, but with the detector set to the 8-amu ion-mass channel, is shown in Fig. 2B. At this channel the monomer peaks are absent, and the dimer peaks are expected to be weak because the probability for a dimer to not fragment upon electron-impact ionization is just a few percent (11). In addition to a specular peak, a diffraction peak is again seen at the calculated dimer diffraction angle, thereby confirming the peak assignments. To confirm that the peaks attributed to dimers are not due to other clusters, we took measurements for various stagnation pressures. A diffrac-

blazed grating

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such that its grooves are almost parallel to the plane spanned by the incident and specular beams. The diffraction experiment takes advantage of the atoms and clusters in the beam having the same mean velocity. Thus, the de Broglie wavelengths and, hence, the diffraction angles are inversely proportional to the particle mass, thereby leading to separate diffraction peaks of atoms, dimers, and larger clusters (7). Figure 2A shows a diffraction pattern for P0 = 1 bar measured with the detector, an electron-

He+-Signal [103 counts/s]

four orders of magnitude larger than the dimer binding energy. Consequently, classical surface reflection of He2 has not been observed in 80 years of He beam scattering since the first experiments by Stern and co-workers, who already used a ruled diffraction grating (9) as well as a crystal surface (10). In our experiment, a collimated helium beam, containing atoms as well as small clusters, scatters under grazing incidence from a blazed diffraction grating (Fig. 1C). The orientation of the grating is

slit 1 skimmer

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Fig. 1. (A) Artist’s view of quantum reflection of a helium dimer at the attractive van der Waals surface potential. (B) The He-He interaction potential (13) (black) and the calculated probability function of 4He2 (red) as a function of internuclear separation. (C) Schematic of the experimental setup. The continuous helium beam is formed by free-jet expansion of pure 4He gas at a stagnation temperature T0 = 8.7 K and pressure P0 = 1 to 2 bar through a 5-mm-diameter orifice into vacuum. In the adiabatic expansion, the gas rapidly cools down to a temperature of ~1 mK, where weakly bound dimers and trimers are formed (12). The mean beam velocity of 300 m/s corresponds to de Broglie wavelengths (l) of He, He2, and He3 of 3.32, 1.66, and 1.11 Å, respectively. At an incidence angle qin = 0.39 mrad, the grating-surface normal component of the velocity is 0.12 m/s. This corresponds to a kinetic energy of He2 along the surface normal of only 0.57 neV. Here the incidence angle and the detection angle q are measured with respect to the grating surface plane. The plane ruled blazed grating (Newport 20RG050-600-1) is made out of 6-mm-thick glass with an aluminum coating and has a surface area of 5 cm by 5 cm. It is characterized by a period d = 20 mm and a blaze angle of 14 mrad. The azimuthal orientation of the grating is set to f = 10 mrad. High angular resolution of about 0.12 mrad is achieved by two 20-mm-wide collimation slits upstream of the grating in combination with a 25-mm-wide detector entrance slit. The nth-order diffraction angle qn follows from the grating equation cosqin − cosqn = n (l/d)sinf. www.sciencemag.org

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Fig. 2. Diffraction patterns of helium beams for two different stagnation pressures and measured with two different mass channels of the detector. (A) The 4-amu mass channel, P0 = 1 bar. (The dark trace represents a fivefold magnification of the intensity.) (B) The 8-amu mass channel, P0 = 1 bar. (C) The 8-amu mass channel, P0 = 2 bar. At 4 amu (A), atoms and, due to fragmentation in the detector, also clusters can be detected, whereas at 8 amu (B and C) the atomic component of the beam is absent. The calculated diffraction angles of He, He2, and He3 are indicated by green dash-dotted, red solid, and blue dashed vertical lines, respectively, each labeled by the diffraction order number. The thin dashed black line indicates the 0th-order peak position.

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REPORTS and trimers to reflect tens of nanometers above the actual surface, where surface-induced forces are too feeble to break up even the fragile He2 bond. References and Notes 1. H. Friedrich, G. Jacoby, C. G. Meister, Phys. Rev. A 65, 032902 (2002). 2. F. Shimizu, Phys. Rev. Lett. 86, 987 (2001). 3. H. Oberst, Y. Tashiro, K. Shimizu, F. Shimizu, Phys. Rev. A 71, 052901 (2005). 4. V. Druzhinina, M. DeKieviet, Phys. Rev. Lett. 91, 193202 (2003). 5. B. S. Zhao, S. Schulz, S. Meek, G. Meijer, W. Schöllkopf, Phys. Rev. A 78, 010902 (2008). 6. F. Luo, G. C. McBane, G. Kim, C. F. Giese, W. R. Gentry, J. Chem. Phys. 98, 3564 (1993). 7. W. Schöllkopf, J. P. Toennies, Science 266, 1345 (1994).

Spin Selectivity in Electron Transmission Through Self-Assembled Monolayers of Double-Stranded DNA B. Göhler,1 V. Hamelbeck,1 T. Z. Markus,2 M. Kettner,1 G. F. Hanne,1 Z. Vager,3 R. Naaman,2* H. Zacharias1 In electron-transfer processes, spin effects normally are seen either in magnetic materials or in systems containing heavy atoms that facilitate spin-orbit coupling. We report spin-selective transmission of electrons through self-assembled monolayers of double-stranded DNA on gold. By directly measuring the spin of the transmitted electrons with a Mott polarimeter, we found spin polarizations exceeding 60% at room temperature. The spin-polarized photoelectrons were observed even when the photoelectrons were generated with unpolarized light. The observed spin selectivity at room temperature was extremely high as compared with other known spin filters. The spin filtration efficiency depended on the length of the DNA in the monolayer and its organization. ouble-stranded DNA (dsDNA) is chiral both because of its primary structure and because of its secondary, double helix, structure. Owing to its broken mirror image symmetry, when a charge moves within a chiral system in one direction, it creates a magnetic field. The direction of spin polarization of photoelectrons emitted from nonmagnetic substrates, which exhibit high spinorbit coupling, depends on the handedness of the circularly polarized light. Photoelectrons emitted from a bare gold substrate upon exposure to linearly polarized light would not be expected to show spin polarization. An organic chiral layer on a nonmagnetic metal surface is not expected to be selfmagnetized, and photoelectrons ejected from such a layer with linearly polarized light would also be unpolarized. However, we observed exceptionally high polarization of electrons ejected from surfaces coated with a self-assembled monolayer of dsDNA, independent of the polarization of the incident light. By directly measuring the spin of the transmitted

D

1 Physikalisches Institut, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. 2Department of Chemical Physics, Weizmann Institute, Rehovot 76100, Israel. 3Department of Particle Physics and Astrophysics, Weizmann Institute, Rehovot 76100, Israel.

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

894

electrons with a Mott polarimeter, we found spin polarizations exceeding 60% at room temperature. This observation establishes the prospect of using dsDNA, or other chiral molecules, as a spin filter. Unconventional magnetic properties affecting spin transport have been reported for inorganicinorganic interfaces (1), topological insulators (2), graphene (3, 4), and organic molecules adsorbed on magnetic substrates (5). Organic molecules would seem unlikely candidates for spin-selective transport properties because of their weak spinorbit coupling. However, studies of photoelectrons ejected from gold surfaces covered with selfassembled, organized monolayers of chiral molecules show that the emission intensity depends on the circular polarization of the exciting light (6) as well as the voltage across the layer and its handedness (7, 8). In these studies, the spin of the transmitted electron was not measured directly, and spin-dependent transmission was inferred from the dependence of the total electron transmission on the circular polarization of the incident photons. Furthermore, those studies could not directly determine whether the ejected electrons are highly polarized when the incident photons are unpolarized, or if the effect results simply from circular dichroism, namely, that the absorption of the system depends on the light circular polarization (9).

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8. R. E. Grisenti et al., Phys. Rev. Lett. 85, 2284 (2000). 9. F. Knauer, O. Stern, Z. Phys. 53, 779 (1929). 10. I. Estermann, O. Stern, Z. Phys. 61, 95 (1930). 11. W. Schöllkopf, J. P. Toennies, J. Chem. Phys. 104, 1155 (1996). 12. L. W. Bruch, W. Schöllkopf, J. P. Toennies, J. Chem. Phys. 117, 1544 (2002). 13. K. T. Tang, J. P. Toennies, C. L. Yiu, Phys. Rev. Lett. 74, 1546 (1995). 14. We thank J. R. Manson for insightful discussions. B.S.Z. acknowledges support by the Alexander von Humboldt Foundation and by the Korea Research Foundation Grant funded by the Korean Government (KRF-2005-214-C00188). 25 November 2010; accepted 12 January 2011 10.1126/science.1200911

The sample preparation is similar to that described in (10). Self-assembled dsDNA monolayers are prepared according to standard procedures by depositing dsDNA, which is thiolated on the 3′ end of one of the DNA strands [see Supporting Online Material (10)] on a clean gold substrate (11) (Fig. 1). Four different lengths of dsDNAwere investigated: 26, 40, 50, and 78 base pairs (bp) long. We used either polycrystalline Au or single-crystal Au(111) as substrates. The monolayers were characterized by various methods that ensure the uniformity and reproducibility of the DNA layer (12). The experiments were carried out under ultrahigh-vacuum conditions. Two photoelectron detection schemes were used: an electron time-of-flight instrument that recorded the kinetic energy distribution of the electrons and a Motttype electron polarimeter for spin analysis (figs. S1 to S3). The photoelectrons were ejected by an ultraviolet (UV) laser pulse with photon energy of 5.84 eV, pulse duration of about 200 ps at 20-kHz repetition rate, and a fluence of 150 pJ/cm2. The laser light was incident normal to the sample and was either linearly or circularly polarized. No damage was observed during the course of the spin polarization measurement (~4 hours). For direct polarization measurements, the photoelectrons were guided by an electrostatic 90°bender and subsequent transport optics. Hence, an initial longitudinal spin polarization is converted into a transverse one for analysis. In the electron polarimeter, an electron spin polarization causes a scattering asymmetry A = (IU – IL)/(IU + IL). Here IU,L denotes the count rates in the upper and lower counter in the Mott polarimeter (fig. S1) (13, 14). The transverse polarization is given by P = A/Seff. The analyzing power, the Sherman function (15), was calibrated to be Seff = −(0.229 T 0.011) (fig. S4). We measured the spin polarization parallel to the sample normal and thus parallel to the initial electron velocity. The spin polarization of photoelectrons from a clean Au(111) single crystal and the sign of its orientation depend on the laser polarization (Fig. 2A). An intensity asymmetry of A = (5.03 T 1.1)% was observed. Combined with the Sherman function Seff, an electron spin polarization of P = −(22 T 5)% was determined for emission from

www.sciencemag.org

Downloaded from www.sciencemag.org on February 17, 2011

tion pattern for P0 = 2 bar recorded at a mass of 8 amu is shown in Fig. 2C. In this measurement, additional peaks are present, and their positions agree nicely with the calculated diffraction angles of the helium trimer. These findings are fully consistent with previous observations that more trimers are formed at increased stagnation pressure (12). In addition, it is known that trimers are more efficiently detected at a mass of 8 amu than dimers (11), further enhancing the trimer peaks relative to those of the dimer. The observed nondestructive scattering of He2 as well as He3 from a reflection grating exemplifies the peculiar nature of a quantum-mechanical impact. Quantum reflection causes helium dimers

REPORTS and trimers to reflect tens of nanometers above the actual surface, where surface-induced forces are too feeble to break up even the fragile He2 bond. References and Notes 1. H. Friedrich, G. Jacoby, C. G. Meister, Phys. Rev. A 65, 032902 (2002). 2. F. Shimizu, Phys. Rev. Lett. 86, 987 (2001). 3. H. Oberst, Y. Tashiro, K. Shimizu, F. Shimizu, Phys. Rev. A 71, 052901 (2005). 4. V. Druzhinina, M. DeKieviet, Phys. Rev. Lett. 91, 193202 (2003). 5. B. S. Zhao, S. Schulz, S. Meek, G. Meijer, W. Schöllkopf, Phys. Rev. A 78, 010902 (2008). 6. F. Luo, G. C. McBane, G. Kim, C. F. Giese, W. R. Gentry, J. Chem. Phys. 98, 3564 (1993). 7. W. Schöllkopf, J. P. Toennies, Science 266, 1345 (1994).

Spin Selectivity in Electron Transmission Through Self-Assembled Monolayers of Double-Stranded DNA B. Göhler,1 V. Hamelbeck,1 T. Z. Markus,2 M. Kettner,1 G. F. Hanne,1 Z. Vager,3 R. Naaman,2* H. Zacharias1 In electron-transfer processes, spin effects normally are seen either in magnetic materials or in systems containing heavy atoms that facilitate spin-orbit coupling. We report spin-selective transmission of electrons through self-assembled monolayers of double-stranded DNA on gold. By directly measuring the spin of the transmitted electrons with a Mott polarimeter, we found spin polarizations exceeding 60% at room temperature. The spin-polarized photoelectrons were observed even when the photoelectrons were generated with unpolarized light. The observed spin selectivity at room temperature was extremely high as compared with other known spin filters. The spin filtration efficiency depended on the length of the DNA in the monolayer and its organization. ouble-stranded DNA (dsDNA) is chiral both because of its primary structure and because of its secondary, double helix, structure. Owing to its broken mirror image symmetry, when a charge moves within a chiral system in one direction, it creates a magnetic field. The direction of spin polarization of photoelectrons emitted from nonmagnetic substrates, which exhibit high spinorbit coupling, depends on the handedness of the circularly polarized light. Photoelectrons emitted from a bare gold substrate upon exposure to linearly polarized light would not be expected to show spin polarization. An organic chiral layer on a nonmagnetic metal surface is not expected to be selfmagnetized, and photoelectrons ejected from such a layer with linearly polarized light would also be unpolarized. However, we observed exceptionally high polarization of electrons ejected from surfaces coated with a self-assembled monolayer of dsDNA, independent of the polarization of the incident light. By directly measuring the spin of the transmitted

D

1 Physikalisches Institut, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. 2Department of Chemical Physics, Weizmann Institute, Rehovot 76100, Israel. 3Department of Particle Physics and Astrophysics, Weizmann Institute, Rehovot 76100, Israel.

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

894

electrons with a Mott polarimeter, we found spin polarizations exceeding 60% at room temperature. This observation establishes the prospect of using dsDNA, or other chiral molecules, as a spin filter. Unconventional magnetic properties affecting spin transport have been reported for inorganicinorganic interfaces (1), topological insulators (2), graphene (3, 4), and organic molecules adsorbed on magnetic substrates (5). Organic molecules would seem unlikely candidates for spin-selective transport properties because of their weak spinorbit coupling. However, studies of photoelectrons ejected from gold surfaces covered with selfassembled, organized monolayers of chiral molecules show that the emission intensity depends on the circular polarization of the exciting light (6) as well as the voltage across the layer and its handedness (7, 8). In these studies, the spin of the transmitted electron was not measured directly, and spin-dependent transmission was inferred from the dependence of the total electron transmission on the circular polarization of the incident photons. Furthermore, those studies could not directly determine whether the ejected electrons are highly polarized when the incident photons are unpolarized, or if the effect results simply from circular dichroism, namely, that the absorption of the system depends on the light circular polarization (9).

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8. R. E. Grisenti et al., Phys. Rev. Lett. 85, 2284 (2000). 9. F. Knauer, O. Stern, Z. Phys. 53, 779 (1929). 10. I. Estermann, O. Stern, Z. Phys. 61, 95 (1930). 11. W. Schöllkopf, J. P. Toennies, J. Chem. Phys. 104, 1155 (1996). 12. L. W. Bruch, W. Schöllkopf, J. P. Toennies, J. Chem. Phys. 117, 1544 (2002). 13. K. T. Tang, J. P. Toennies, C. L. Yiu, Phys. Rev. Lett. 74, 1546 (1995). 14. We thank J. R. Manson for insightful discussions. B.S.Z. acknowledges support by the Alexander von Humboldt Foundation and by the Korea Research Foundation Grant funded by the Korean Government (KRF-2005-214-C00188). 25 November 2010; accepted 12 January 2011 10.1126/science.1200911

The sample preparation is similar to that described in (10). Self-assembled dsDNA monolayers are prepared according to standard procedures by depositing dsDNA, which is thiolated on the 3′ end of one of the DNA strands [see Supporting Online Material (10)] on a clean gold substrate (11) (Fig. 1). Four different lengths of dsDNAwere investigated: 26, 40, 50, and 78 base pairs (bp) long. We used either polycrystalline Au or single-crystal Au(111) as substrates. The monolayers were characterized by various methods that ensure the uniformity and reproducibility of the DNA layer (12). The experiments were carried out under ultrahigh-vacuum conditions. Two photoelectron detection schemes were used: an electron time-of-flight instrument that recorded the kinetic energy distribution of the electrons and a Motttype electron polarimeter for spin analysis (figs. S1 to S3). The photoelectrons were ejected by an ultraviolet (UV) laser pulse with photon energy of 5.84 eV, pulse duration of about 200 ps at 20-kHz repetition rate, and a fluence of 150 pJ/cm2. The laser light was incident normal to the sample and was either linearly or circularly polarized. No damage was observed during the course of the spin polarization measurement (~4 hours). For direct polarization measurements, the photoelectrons were guided by an electrostatic 90°bender and subsequent transport optics. Hence, an initial longitudinal spin polarization is converted into a transverse one for analysis. In the electron polarimeter, an electron spin polarization causes a scattering asymmetry A = (IU – IL)/(IU + IL). Here IU,L denotes the count rates in the upper and lower counter in the Mott polarimeter (fig. S1) (13, 14). The transverse polarization is given by P = A/Seff. The analyzing power, the Sherman function (15), was calibrated to be Seff = −(0.229 T 0.011) (fig. S4). We measured the spin polarization parallel to the sample normal and thus parallel to the initial electron velocity. The spin polarization of photoelectrons from a clean Au(111) single crystal and the sign of its orientation depend on the laser polarization (Fig. 2A). An intensity asymmetry of A = (5.03 T 1.1)% was observed. Combined with the Sherman function Seff, an electron spin polarization of P = −(22 T 5)% was determined for emission from

www.sciencemag.org

Downloaded from www.sciencemag.org on February 17, 2011

tion pattern for P0 = 2 bar recorded at a mass of 8 amu is shown in Fig. 2C. In this measurement, additional peaks are present, and their positions agree nicely with the calculated diffraction angles of the helium trimer. These findings are fully consistent with previous observations that more trimers are formed at increased stagnation pressure (12). In addition, it is known that trimers are more efficiently detected at a mass of 8 amu than dimers (11), further enhancing the trimer peaks relative to those of the dimer. The observed nondestructive scattering of He2 as well as He3 from a reflection grating exemplifies the peculiar nature of a quantum-mechanical impact. Quantum reflection causes helium dimers

REPORTS ization for electrons emitted from the molybdenum sample holder was zero for circular as well as linear laser polarization (fig. S5).

linear polarized laser radiation

polarized electrons transmitted through chiral layer

monolayer of dsDNA

unpolarized photoelectrons (linear polarized radiation)

The spin polarization of electrons from the same Au(111) single crystal, transmitted through a self-assembled monolayer of 50-bp dsDNA, was measured for excitation with clockwise (cw) (Fig. 2B) and counterclockwise (ccw) (Fig. 2D) circularly polarized light, and linearly (Fig. 2C) polarized light. Strongly spin-polarized electrons with average polarizations of –(35 T 3)%, −(29 T 3)%, and –(31 T 4)%, respectively, were observed, and all the spin polarizations showed the same negative sign, independent of the light polarization. The ordered monolayer of dsDNA acts as a spin filter for photoelectrons emitted from the gold surface. A systematic study of the dependence of the degree of electron spin polarization on the length of the dsDNA was carried out on polycrystalline gold substrates (fig. S7). For a bare polycrystralline gold substrate, a spin polarization of essentially zero is observed (fig. S8). When a polycrystalline substrate was coated with a self-assembled monolayer of dsDNA, a strong spin polarization was observed. The spin polarization observed for electrons transmitted through a monolayer of 78-bp DNA is shown for cw (Fig. 3A) and ccw (Fig. 3C) circularly polarized light, and for linearly (Fig. 3B) polarized light. The average polarizations are P = −(55 T 7)%, −(61 T 6)%, and −(57 T 6) %, respectively, again nearly independent of the light polarization. The observed spin polarization of −61% translates into

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the single-crystal substrate. As expected, when the laser was polarized linearly, no spin polarization was observed. For reference, the spin polar-

gold substrate

Fig. 1. A scheme describing the monolayer of dsDNA as spin filter. Unpolarized electrons are ejected from the gold substrate by a linearly polarized light. Most of the electrons transmitted through the DNA are polarized with their spin aligned antiparallel to their velocity. The electrons that are not transmitted are captured by the DNA and tunnel back to the grounded substrate within the time period between two laser pulses.

Fig. 2. (A) The polarized electron signal measured for electrons ejected from bare Au(111) with clockwise (cw) (green) and counterclockwise (ccw) (red) circularly polarized light, and with linearly polarized light (blue). The polarization is −22, +22, and 0%, respectively. The longitudinal spin polarization is measured; the spin is oriented parallel (green) or antiparallel (red) to the direction of propagation of the electron. Spin polarization of electrons transmitted through 50-bp dsDNA/Au(111). The polarizations were −35, −31, and −29% for light polarized cw circularly [(B), green], linearly [(C), blue], and ccw circularly [(D), red], respectively.

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Fig. 3. The photoelectron polarization as measured for electrons ejected from a poly(Au)-coated substrate with a monolayer of 78-bp dsDNA. For the cw circularly polarized light, the electron polarization is –(54.5 T 7.0)% [(A), green]; for the linearly polarized light, the polarization is –(57.2 T 5.9)% [(B), blue]; and for the ccw polarized laser, the electron polarization is –(60.8 T 5.8)% [(C), red]. SCIENCE

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Longitudinal spin polarization (%)

Fig. 4. The electron spin Helical turns (approx.) polarization for various 0 1 2 3 4 5 6 7 8 monolayers of DNA, consisting of different numccw circ. pol. light bers of double-stranded 40 linear pol. light base pairs (filled circles), cw circ. pol. light monolayers of ssDNA (open diamonds), sample of dsDNA damaged by UV light 20 (open triangles), and uncleaned bare polycrystalline gold substrate (open circles). Measurements 0 were conducted with cw, ccw, and linearly polarized light (green, red, and blue, respectively). For clarity, -20 the symbols for the different light polarizations are offset by plus and minus 1 bp. Whereas for the -40 dsDNA, on average, the polarization increases with the length of the DNA, no polarization is obtained -60 for the ssDNA. For the dsDNA, the polarization increases slightly when the electrons are injected with -80 ccw circularly polarized 0 10 20 30 40 50 60 70 80 light, because the elecNumber of base pairs trons injected into the layer are spin polarized with polarization that coincides with the spin selectivity of the monolayer. The error bars represent the SD resulting from variation between the different monolayers measured. a ratio of 4.1:1 for left- to right-handed spins in the transmitted electron flux. In Fig. 4, the electron spin polarization is presented for four different monolayers of dsDNA of different lengths. The results of 40 different experiments on 12 different samples are shown. Increasing the length of the dsDNA tends to increase the absolute value of electron spin polarization. Also shown in Fig. 4 are polarization results when the sample was damaged by UV radiation and when the gold was coated by a monolayer of single-stranded DNA. In these latter cases, no net spin selectivity could be observed. The results presented here indicate that wellorganized self-assembled monolayers of dsDNA on Au act as very efficient spin filters. The spin selectivity depends on the organization of the molecules, and within the range of DNA length studied, the selectivity increases with its length. Even the longest molecules that we used are still shorter than the persistence length of the DNA, which is the minimal length where notable changes from rigid rods are observed. Hence, the DNA oligomers studied here are rigid and the monolayer can be visualized as consisting of rigid chiral rods closely packed together (Fig. 1). In the case of a monolayer made from single-stranded DNA (ssDNA), the molecules are more floppy and do not form rigid close-packed monolayers (8), and indeed no spin selectivity is observed. Because the photon energy is lower than the ionization energy of the DNA and the laser intensity is low, the photoelectrons all originate from

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the gold substrate. In addition, less than 0.1% of the incident light is absorbed in the layer, even under resonance conditions. The low intensities and weak absorbance ensure further that nonlinear excitation processes do not occur in the DNA layer. Several groups have reported on experiments in which electrons were transmitted through freestanding (16, 17) or supported (18–21) thin ferromagnetic films that acted as a spin filter. In these cases and for low-energy electrons, the selectivity was about 25% (17). In these experiments, the spin polarization can be explained by inelastic electron scattering involving unoccupied d states above the Fermi level. The scattering rate for minority spin electrons is then enhanced with respect to that of majority spin electrons because of an excess of minority spin holes (22, 23). In those experiments, the polarization decreased sharply as a function of collision energies, owing to the spin dilution by secondary electrons (20). In the present work, the polarization is energy independent within the energy range (Ekin = 0 to 1.2 eV) studied. Although no extensive optimization has been performed and polarized light is not needed, the polarization achieved in the present system is almost as high as that obtained by photoemission with circularly polarized light from artificially strained GaAs substrate (70 to 80%) (24). The mechanism of how charge transport or charge redistribution through chiral systems generates a magnetic field is elementary; however,

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this magnetism is transient and ends when the charge flow stops. A possible way to transform transient charge flow into permanent magnetism is by spin-orbit coupling that converts the angular momenta of the electrons into spin alignment. Spin-orbit coupling in hydrocarbons is commonly believed to be very weak, and no appreciable spin alignment is expected. Indeed, the interaction of spin-polarized electrons with chiral molecules has been studied (25). When these electrons were scattered from the gas phase and thus randomly oriented chiral molecules, only a very small preference on the order of 10−4 of one spin orientation over the other was found, and only when a heavy metal atom with substantial spin-orbit interaction was present in the molecules. In contrast to these gas-phase studies, electrons transmitted through organized monolayers of dipolar-chiral molecules display a large dependence on the handedness of the molecules (6–8). Several models have been presented for explaining those observations; however, they failed to provide quantitative agreement with the experimental observations (26, 27). If the effect described here is caused by a pseudo-magnetic field within the monolayer, it means that a field exceeding a few hundred Tesla must be present. Similar arguments have recently been given for the observations in strained graphene (4). Hence, this study identifies the need to revise our understanding of the interaction of electrons and their spin with chiral organic monolayers. The electrons that are filtered out by the layer are captured by the DNA and tunnel back to the grounded substrate. The present study indicates that the capturing of low-energy electrons by DNA is highly spin selective. The role of spin in electron-biomolecule interactions has usually been ignored. Our results indicate that the spin may play an important role in electron-DNA interactions in biological systems and open new possibilities for explaining the origin of enantioselectivity in nature. Finally, based on the phenomenon observed, it is possible to apply self-assembled monolayers of chiral molecules as very efficient spin filters at room temperature for spintronic applications (28–30). References and Notes 1. A. Brinkman et al., Nat. Mater. 6, 493 (2007). 2. X.-L. Qi, S.-C. Zhang, Phys. Today 63, 33 (2010). 3. Y.-W. Son, M. L. Cohen, S. G. Louie, Nature 444, 347 (2006). 4. N. Levy et al., Science 329, 544 (2010). 5. J. Brede et al., Phys. Rev. Lett. 105, 047204 (2010). 6. K. Ray, S. P. Ananthavel, D. H. Waldeck, R. Naaman, Science 283, 814 (1999). 7. R. Naaman, Z. Vager, MRS Bull. 35, 429 (2010). 8. S. G. Ray, S. S. Daube, G. Leitus, Z. Vager, R. Naaman, Phys. Rev. Lett. 96, 036101 (2006). 9. We chose to investigate in this study longer DNA because of its improved stability, as double helix, as compared to the shorter oligomers studied in (8). 10. T. Aqua, R. Naaman, S. S. Daube, Langmuir 19, 10573 (2003). 11. H. Ron, S. Matlis, I. Rubinstein, Langmuir 14, 1116 (1998). 12. T. Z. Markus et al., J. Am. Chem. Soc. 131, 89 (2009). 13. N. F. Mott, Proc. R. Soc. A 124, 425 (1929). 14. N. F. Mott, Proc. R. Soc. A 135, 429 (1932). 15. A. Gellrich, J. Kessler, Phys. Rev. A 43, 204 (1991). 16. D. Oberli, R. Burgermeister, S. Riesen, W. Weber, H. C. Siegmann, Phys. Rev. Lett. 81, 4228 (1998).

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REPORTS

REPORTS 23. 24. 25. 26. 27. 28. 29. 30.

H. J. Drouhin, Phys. Rev. B 56, 14886 (1997). T. Maruyama et al., Phys. Rev. Lett. 66, 2376 (1991). S. Mayer, J. Kessler, Phys. Rev. Lett. 74, 4803 (1995). S. S. Skourtis, D. N. Beratan, R. Naaman, A. Nitzan, D. H. Waldeck, Phys. Rev. Lett. 101, 238103 (2008). S. Yeganeh, M. A. Ratner, E. Medina, V. Mujica, J. Chem. Phys. 131, 014707 (2009). G. A. Prinz, Science 282, 1660 (1998). S. A. Wolf et al., Science 294, 1488 (2001). D. D. Awschalom, M. E. Flatté, Nat. Phys. 3, 153 (2007).

Capillary Forces in Suspension Rheology Erin Koos* and Norbert Willenbacher The rheology of suspensions (solid particles dispersed in a fluid) is controlled primarily through the volume fraction of solids. We show that the addition of small amounts of a secondary fluid, immiscible with the continuous phase of the suspension, causes agglomeration due to capillary forces and creates particle networks, dramatically altering the bulk rheological behavior from predominantly viscous or weakly elastic to highly elastic or gel-like. This universal phenomenon is observed for a rich variety of particle/liquid systems, independent of whether the second liquid wets the particles better or worse than the primary liquid. These admixtures form stable suspensions where settling would otherwise occur and may serve as a precursor for microporous polymer foams, or lightweight ceramics. he rheology and flow of suspensions is usually controlled by the interplay between the attractive van der Waals forces, repulsive electrostatic forces, or steric interactions among particles hydrodynamic interactions and Brownian forces (1–6). Capillary forces, which play a dominant role in wet granular materials, can also be an important factor in suspensions. In granular media, the addition of water, either directly or due to aging in a humid environment (7, 8), is associated with an increase in the angle of repose and grain cohesiveness (9–11). The water wets the grains, creating a network of grains connected by pendular bridges, and allows, for example, the creation of complex structures such as sandcastles (12, 13). In suspensions, this behavior can be reproduced through the addition of small amounts of a second immiscible fluid that preferentially wets the solid particles (14–16) and creates pendular bridges between particles causing the agglomeration of individual particles and, if the volume fraction of solids is sufficient, creates a network of particles within the bulk fluid. The addition of this secondary “binder” fluid will cause an increase in sedimentation volume (17–19), indicating network formation within the suspension. This agglomeration of particles has been used to separate solids from bulk fluid (20) in coal and ore preparation (21, 22), to separate oil sands, and for dye-pigment preparation (23). This state, in which the secondary fluid preferentially wets the particles, is termed the “pendular” state because of the pendular bridges formed between particles. This

T

Institute for Mechanical Process Engineering and Mechanics, Karlsruhe Institute of Technology, Gotthard-Franz-Straße 3, Building 50.31, 76131 Karlsruhe, Germany. *To whom correspondence should be addressed. E-mail: [email protected]

state is analogous to the pendular state in wet granular media in which the fluid saturation is small and is primarily responsible for the granulation of powders (24, 25). In addition to particle agglomeration caused by the addition of a binder fluid in the pendular state, one can imagine a situation where the second immiscible fluid does not preferentially wet the particles. In this state, though the second fluid is attached to the particles and agglomeration still

31. T.Z.M. and R.N. acknowledge the partial support from the Israel Science Foundation.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/894/DC1 Materials and Methods Figs. S1 to S8 References 20 October 2010; accepted 20 December 2010 10.1126/science.1199339

occurs, there is no pendular bridge formed between the particles. The addition of the secondary fluid is able to agglomerate the particles and create sample-spanning network structures due to the strong capillary force from the bulk wetting fluid. This state is analogous to the capillary state in wet granular media, where almost all of the pores between particles are filled with water (or another wetting fluid) (26, 27). In wet granular materials, strong cohesive strength is observed slightly below complete saturation of the solids by the wetting fluid. In these suspensions, the secondary, preferentially nonwetting fluid is playing the part of the unfilled voids in wet granular materials where the saturation by the preferentially wetting fluid is high. In our analogy to wet granular materials, we term these admixtures “capillary” suspensions. Despite how well the analogy describes the behavior—and the use of capillary forces in suspensions for solid/liquid separation for more than a century (20)—this phenomenon has not been considered with respect to rheology and formulation of stable suspensions at particle loadings substantially lower than dense pack-

0.00% wt. S = 1.000

0.10% wt. S = 0.999

0.20% wt. S = 0.997

0.30% wt. S = 0.996

0.40 % wt. S = 0.995

0.50% wt. S = 0.993

Downloaded from www.sciencemag.org on February 17, 2011

17. Y. Lassailly, H. Drouhin, A. van der Sluijs, G. Lampel, C. Marliere, Phys. Rev. B 50, 13054 (1994). 18. D. P. Pappas et al., Phys. Rev. Lett. 66, 504 (1991). 19. C. Gröbli, D. Guarisco, S. Frank, F. Meier, Phys. Rev. B 51, 2945 (1995). 20. M. Getzlaff, J. Bansmann, G. Schönhense, Solid State Commun. 87, 467 (1993). 21. C. Cacho, Y. Lassailly, H.-J. Drouhin, G. Lampel, J. Peretti, Phys. Rev. Lett. 88, 066601 (2002). 22. G. Schönhense, H. C. Siegmann, Ann. Phys. (Leipzig) 2, 465 (1993).

Fig. 1. The transition from weakly elastic, fluidlike behavior to highly elastic, gel-like behavior is visible with the addition of small amounts of water to a suspension of hydrophobically modified calcium carbonate (Socal, r = 0.8 mm, f = 0.111) in DINP. The wetting angle between the solid and water in DINP is q = 139.2°, and S is the percentage of the total liquid volume occupied by preferentially wetting fluid DINP (31).

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REPORTS 23. 24. 25. 26. 27. 28. 29. 30.

H. J. Drouhin, Phys. Rev. B 56, 14886 (1997). T. Maruyama et al., Phys. Rev. Lett. 66, 2376 (1991). S. Mayer, J. Kessler, Phys. Rev. Lett. 74, 4803 (1995). S. S. Skourtis, D. N. Beratan, R. Naaman, A. Nitzan, D. H. Waldeck, Phys. Rev. Lett. 101, 238103 (2008). S. Yeganeh, M. A. Ratner, E. Medina, V. Mujica, J. Chem. Phys. 131, 014707 (2009). G. A. Prinz, Science 282, 1660 (1998). S. A. Wolf et al., Science 294, 1488 (2001). D. D. Awschalom, M. E. Flatté, Nat. Phys. 3, 153 (2007).

Capillary Forces in Suspension Rheology Erin Koos* and Norbert Willenbacher The rheology of suspensions (solid particles dispersed in a fluid) is controlled primarily through the volume fraction of solids. We show that the addition of small amounts of a secondary fluid, immiscible with the continuous phase of the suspension, causes agglomeration due to capillary forces and creates particle networks, dramatically altering the bulk rheological behavior from predominantly viscous or weakly elastic to highly elastic or gel-like. This universal phenomenon is observed for a rich variety of particle/liquid systems, independent of whether the second liquid wets the particles better or worse than the primary liquid. These admixtures form stable suspensions where settling would otherwise occur and may serve as a precursor for microporous polymer foams, or lightweight ceramics. he rheology and flow of suspensions is usually controlled by the interplay between the attractive van der Waals forces, repulsive electrostatic forces, or steric interactions among particles hydrodynamic interactions and Brownian forces (1–6). Capillary forces, which play a dominant role in wet granular materials, can also be an important factor in suspensions. In granular media, the addition of water, either directly or due to aging in a humid environment (7, 8), is associated with an increase in the angle of repose and grain cohesiveness (9–11). The water wets the grains, creating a network of grains connected by pendular bridges, and allows, for example, the creation of complex structures such as sandcastles (12, 13). In suspensions, this behavior can be reproduced through the addition of small amounts of a second immiscible fluid that preferentially wets the solid particles (14–16) and creates pendular bridges between particles causing the agglomeration of individual particles and, if the volume fraction of solids is sufficient, creates a network of particles within the bulk fluid. The addition of this secondary “binder” fluid will cause an increase in sedimentation volume (17–19), indicating network formation within the suspension. This agglomeration of particles has been used to separate solids from bulk fluid (20) in coal and ore preparation (21, 22), to separate oil sands, and for dye-pigment preparation (23). This state, in which the secondary fluid preferentially wets the particles, is termed the “pendular” state because of the pendular bridges formed between particles. This

T

Institute for Mechanical Process Engineering and Mechanics, Karlsruhe Institute of Technology, Gotthard-Franz-Straße 3, Building 50.31, 76131 Karlsruhe, Germany. *To whom correspondence should be addressed. E-mail: [email protected]

state is analogous to the pendular state in wet granular media in which the fluid saturation is small and is primarily responsible for the granulation of powders (24, 25). In addition to particle agglomeration caused by the addition of a binder fluid in the pendular state, one can imagine a situation where the second immiscible fluid does not preferentially wet the particles. In this state, though the second fluid is attached to the particles and agglomeration still

31. T.Z.M. and R.N. acknowledge the partial support from the Israel Science Foundation.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/894/DC1 Materials and Methods Figs. S1 to S8 References 20 October 2010; accepted 20 December 2010 10.1126/science.1199339

occurs, there is no pendular bridge formed between the particles. The addition of the secondary fluid is able to agglomerate the particles and create sample-spanning network structures due to the strong capillary force from the bulk wetting fluid. This state is analogous to the capillary state in wet granular media, where almost all of the pores between particles are filled with water (or another wetting fluid) (26, 27). In wet granular materials, strong cohesive strength is observed slightly below complete saturation of the solids by the wetting fluid. In these suspensions, the secondary, preferentially nonwetting fluid is playing the part of the unfilled voids in wet granular materials where the saturation by the preferentially wetting fluid is high. In our analogy to wet granular materials, we term these admixtures “capillary” suspensions. Despite how well the analogy describes the behavior—and the use of capillary forces in suspensions for solid/liquid separation for more than a century (20)—this phenomenon has not been considered with respect to rheology and formulation of stable suspensions at particle loadings substantially lower than dense pack-

0.00% wt. S = 1.000

0.10% wt. S = 0.999

0.20% wt. S = 0.997

0.30% wt. S = 0.996

0.40 % wt. S = 0.995

0.50% wt. S = 0.993

Downloaded from www.sciencemag.org on February 17, 2011

17. Y. Lassailly, H. Drouhin, A. van der Sluijs, G. Lampel, C. Marliere, Phys. Rev. B 50, 13054 (1994). 18. D. P. Pappas et al., Phys. Rev. Lett. 66, 504 (1991). 19. C. Gröbli, D. Guarisco, S. Frank, F. Meier, Phys. Rev. B 51, 2945 (1995). 20. M. Getzlaff, J. Bansmann, G. Schönhense, Solid State Commun. 87, 467 (1993). 21. C. Cacho, Y. Lassailly, H.-J. Drouhin, G. Lampel, J. Peretti, Phys. Rev. Lett. 88, 066601 (2002). 22. G. Schönhense, H. C. Siegmann, Ann. Phys. (Leipzig) 2, 465 (1993).

Fig. 1. The transition from weakly elastic, fluidlike behavior to highly elastic, gel-like behavior is visible with the addition of small amounts of water to a suspension of hydrophobically modified calcium carbonate (Socal, r = 0.8 mm, f = 0.111) in DINP. The wetting angle between the solid and water in DINP is q = 139.2°, and S is the percentage of the total liquid volume occupied by preferentially wetting fluid DINP (31).

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REPORTS

101 0.00% 0.05% 0.08%

100 10-2

10-1 100 101 ω (rad s-1)

0.10% 0.13% 0.15% 0.20% 102

103

101

100 0.988

φ = 0.112 φ = 0.144 φ = 0.173 φ = 0.201 φ = 0.227 φ = 0.251 φ = 0.295 0.992 0.996 Fraction of wetting liquid, S

Fig. 2. Effect of added water on an oil-based suspension (capillary state). (A) Magnitude of the complex shear modulus for Socal in silicone oil (AK1000, f = 0.173) for varying weight percentages of water showing the transition from fluidlike to highly elastic, gel-like behavior. |G*|, magnitude of the complex shear modulus; w, angular frequency; rad, radians. (B) Normalized yield stress ing. Such capillary suspensions, which include a small fraction of a preferentially nonwetting fluid, have recently been investigated because of their influence on the aging of polyvinyl chloride (PVC) plastisol mixtures [PVC particles suspended in a plasticizer such as diisononyl phthalate (DINP)] caused by the uptake of water (28–30), but without a clear understanding that capillary forces were the cause of the observed transition. These capillary suspensions are quite sensitive to the addition of the secondary fluid, as seen in Fig. 1 and movie S1. The transition from a fluidlike state to a gel-like state occurs with as little as 0.2 weight percent (wt %) of the second fluid in suspensions with a volume of solids f as low as 10%. We took measurements with the use of several different fluid/particle combinations with particles of a wide variety of sizes and shapes, as shown in fig. S1 and described in table S1 (31). We measured contact angles for each fluid in air and then used the results to calculate the contact angle that the secondary fluid makes against the solid particles in the bulk fluid environment using surface energy components (31–33). These contact angles fall into two groups: (i) those smaller than 90°, where the mixture is in the pendular state, and (ii) those greater than 90°, where the mixture is in the capillary state. In the capillary state, we used the hydrophobically modified calcium carbonate (Socal, Solvay Advanced Functional Minerals, Salin de Giraud, France) in DINP or silicone oil as a model system. The transition between the weakly elastic, fluidlike behavior to highly elastic, gellike behavior can be observed through measurements of the shear modulus conducted using a small-amplitude oscillatory shear (Fig. 2A). Without added water, the magnitude of the complex shear modulus increases linearly with increasing frequency, but as small weight percentages of water are added to the suspension of Socal in silicone oil, the shear modulus begins to increase at low frequencies. At 0.20 wt % of added water, the mixture becomes highly elastic, and

898

101

10

0.988

1

104

φ = 0.227 φ = 0.251 φ = 0.295

0.992 0.996 Fraction of wetting liquid, S

1

the complex shear modulus is nearly constant with frequency. We varied the amount of water (the secondary fluid) between 0.0 and 0.8% by mass, corresponding to a saturation S of the wetting fluid between 1.00 and 0.99. Each mixture was characterized in steady shear by measuring the yield stress (31) and steady-state viscosity, as shown in Fig. 2, B and C, respectively. In Fig. 2B, the yield stress when water was present, normalized against the yield stress measured when no water is present (S = 1), is displayed as a function of the wetting-liquid fraction. These mixtures are very sensitive to the addition of water, increasing the yield stress by more than two orders of magnitude over the singlefluid suspensions. This increase in the normalized yield stress is independent of the volume fraction of solids. Each volume-fraction experiment shows a characteristic curve with the most rapid change in yield stress occurring at a fraction of 0.997. Below a wetting fluid fraction of 0.995, the curves plateau. As with the yield stress, the viscosity (Fig. 2C) evaluated at a single shear rate (˙g ¼ 1 s−1) also increases by more than one order of magnitude from the S = 1 value. The transition from the lowviscosity, fluidlike behavior to the high-viscosity, gel-like behavior occurs at a saturation of the SCIENCE

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and (C) normalized viscosity at a single shear rate of g˙ = 1 s−1, for various volume fractions of Socal in DINP. The yield stress in (B) and viscosity in (C) are normalized by the value at S = 1, where only DINP is present. The wetting angles for the water are q = 122.8° (A) and q = 139.2° [(B) and (C)]; see table S1. Error bars in (B) indicate repeatability error.

Fig. 3. Dependence of the yield stress on the fraction of the wetting liquid. For this PVC (Vinnolit C12/62V), the increase in yield stress is greatest in the capillary state, in which water is the primary fluid and DINP is the secondary fluid. For the hematite, the increase in yield stress is greatest in the pendular state, in which DINP is the primary fluid and water is the secondary fluid. The saturation is defined as the fraction of total fluid volume occupied by the preferentially wetting fluid. Error bars indicate repeatability error.

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φ = 0.112 φ = 0.144 φ = 0.173 φ = 0.201

PVC in water/DINP Hematite in DINP/water

0.2 0.4 0.6 0.8 Fraction of wetting liquid, S

1

wetting fluid of 0.997. This transition in viscosity occurs at the same saturation point as the yield stress shows a dramatic increase. Furthermore, the viscosity measurements show the same coincidence of the curves with varying volume fraction. This change in the normalized viscosity is only slightly more than one order of magnitude, so it is less pronounced than the increase in the normalized yield stress. The gel-like transition in capillary suspensions dramatically increases the yield stress and viscosity above the corresponding values from the single-fluid suspensions. The full dependence of yield stress on the saturation, the fraction of available volume filled by the wetting fluid, is shown in Fig. 3 for PVC (C12/62V, Vinnolit, Ismaning, Germany) and hematite, both in various percentages of water and DINP. For the PVC system, the yield stress increases slightly in the pendular state (where DINP is the primary fluid) and increases greatly in the capillary state (where water is the primary fluid). The greatest increase in the yield stress occurs at a saturation of S = 0.7, in the capillary state, for the PVC as well as many of the other liquid/particle combinations that we tested (for example, Socal and hydrophobic glass). For hematite and similar systems (for instance, silica and hydrophilic glass), the transition in the pendular state is more pronounced

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102

102

Normalized Viscosity

|G*| (Pa)

103

Yield Stress (Pa)

104

10-1 -3 10

C 102

B 103 Normalized Yield Stress

A 105

REPORTS

A

B

40 µm

40 µm

C

300 µm

Fig. 5. PVC foam and solid. Mixture of PVC in water with added DINP (f = 0.20, S = 0.973) shown (A) without any treatment and (B) after the water is removed under vacuum (100 mbar for 24 hours) with additional heating (70°C for 6 hours). (C) Micrograph of the heated (fused) foam. with the highest yield stresses observed for lowsaturation values around S = 0.2. Particles can create stable flocculated networks in suspensions through the effects of the van der Waals forces; they are much smaller than the capillary force between particles considered here (34). Typical values of the force due to a capillary bridge between two particles in contact (Fc) are given by Fc ¼ 2prGcosq ≈ 300 nN

ð1Þ

−6

where the particle radius r is 10 m, the surface tension G is 50 × 10−3 N·m−1, and the wetting angle q is small (cosq ≈ 1). The typical value for the van der Waals force (FvdW) is much smaller FvdW ¼

AH r ≈ 1 nN 12 h2

ð2Þ

where the particle separation is given by the surface roughness h = 10−9 m and Hamaker’s coefficient AH has a value of 10−20 N·m. The capillary force serves as the dominant factor influencing the agglomeration of particles. Although the magnitude of the capillary force may be reduced (e.g., due to particle separation, variations in wetting angles, or small pendular bridge volume), capillary forces due to a second immiscible fluid have a great potential for increasing or strengthening the particle network formation within suspensions.

There are other factors that may cause the agglomeration of particles and the observed increase in gel strength. One such factor is the displaced volume caused by the addition of the second immiscible fluid. The effective volume fraction for solid particles and drops of the secondary fluid contained within the bulk fluid may approach the random close-packing volume fraction, causing a substantial rise in the viscosity. This explanation, however, does not account for the transition observed at low-volume fractions (as low as f = 0.1 for the DINP-calcium carbonate mixtures). The very small addition (0.2 wt %) of a secondary fluid does not increase the volume fraction of the dispersed phase significantly. Cavalier and Larché suggested that hydrogen bonds between water molecules might explain the transition in which water is the secondary fluid (as it was in their dioctyl phthalate–calcium carbonate mixtures) (30). However, we observe a similar capillary-state transition using PVC particles suspended in water with added DINP. In this case, the lack of any available H-bonding protons in the molecule makes hydrogen bonding an unlikely mechanism for the transition. We used a microscope to image suspensions of glass beads with hydrophilic and hydrophobic surface treatments in DINP with and without the

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addition of water as a secondary fluid (31). We used a fluorescent dye to mark the location of the water in these images, which are shown in Fig. 4B for the pendular state and Fig. 4D for the capillary state. Corresponding images of the suspensions without the addition of a secondary fluid are shown in Fig. 4, A and C. In the pendular state, the water preferentially wets the glass beads, and the circular outlines of capillary bridges are clearly visible joining several of these particles tightly together. In the capillary state, the water does not preferentially wet the treated glass. In this state, the water is present as globular drops in the center of the glass agglomerates. We investigated the effects of a second immiscible fluid on the flow of particle suspensions. We find that the addition of this second fluid can dramatically change the rheological properties of the mixture; the second fluid creates a network of interconnected particles leading to a transition to a highly elastic, gel-like state. Most notably, this effect exists—and is quite strong—even when the second fluid wets the particles less well than the primary fluid. The addition of a second immiscible fluid has several different effects, all of which are important in industrial applications. For suspensions where settling would occur, the addition of a second fluid allows for the creation of extremely stable suspensions (as shown in movie S2) through the creation of a sample-spanning network structure. For example, both the PVC and glass particles would settle to the bottom of a container in a single-fluid suspension within a few hours, but with the addition of a secondary fluid, suspensions become so stable that they do not phaseseparate when left for many weeks. This effect can be used to keep mixtures homogeneous as they are stored and transported. We also show that this process can dramatically change the yield stress and viscosity of a suspension, allowing for the creation of fluids with tunable flow or processing behavior that may be adjusted by temperature (see supporting online material) or the addition of surfactants. Most importantly, this effect is completely reversible and allows for the yield stress or viscosity to be varied to meet the needs of each step in an industrial process. Finally, the addition of a second immiscible fluid creates a strong network of particles at lower volume fractions than could be obtained otherwise. This particle network may be used as a precursor for porous ceramics or foams, such as that shown in Fig. 5. The PVC solid, which consists of only fused PVC particles, has a bulk density of 0.4 g·ml−1, indicating that some compaction has occurred so that the actual volume fraction of solids is f = 0.3 compared with the starting volume fraction of f = 0.2. Without the secondary fluid (DINP), the creation of these light foams would be impossible, as the PVC particles would settle in the water to a much larger volume fraction, near the maximum randompacked volume fraction of f = 0.6. The creation of these foams opens up a new route to material

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Fig. 4. Images of the A B pendular and capillary states. Clean glass (hydrophilic, q = 49.4°, r = 12.3 mm, f = 0.3; see table S1) in DINP with (A) no added water and (B) with 1 wt % added water. Treated glass (hydropho40 µm bic, q = 99.3°) in DINP with (C) no added water C D and (D) 1 wt % added water. The composite images with added water [(B) and (D)] in the pendular and capillary states, respectively, are composed of the fluorescent dye image 40 µm in yellow, used to highlight the secondary fluid, merged onto the corresponding real light image (see fig. S4).

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REPORTS

References and Notes 1. C. Allain, M. Cloitre, M. Wafra, Phys. Rev. Lett. 74, 1478 (1995). 2. R. Buscall et al., J. Non-Newt. Fluid Mech. 24, 183 (1987). 3. S. C. Tsai, K. Zammouri, J. Rheol. 32, 737 (1988). 4. M. Dijkstra, Curr. Opin. Colloid Interface Sci. 6, 372 (2001). 5. Z. Zhou, P. J. Scales, D. V. Boger, Chem. Eng. Sci. 56, 2901 (2001). 6. P. J. Lu et al., Nature 453, 499 (2008). 7. L. Bocquet, E. Charlaix, S. Ciliberto, J. Crassous, Nature 396, 735 (1998). 8. N. Fraysse, H. Thomé, L. Petit, Eur. Phys. J. B 11, 615 (1999). 9. T. C. Halsey, A. J. Levine, Phys. Rev. Lett. 80, 3141 (1998). 10. T. G. Mason, A. J. Levine, D. Ertaş, T. C. Halsey, Phys. Rev. E 60, R5044 (1999). 11. G. D’Anna, Phys. Rev. E 62, 982 (2000). 12. S. Herminghaus, Adv. Phys. 54, 221 (2005). 13. N. Mitarai, F. Nori, Adv. Phys. 55, 1 (2006). 14. H. R. Kruyt, F. G. Van Selms, Recl. Trav. Chim. Pays-Bas 62, 415 (1943).

15. A. E. J. Eggleton, I. E. Puddington, Can. J. Chem. 32, 86 (1954). 16. S. Van Kao, L. E. Nielsen, C. T. Hill, J. Colloid Interface Sci. 53, 367 (1975). 17. W. Ostwald, W. Haller, Kolloid (Beih.) 29, 354 (1929). 18. C. R. Bloomquist, R. S. Shutt, Ind. Eng. Chem. 32, 827 (1940). 19. P. G. Howe, D. P. Benton, I. E. Puddington, Can. J. Chem. 33, 1189 (1955). 20. A. E. Cattermole, “Classification of the metallic constituents of ores,” U.S. Patent 763259 (1904). 21. G. J. Perrot, S. P. Kinney, Chem. Metall. Eng. 25, 182 (1921). 22. J. R. Farnand, I. E. Puddington, Can. Min. Metall. Bull. 62, 267 (1969). 23. T. Langstroth, in Pigment Handbook, P. A. Lewis, Ed. (Wiley, New York, 1988), vol. 3, chap. 6. 24. B. J. Ennis, J. Li, G. I. Tardos, R. Pfeffer, Chem. Eng. Sci. 45, 3071 (1990). 25. B. J. Ennis, G. I. Tardos, R. Pfeffer, Powder Technol. 65, 257 (1991). 26. W. Pietsch, H. Rumpf, Chem. Ing. Tech. 39, 885 (1967). 27. H. Schubert, Kapillarita¨t in Poro¨sen Feststoffsystemen (Springer, Berlin, 1982). 28. B. Hochstein, N. Willenbacher, AIP Conf. Proc. 1027, 761 (2008). 29. L. Tanzil, B. Hochstein, N. Willenbacher, in Produktgestaltung in der Partikeltechnologie, U. Teipel, Ed. (Fraunhofer

Steric Control of the Reaction of CH Stretch–Excited CHD3 with Chlorine Atom Fengyan Wang,1 Jui-San Lin,1 Kopin Liu1,2* Exciting the CH-stretching mode of CHD3 (where D is deuterium) is known to promote the C-H bond’s reactivity toward chlorine (Cl) atom. Conventional wisdom ascribes the vibrational-rate enhancement to a widening of the cone of acceptance (i.e., the collective Cl approach trajectories that lead to reaction). A previous study of this reaction indicated an intriguing alignment effect by infrared laser–excited reagents, which on intuitive grounds is not fully compatible with the above interpretation. We report here an in-depth experimental study of reagent alignment effects in this reaction. Pronounced impacts are evident not only in total reactivity but also in product state and angular distributions. By contrasting the data with previously reported stereodynamics in reactions of unpolarized, excited CHD3 with fluorine (F) and O(3P), we elucidate the decisive role of long-range anisotropic interactions in steric control of this chemical reaction. he directional nature of chemical bonds generally renders intermolecular interactions and the Born-Oppenheimer potential energy surfaces (PESs) that describe such interactions anisotropic. In the course of reactions that form and break those bonds such anisotropy will tend to dynamically steer the scattering trajectories either toward or away from the transition state (a critical configuration along the reaction path, serving as a bottleneck to reaction), thereby promoting or suppressing reactivity. This steering or reorientation effect has long been recognized by chemists, and mechanistic insights at the molecular level have been garnered for a number of elementary reactions (1–8). Particularly relevant

T

1 Institute of Atomic and Molecular Sciences (IAMS), Academia Sinica, Taipei, Taiwan 10617. 2Department of Physics, National Taiwan University, Taipei, Taiwan 10617 and Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300.

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

900

to this work are two recent studies on the reactions of CH stretch–excited, unpolarized CHD3 (v1 = 1) with F atom (9) and O atom (10). The reaction of F + CHD3 is highly exothermic (change in enthalpy DH0 = −31.3 kcal/mol) with a small early barrier, for which the transitionstate structure is reactant-like. Our group found that one quantum excitation of the CH stretch (v1 = 1) in CHD3 inhibits C-H bond cleavage, resulting in a deceleration of the overall reaction rate (9). This unexpected finding was conjectured, and later calculated theoretically (11), to be a result of steering or deflection of the approaching F atom away from the targeted H atom. By contrast, an opposite effect was discovered in the O + CHD3 reaction (10), which is slightly endothermic (DH0 = 2.1 kcal/mol) with a barrier height of ~9.6 kcal/mol. The location of the barrier is nearly at the midpoint of the pathway; namely, the transition-state structure is neither reactant-like nor product-like. CH-stretching excitation led to a substantial rate

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Institut für Chemische Technologie, Pfinztal, Germany, 2008), vol. 4, pp. 67–75. K. Cavalier, F. Larche, Colloids Surf. A Physicochem. Eng. Asp. 197, 173 (2002). Materials and methods are available as supporting material on Science Online. R. Aveyard, B. P. Binks, J. H. Clint, Adv. Colloid Interface Sci. 100-102, 503 (2003). B. P. Binks, J. H. Clint, Langmuir 18, 1270 (2002). J. P. K. Seville, C. D. Willett, P. C. Knight, Powder Technol. 113, 261 (2000). We thank H. J. Butt for discussions and the German Research Foundation (Deutsche Forschungsgemeinschaft grant no. WI3138/6-1) for funding. We would also like to acknowledge the generous donation of materials we received from Henkel and Potters Europe.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/897/DC1 Materials and Methods SOM Text Figs. S1 to S4 Table S1 References Movies S1 and S2 18 October 2010; accepted 6 January 2011 10.1126/science.1199243

promotion at fixed collision energy (Ec). Moreover, the product angular distribution broadened markedly from backward peaking to sideways dominant, suggesting that the vibrational enhancement operates by extending the range of impact parameters, thus opening up the cone of acceptance to reaction; this mechanism has been theoretically predicted to be particularly prominent in thermoneutral atom + diatom reactions (6). The driving force behind this vibrationally induced steric mechanism was further attributed to long-range anisotropic interactions, which pull or focus the trajectories toward the transition state (10, 12). A deeper implication of these two studies is that in a reaction with strong anisotropic inter-

Fig. 1. Experimental setup for state-selected, aligned molecular beam scattering experiments. A linearly polarized IR laser directed perpendicularly to the relative velocity vector of the two molecular beams prepares the vibrationally excited CHD3 at the scattering center. Reagent alignment is controlled by IR laser polarization direction: “//” refers to an endon attack and “⊥” to a side-on approach. Time-sliced velocity-map imaging reveals the alignment effects on product pair–correlated distributions.

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design, aiming at lightweight construction materials, catalyst carriers with a large inner surface, or microporous foams with superior insulation properties.

REPORTS

References and Notes 1. C. Allain, M. Cloitre, M. Wafra, Phys. Rev. Lett. 74, 1478 (1995). 2. R. Buscall et al., J. Non-Newt. Fluid Mech. 24, 183 (1987). 3. S. C. Tsai, K. Zammouri, J. Rheol. 32, 737 (1988). 4. M. Dijkstra, Curr. Opin. Colloid Interface Sci. 6, 372 (2001). 5. Z. Zhou, P. J. Scales, D. V. Boger, Chem. Eng. Sci. 56, 2901 (2001). 6. P. J. Lu et al., Nature 453, 499 (2008). 7. L. Bocquet, E. Charlaix, S. Ciliberto, J. Crassous, Nature 396, 735 (1998). 8. N. Fraysse, H. Thomé, L. Petit, Eur. Phys. J. B 11, 615 (1999). 9. T. C. Halsey, A. J. Levine, Phys. Rev. Lett. 80, 3141 (1998). 10. T. G. Mason, A. J. Levine, D. Ertaş, T. C. Halsey, Phys. Rev. E 60, R5044 (1999). 11. G. D’Anna, Phys. Rev. E 62, 982 (2000). 12. S. Herminghaus, Adv. Phys. 54, 221 (2005). 13. N. Mitarai, F. Nori, Adv. Phys. 55, 1 (2006). 14. H. R. Kruyt, F. G. Van Selms, Recl. Trav. Chim. Pays-Bas 62, 415 (1943).

15. A. E. J. Eggleton, I. E. Puddington, Can. J. Chem. 32, 86 (1954). 16. S. Van Kao, L. E. Nielsen, C. T. Hill, J. Colloid Interface Sci. 53, 367 (1975). 17. W. Ostwald, W. Haller, Kolloid (Beih.) 29, 354 (1929). 18. C. R. Bloomquist, R. S. Shutt, Ind. Eng. Chem. 32, 827 (1940). 19. P. G. Howe, D. P. Benton, I. E. Puddington, Can. J. Chem. 33, 1189 (1955). 20. A. E. Cattermole, “Classification of the metallic constituents of ores,” U.S. Patent 763259 (1904). 21. G. J. Perrot, S. P. Kinney, Chem. Metall. Eng. 25, 182 (1921). 22. J. R. Farnand, I. E. Puddington, Can. Min. Metall. Bull. 62, 267 (1969). 23. T. Langstroth, in Pigment Handbook, P. A. Lewis, Ed. (Wiley, New York, 1988), vol. 3, chap. 6. 24. B. J. Ennis, J. Li, G. I. Tardos, R. Pfeffer, Chem. Eng. Sci. 45, 3071 (1990). 25. B. J. Ennis, G. I. Tardos, R. Pfeffer, Powder Technol. 65, 257 (1991). 26. W. Pietsch, H. Rumpf, Chem. Ing. Tech. 39, 885 (1967). 27. H. Schubert, Kapillarita¨t in Poro¨sen Feststoffsystemen (Springer, Berlin, 1982). 28. B. Hochstein, N. Willenbacher, AIP Conf. Proc. 1027, 761 (2008). 29. L. Tanzil, B. Hochstein, N. Willenbacher, in Produktgestaltung in der Partikeltechnologie, U. Teipel, Ed. (Fraunhofer

Steric Control of the Reaction of CH Stretch–Excited CHD3 with Chlorine Atom Fengyan Wang,1 Jui-San Lin,1 Kopin Liu1,2* Exciting the CH-stretching mode of CHD3 (where D is deuterium) is known to promote the C-H bond’s reactivity toward chlorine (Cl) atom. Conventional wisdom ascribes the vibrational-rate enhancement to a widening of the cone of acceptance (i.e., the collective Cl approach trajectories that lead to reaction). A previous study of this reaction indicated an intriguing alignment effect by infrared laser–excited reagents, which on intuitive grounds is not fully compatible with the above interpretation. We report here an in-depth experimental study of reagent alignment effects in this reaction. Pronounced impacts are evident not only in total reactivity but also in product state and angular distributions. By contrasting the data with previously reported stereodynamics in reactions of unpolarized, excited CHD3 with fluorine (F) and O(3P), we elucidate the decisive role of long-range anisotropic interactions in steric control of this chemical reaction. he directional nature of chemical bonds generally renders intermolecular interactions and the Born-Oppenheimer potential energy surfaces (PESs) that describe such interactions anisotropic. In the course of reactions that form and break those bonds such anisotropy will tend to dynamically steer the scattering trajectories either toward or away from the transition state (a critical configuration along the reaction path, serving as a bottleneck to reaction), thereby promoting or suppressing reactivity. This steering or reorientation effect has long been recognized by chemists, and mechanistic insights at the molecular level have been garnered for a number of elementary reactions (1–8). Particularly relevant

T

1 Institute of Atomic and Molecular Sciences (IAMS), Academia Sinica, Taipei, Taiwan 10617. 2Department of Physics, National Taiwan University, Taipei, Taiwan 10617 and Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300.

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

900

to this work are two recent studies on the reactions of CH stretch–excited, unpolarized CHD3 (v1 = 1) with F atom (9) and O atom (10). The reaction of F + CHD3 is highly exothermic (change in enthalpy DH0 = −31.3 kcal/mol) with a small early barrier, for which the transitionstate structure is reactant-like. Our group found that one quantum excitation of the CH stretch (v1 = 1) in CHD3 inhibits C-H bond cleavage, resulting in a deceleration of the overall reaction rate (9). This unexpected finding was conjectured, and later calculated theoretically (11), to be a result of steering or deflection of the approaching F atom away from the targeted H atom. By contrast, an opposite effect was discovered in the O + CHD3 reaction (10), which is slightly endothermic (DH0 = 2.1 kcal/mol) with a barrier height of ~9.6 kcal/mol. The location of the barrier is nearly at the midpoint of the pathway; namely, the transition-state structure is neither reactant-like nor product-like. CH-stretching excitation led to a substantial rate

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Institut für Chemische Technologie, Pfinztal, Germany, 2008), vol. 4, pp. 67–75. K. Cavalier, F. Larche, Colloids Surf. A Physicochem. Eng. Asp. 197, 173 (2002). Materials and methods are available as supporting material on Science Online. R. Aveyard, B. P. Binks, J. H. Clint, Adv. Colloid Interface Sci. 100-102, 503 (2003). B. P. Binks, J. H. Clint, Langmuir 18, 1270 (2002). J. P. K. Seville, C. D. Willett, P. C. Knight, Powder Technol. 113, 261 (2000). We thank H. J. Butt for discussions and the German Research Foundation (Deutsche Forschungsgemeinschaft grant no. WI3138/6-1) for funding. We would also like to acknowledge the generous donation of materials we received from Henkel and Potters Europe.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/897/DC1 Materials and Methods SOM Text Figs. S1 to S4 Table S1 References Movies S1 and S2 18 October 2010; accepted 6 January 2011 10.1126/science.1199243

promotion at fixed collision energy (Ec). Moreover, the product angular distribution broadened markedly from backward peaking to sideways dominant, suggesting that the vibrational enhancement operates by extending the range of impact parameters, thus opening up the cone of acceptance to reaction; this mechanism has been theoretically predicted to be particularly prominent in thermoneutral atom + diatom reactions (6). The driving force behind this vibrationally induced steric mechanism was further attributed to long-range anisotropic interactions, which pull or focus the trajectories toward the transition state (10, 12). A deeper implication of these two studies is that in a reaction with strong anisotropic inter-

Fig. 1. Experimental setup for state-selected, aligned molecular beam scattering experiments. A linearly polarized IR laser directed perpendicularly to the relative velocity vector of the two molecular beams prepares the vibrationally excited CHD3 at the scattering center. Reagent alignment is controlled by IR laser polarization direction: “//” refers to an endon attack and “⊥” to a side-on approach. Time-sliced velocity-map imaging reveals the alignment effects on product pair–correlated distributions.

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design, aiming at lightweight construction materials, catalyst carriers with a large inner surface, or microporous foams with superior insulation properties.

REPORTS that the relative velocity vector lay parallel to the ultraviolet (UV) probe laser direction. The infrared (IR) excitation laser, tuned to the R(0) transition of the CHD3(v1 = 1 ← 0) band at 2999.21 cm−1 (20), was directed perpendicularly to the UV laser. Thus, a single rovibrationally excited reactant CHD3(v1 = 1, |JKM ≥ |100〉) was prepared, where the quantum numbers J, K, and M denote the rotational angular momentum, the projection of J onto the top axis, and the projection of J onto the IRpolarization axis, respectively. A variable waveplate was used to control the linear polarization axis of the IR laser. The // and ⊥ symbols refer, respectively, to parallel and perpendicular orientation of the IR-polarization axis relative to the initial relative velocity vector of the collision system. The UV probe laser was fired 3.5 ms after the IR excitation pulse to allow product buildup and to maximize the differential signals of the // and ⊥ configurations. To unravel the stereodynamics, we acquired four product images: IR-off, IR-//, IR-⊥, and IR-aM [i.e., an IR polarization at the magic angle, 54.7°, with respect to the relative velocity vector axis (21)]. Experimentally, imaging data at each configuration were accumulated for 5 min in sequence, and this cycle was then repeated, typically >20 times, to generate the final set of product images. Figure 2 presents raw images of the probed CD3(v = 0) products at Ec = 3.84 kcal/mol. Consistent with a previous report (17), no IR-generated signals for CHD2 products could be detected at this energy (fig. S1). The IR-off and IR-aM images are almost identical to those reported previously (16, 22). The IR-off image features two backscattered structures, labeled as (00, 0)g and (00, 0)b (see Fig. 2 legend), respectively, whereas the IR-aM image exhibits distinct, additional ringlike structures reflecting the impact of C-H stretch excitation on the reaction dynamics. On energetic grounds, the sharp forward peak in the inner ring can be assigned to the (00, 1)s product pair, whereas the back-scattered outer ring is ascribed to the (00, 0)s pair. Compared to the IR-aM image, even casual inspection reveals that the relative intensities of the sharp forward peak and the outer-ring feature show

Fig. 2. Four CD3(v = 0) product images with a superimposed axis indicating the scattering directions; the 0° angle refers to the initial CHD3 beam direction in the centerof-mass frame. The pair-correlated labeling is defined as follows: The numbers in the parentheses denote the quanta of vibrational excitation in the modes of CD3 (left) and HCl (right) products; the outer subscript indicates the reactant state (“g” for ground-state CHD3, “b” for bend-excited CHD3, and “s” for stretch-excited CHD3). aM indicates magic angle polarization.

opposite propensities as the IR-polarization axis flips from // to ⊥. To map out the polarization-angle dependence of the alignment effect, we acquired images at many intermediate polarization angles. The results are summarized in Fig. 3 (top). For the (00, 0)s pair the total intensity (I ) was included, whereas for (00, 1)s, only the forward signals within a 70° cone (i.e., excluding the IR-off feature) were counted. Notably, the two product channels display distinct, out-of-phase modulations in signals. The polarization-angle dependences can be fitted (solid lines) with I(a) = Iiso [1 + bP2(cosa)] (Eq. 1), analogous to the form that describes the onephoton excited, aligned CHD3 reactant (13, 23): Iiso is proportional to the product-state–specific reaction cross section from an unpolarized reagent, b characterizes the stereoselective reactivity with values ranging from +2 to −1, and P2(cosa) = (3cos2a − 1)/2 is the second-order Legendre polynomial with a denoting the IR polarization angle with respect to the initial relative velocity. After full image analysis accounting for the density-to-flux transformation (18) and the hyperfine depolarization

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actions, prealigning the reagents will have little impact on reactivity because of the reorientation effects en route to the barrier. That is, only reactions with weak steering interactions are viable candidates for steric control (active influence over the outcome by prealignment). In this regard, a previous report on the Cl + CHD3(v1 = 1) reaction, in which a reagent alignment effect on reactivity was observed (13), raises puzzling questions. That study probed a single rovibrational product state, for which Cl atoms approaching perpendicularly to the stretch-excited C-H bond preferentially yielded forward-scattered HCl(v = 1, j = 1) product. Here, v refers to vibrational excitation and j refers to rotational excitation. The Cl + CHD3 reaction is slightly endothermic (DH0 = 1.8 kcal/mol) with a moderate barrier height of ~4 kcal/mol. The transitionstate structure is more product-like (14, 15) than in the O(3P) reaction. A detailed study of this reaction indeed demonstrated (16, 17) an appreciable rate promotion upon CH-stretching excitation, as observed in the O(3P) + CHD3(v1 = 1) case. Does the vibrationally induced steric mechanism also operate in the Cl + CHD3(v1 = 1) system to promote reactivity? If so, how can the CHD3(v1 = 1) remain asymptotically aligned, as is necessary to explain the reagent alignment effect (13), given the anisotropic steering force that underlies the vibrationally induced steric mechanism (6)? We report here an in-depth study of the stereodynamics of Cl + CHD3(v1 = 1), aimed at clarifying this apparent paradox and shedding more light on the general tug-of-war between the reorientation effect exerted by the interaction potential and the stereoselectivity induced by prealigning the reagents. We performed a crossed-beam scattering experiment under single-collision conditions. The details of the apparatus have been described previously (16–19), except for the preparation of aligned CHD3(v1 = 1) reagents, which we performed in this work at the molecular beam crossing region [rather than in the source chamber (16, 17)]. Figure 1 depicts the experimental setup and time-sliced, velocity-map imaging detection scheme. Two molecular beams were rotated so

Fig. 3. (Top) Polarization-angle dependence of the probed signals from the stretch-excited reaction. The v = 0 data correspond to the outer-ring feature labeled (00,0)s in Fig. 2; for the v = 1 data [the (00, 1)s pair], only the forward signals within a 70° cone are counted. Two sets of data are normalized by I(aM) = 1 for display. Solid lines are best fits based on Eq. 1. (Bottom) Temporal profiles of the statespecific signals in the top panel were monitored to determine the degree of hyperfine depolarization. Imin (Imax) denotes I// (I⊥) for v = 1, and I⊥ (I//) for v = 0. The results for each of the two product states agree within 10%; averaging with another set of v = 0,1 measurements yields C/I(aM) = 0.32 T 0.02 at Dt = 3.5 ms, which is shown by the shaded area in the top figure. By subtracting the depolarization component C from the data shown in the top panel, the genuine b parameters can then be determined. www.sciencemag.org

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Fig. 4. (Top) Polarization-dependent angular distributions of HCl(v = 0) and (v = 1) states formed concomitantly with the probed CD3(v = 0) products. A large disparity is evident for different IR-polarizations. (Middle) Polarized integral cross sections presented graphically (see table S2 for the numerical data). All data are normalized to stotal at aM, and the error represents two SDs. Previous study of Cl reaction with an unpolarized CHD3(v1 = 1) demonstrated a vibrational enhancement factor of 10 relative to the CHD3(v = 0) cross section at Ec = 3.84 kcal/mol (16). (Bottom) Molecular cartoons illustrating the stereodynamics of the scattering directions of the HCl(v = 0) product channel.

(see below), the resulting anisotropy parameters b could be extracted (table S1). For R(0) rotational transition pathways, these were 1.38 T 0.24 for HCl(v = 0) and −0.38 T 0.05 for the forwardscattered HCl(v = 1). A negative b value (signifying that the ⊥-polarization is favored) for forwardHCl(v = 1) corroborates the previous finding (13). As alluded to earlier, the products were probed 3.5 ms after the IR excitation to build up product yield. During this time, recoupling of the randomly distributed nuclear spins in CHD3 with the initially aligned rotational angular momentum leads to a dephasing of coherently excited hyperfine components (13, 24), which scrambles (or depolarizes) the initial alignment and contaminates the observed product signals. That is, if there were no hyperfine depolarization, the observed alignment effects would have been more pronounced. To determine the degree of such depolarization, we measured the temporal profiles of I// and I⊥ with three IR laser polarizations (0°, 54.7°, and 90°) at different time delays (Dt) between two lasers. As shown in Fig. 3 (bottom), a linear dependence of Imin/Imax on Dt is evident. By reexpressing the polarization-angle dependence as I = I′iso [1 + bP2(cosa)] + C, one can deduce the depolarization component C at Dt using the extrapolated Imin/Imax value at Dt = 0. The results of four independent determinations yielded C/I(aM) = 0.32 T 0.02 at Dt = 3.5 ms, which is sketched as the shaded area in Fig. 3 (top) and is to be subtracted to derive the genuine b values. The image analysis followed the previous procedures (16–19); the resulting pair-correlated product angular and speed distributions are presented in figs. S2 and S3. With the depolarized contributions subtracted out from the measured // and ⊥

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distributions, the final product angular distributions [Fig. 4 (top)] and speed distributions (fig. S4) then represent the genuine stereodynamics of the reaction from an optically aligned CHD3(v1 = 1, |100〉) reactant. From the product speed distribution (fig. S4), the polarization-dependent cross sections are obtained and summarized in Fig. 4 (bottom) (see table S2 for the numerical data). All polarization-dependent cross sections presented in Fig. 4 are normalized to stotal(aM). The //-polarized CHD3(v1 = 1) yields a higher total reactivity, s///s⊥ = 2.7, and this preference for //-polarization originates solely from the HCl(v = 0) product channel (s///s⊥ = 7.71); HCl(v = 1) shows an opposite propensity slightly in favor of ⊥-polarization, s///s⊥= 0.87. In terms of the correlated HCl vibrational branching ratio, s(v = 1)/s(v = 0) changes from a cold distribution of 0.31 for //- polarization to an inverted distribution, 2.71, for ⊥-polarization—a marked manifestation of stereoselectivity on product state populations. Although the state-specific steric effects are pronounced, their mechanistic origin is less obvious. An optically prepared CHD3 reagent can yield alignments from both the rotational angular momentum and the vibrational transition dipole moment (13). The former arises from unequal population of (2J + 1)-fold degenerate mJ sublevels induced by optical excitation, whereas the latter, relying on a parallel transition for v1 = 1 ← 0, dictates that the C-H bond axis lie along the polarization direction of the IR radiation. These two types of alignment can be distinguished by means of different rotational-branch excitations (13): The alignment of the C-H bond will always peak along the laser polarization axis for all

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branches, whereas the rotational alignment reverses when switching from R- to Q-branch excitation. Hence, to unravel the origin of the observed alignment effects, we also determined the b values via the Q(1) and P(1) excitations. Quantitative comparisons of the resulting b values (table S1) lead us to conclude that the observed polarization dependence for the (00, 0)s product pair is due primarily to the C-H bond axis alignment, whereas the forward-scattered (00, 1)s pair is subject to both the bond axis and rotational angular momentum alignments. The need to consider both alignment origins complicates physical interpretations. More sophisticated probes will be required to disentangle the two alignment effects. With this caveat in mind, we examine the polarization-dependent angular distributions. The trajectories of the (00, 0)s pair from an unpolarized CHD3(v1 = 1) at aM are mainly back-scattered, suggestive of a direct rebound reaction mechanism at this Ec (16, 25). Moreover, all polarized angular distributions for HCl(v = 0) products span the same angular range as that of the ground-state reaction (fig. S5). We surmise that neither the vibrational enhancement nor alignment preference can be attributed to enlarging the range of reactive impact parameters; rather, they are ascribed to the increase of reaction probabilities at fixed impact parameters. This conclusion is in sharp contrast to the findings in studying the O(3P) + CHD3(v1 = 1) reaction, for which a greater range of impact parameters contribute to reaction upon vibrational excitation, yielding a much broader product angular distribution than the ground-state reaction (10). A closer inspection of the HCl(v = 0) angular distributions for the //- and ⊥-configurations reveals that whereas the former remains backward-peaked, the latter, with lower reactivity, appears sidewaysdominant. Qualitatively, the result for (00, 0)s is in keeping with a direct reaction with collinear transition state, in which a //-aligned CHD3 favors small– impact parameter, end-on collisions (26), whereas the ⊥-aligned CH bond prefers larger–impact parameter, side-on attack of Cl atoms (27). This intuitive classical picture implicitly assumes little reorientation during the approach motion to the barrier. The reactive fluxes into the (00, 1)s pair show a slight preference for ⊥-polarized CHD3(v1 = 1). Both polarization-dependent angular distributions cover the full angular range. The observed propensity is, however, evident only in a very narrow range of forward-scattering angles; little differential reactivity is discernible at larger angles. The origin of this channel’s dynamics is more subtle owing to the presence of both bond-axis and rotational angular momentum alignment effects, as mentioned above. Mechanistically, the formation of HCl(v = 1) is likely mediated by a reactive resonance pathway (16, 17, 28), rather than a direct reaction, further complicating the issue. Irrespective of the underlying mechanism, the marked state-specific stereoselectivity reported here is a testament to the conservation of reactant prealignment and corresponding absence of substantial steering effects in this reaction—in sharp

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REPORTS

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References and Notes F. F. Crim, Proc. Natl. Acad. Sci. U.S.A. 105, 12654 (2008). W. L. Hase, Science 266, 998 (1994). D. C. Clary, Annu. Rev. Phys. Chem. 41, 61 (1990). A. J. Orr-Ewing, R. N. Zare, Annu. Rev. Phys. Chem. 45, 315 (1994). 5. D. Skouteris et al., Science 286, 1713 (1999). 6. R. D. Levine, J. Phys. Chem. 94, 8872 (1990). 7. A. J. Alexander, M. Brouard, K. S. Kalogerakis, J. P. Simons, Chem. Soc. Rev. 27, 405 (1998).

1. 2. 3. 4.

8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21.

B. L. Yoder, R. Bisson, R. D. Beck, Science 329, 553 (2010). W. Zhang, H. Kawamata, K. Liu, Science 325, 303 (2009). F. Wang, K. Liu, Chem. Sci. 1, 126 (2010). G. Czakó, J. M. Bowman, J. Am. Chem. Soc. 131, 17534 (2009). The contrasting behaviors of O and F can also be qualitatively rationalized from the transition-state theory perspective. The transition state in F + CHD3 possesses a reactant-like structure, and thus a stretched or compressed C-H bond of CHD3 deviates from the transition-state structure, disfavoring the reaction. By contrast, ab initio calculations predicted that in O(3P) + CHD3, both the breaking C-H bond and forming O-H bond are elongated. Excitation of the C-H bond of CHD3 therefore helps attain the transition-state structure. W. R. Simpson, T. P. Rakitzis, S. A. Kandel, A. J. Orr-Ewing, R. N. Zare, J. Chem. Phys. 103, 7313 (1995). W. T. Duncan, T. N. Truong, J. Chem. Phys. 103, 9642 (1995). J. C. Corchado, D. G. Truhlar, J. Espinosa-Garcia, J. Chem. Phys. 112, 9375 (2000). S. Yan, Y.-T. Wu, B. Zhang, X.-F. Yue, K. Liu, Science 316, 1723 (2007). S. Yan, Y.-T. Wu, K. Liu, Proc. Natl. Acad. Sci. U.S.A. 105, 12667 (2008). J. J. Lin, J. Zhou, W. Shiu, K. Liu, Rev. Sci. Instrum. 74, 2495 (2003). J. J. Lin, J. Zhou, W. Shiu, K. Liu, Science 300, 966 (2003). O. N. Ulenikov et al., Mol. Phys. 108, 1209 (2010). Magic angle imaging serves two purposes. First, at this polarization angle, the approaching Cl atom encounters a practically unpolarized CHD3(v1 = 1, j = 1) reagent. Previously, when we prepared CHD3(v1 = 1) in the source chamber (16, 17), the excited reagents traveled for >100 ms before reacting, which we presumed was long enough to depolarize initially aligned CHD3(v1 = 1) molecules by hyperfine interactions. Indeed, the results from the acquired aM image agree broadly with the previous findings. Second, the signals from the

The Magnitude and Duration of Late Ordovician–Early Silurian Glaciation Seth Finnegan,1* Kristin Bergmann,1 John M. Eiler,1 David S. Jones,2 David A. Fike,2 Ian Eisenman,1,3 Nigel C. Hughes,4 Aradhna K. Tripati,1,5 Woodward W. Fischer1 Understanding ancient climate changes is hampered by the inability to disentangle trends in ocean temperature from trends in continental ice volume. We used carbonate “clumped” isotope paleothermometry to constrain ocean temperatures, and thereby estimate ice volumes, through the Late Ordovician–Early Silurian glaciation. We find tropical ocean temperatures of 32° to 37°C except for short-lived cooling by ~5°C during the final Ordovician stage. Evidence for ice sheets spans much of the study interval, but the cooling pulse coincided with a glacial maximum during which ice volumes likely equaled or exceeded those of the last (Pleistocene) glacial maximum. This cooling also coincided with a large perturbation of the carbon cycle and the Late Ordovician mass extinction. arth history is punctuated by glacial episodes that vary widely in their magnitude and duration (1), as well as in their effects on global biodiversity (2). Far more is known

E

1 Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125, USA. 2Department of Earth and Planetary Sciences, Washington University, St. Louis, MO 63130, USA. 3Department of Atmospheric Sciences, University of Washington, Seattle, WA 98195, USA. 4Department of Earth Sciences, University of California, Riverside, CA 92521, USA. 5Department of Earth and Space Sciences and Department of Atmospheric and Oceanic Sciences, Institute of Geophysics and Planetary Physics, University of California, Los Angeles, CA 90095, USA.

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

about the most recent glacial age in the Pleistocene than about older glacial episodes. The Late Ordovician–Early Silurian glaciation of the southern supercontinent of Gondwana (Fig. 1A) is unusual because it occurred during a period when atmospheric partial pressure of CO2 ( pCO2) was generally higher [perhaps 8 to 16 times higher (3)] than today’s pCO2, was short-lived compared to subsequent Gondwanan glaciations (1), and is the only glacial episode that appears to have coincided with a major mass extinction of marine life (4) (Fig. 1B). These observations have led to suggestions that the Late Ordovician–Early Silurian icehouse represents a climate mode distinct from more recent glaciations (5), but fundamental

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22. 23. 24. 25.

26.

27.

28. 29. 30. 31.

stretch-excited reagents under the three polarization angles are related by (I// + 2I⊥) = 3IaM, which provides a stringent check (within T1% in this work) of the consistency of the data. G. Nyman, J. Zhou, B. Zhang, K. Liu, Isr. J. Chem. 47, 1 (2007). R. N. Zare, Ber. Bunsenges. Phys. Chem 86, 422 (1982). R. Altkorn, R. N. Zare, C. H. Greene, Mol. Phys. 55, 1 (1985). R. D. Levine, R. B. Bernstein, Molecular Reaction Dynamics and Chemical Reactivity (Oxford Univ. Press, Oxford, 1987). In an optically aligned (not oriented) CHD3 molecule, the H atom of the aligned C-H bond can point either toward or away from the approaching Cl atom; thus, no distinction of the head-versus-tail dynamics can be made. In a direct reaction, the forward-scattered product is normally associated with large–impact parameter collisions, for which there is a purely kinematic smearing of the alignment effect even for a perfectly aligned reagent (30). R. Martínez, M. González, P. Defazio, C. Petrongolo, J. Chem. Phys. 127, 104302 (2007). J. Zhou, B. Zhang, J. J. Lin, K. Liu, Mol. Phys. 103, 1757 (2005). I. Schetchter, R. D. Levine, J. Chem. Soc. Faraday Trans. II 85, 1059 (1989). We are indebted to S. Yan for earlier attempts of this project and to J. Lam for help with the experiment. This work was supported by National Science Council (NSC-99-2113-M-011-016), Academia Sinica, and the Air Force Office of Scientific Research (AOARD-10-4034).

Downloaded from www.sciencemag.org on February 17, 2011

contrast to the analogous reactions of CHD3 (v1 = 1) with F and O(3P) atoms (9, 10). In retrospect, this implication may not be too surprising. It has been demonstrated (22, 29) that in the ground-state Cl + CHD3 reaction, the product angular distribution essentially mirrors the opacity function (the reaction probability as a function of impact parameters) in a one-to-one correspondent manner [see figures 2 and 5 of (22)]. In accounting for enforcement of such a mirror-like correspondence, a rather weak anisotropic PES in the entrance valley could have been inferred. The structure of the transition state is product-like, and thus the reaction barrier is recessed in the exit valley. Upon vibrational excitation of CHD3(v1 = 1), the elongation of the C-H bond can enlarge the range of attack angles at the reaction barrier, thereby increasing the reaction probability at fixed impact parameters, but the resulting anisotropic interactions may not extend into the entrance valley far enough to appreciably steer the prealigned reagents.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/900/DC1 Figs. S1 to S5 Tables S1 and S2 References 29 October 2010; accepted 13 January 2011 10.1126/science.1199771

questions about the event are still largely unresolved: Estimates of peak ice sheet volume range from ~50 to more than 250 million km3 (6) (Fig. 1A), estimates of its duration range from 35 million years (1) to less than 1 million years (5) (Fig. 1B), and it is unclear how much sea surface temperatures (SSTs) cooled in the tropical environments that hosted much of Late Ordovician biodiversity (7–9). These uncertainties persist because few climate proxies can be reliably applied to Paleozoic rocks. Oxygen isotope ratios (d18O) in wellpreserved marine carbonate and phosphate minerals provide a useful proxy and have been widely applied in Paleozoic climate reconstruction (5, 7, 8) but suffer a fundamental limitation: The d18O value of a mineral is influenced by both the temperature and the isotopic composition of the water reservoir from which it precipitates (d18Owater). Consequently, without independent constraints on one or the other of these variables, interpreting d18O trends in the stratigraphic record poses an underdetermined problem. This issue has been addressed for the Pleistocene last glacial maximum (LGM)—for example, using sediment porewater profiles (10)—but remains largely unresolved for older glaciations. We used carbonate clumped isotope paleothermometry (11, 12) to constrain the precipitation temperatures of a suite of Late Ordovician– Early Silurian carbonates. This approach measures the state of ordering of heavy isotopes (D47) in carbonate minerals and is independent of the

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References and Notes F. F. Crim, Proc. Natl. Acad. Sci. U.S.A. 105, 12654 (2008). W. L. Hase, Science 266, 998 (1994). D. C. Clary, Annu. Rev. Phys. Chem. 41, 61 (1990). A. J. Orr-Ewing, R. N. Zare, Annu. Rev. Phys. Chem. 45, 315 (1994). 5. D. Skouteris et al., Science 286, 1713 (1999). 6. R. D. Levine, J. Phys. Chem. 94, 8872 (1990). 7. A. J. Alexander, M. Brouard, K. S. Kalogerakis, J. P. Simons, Chem. Soc. Rev. 27, 405 (1998).

1. 2. 3. 4.

8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21.

B. L. Yoder, R. Bisson, R. D. Beck, Science 329, 553 (2010). W. Zhang, H. Kawamata, K. Liu, Science 325, 303 (2009). F. Wang, K. Liu, Chem. Sci. 1, 126 (2010). G. Czakó, J. M. Bowman, J. Am. Chem. Soc. 131, 17534 (2009). The contrasting behaviors of O and F can also be qualitatively rationalized from the transition-state theory perspective. The transition state in F + CHD3 possesses a reactant-like structure, and thus a stretched or compressed C-H bond of CHD3 deviates from the transition-state structure, disfavoring the reaction. By contrast, ab initio calculations predicted that in O(3P) + CHD3, both the breaking C-H bond and forming O-H bond are elongated. Excitation of the C-H bond of CHD3 therefore helps attain the transition-state structure. W. R. Simpson, T. P. Rakitzis, S. A. Kandel, A. J. Orr-Ewing, R. N. Zare, J. Chem. Phys. 103, 7313 (1995). W. T. Duncan, T. N. Truong, J. Chem. Phys. 103, 9642 (1995). J. C. Corchado, D. G. Truhlar, J. Espinosa-Garcia, J. Chem. Phys. 112, 9375 (2000). S. Yan, Y.-T. Wu, B. Zhang, X.-F. Yue, K. Liu, Science 316, 1723 (2007). S. Yan, Y.-T. Wu, K. Liu, Proc. Natl. Acad. Sci. U.S.A. 105, 12667 (2008). J. J. Lin, J. Zhou, W. Shiu, K. Liu, Rev. Sci. Instrum. 74, 2495 (2003). J. J. Lin, J. Zhou, W. Shiu, K. Liu, Science 300, 966 (2003). O. N. Ulenikov et al., Mol. Phys. 108, 1209 (2010). Magic angle imaging serves two purposes. First, at this polarization angle, the approaching Cl atom encounters a practically unpolarized CHD3(v1 = 1, j = 1) reagent. Previously, when we prepared CHD3(v1 = 1) in the source chamber (16, 17), the excited reagents traveled for >100 ms before reacting, which we presumed was long enough to depolarize initially aligned CHD3(v1 = 1) molecules by hyperfine interactions. Indeed, the results from the acquired aM image agree broadly with the previous findings. Second, the signals from the

The Magnitude and Duration of Late Ordovician–Early Silurian Glaciation Seth Finnegan,1* Kristin Bergmann,1 John M. Eiler,1 David S. Jones,2 David A. Fike,2 Ian Eisenman,1,3 Nigel C. Hughes,4 Aradhna K. Tripati,1,5 Woodward W. Fischer1 Understanding ancient climate changes is hampered by the inability to disentangle trends in ocean temperature from trends in continental ice volume. We used carbonate “clumped” isotope paleothermometry to constrain ocean temperatures, and thereby estimate ice volumes, through the Late Ordovician–Early Silurian glaciation. We find tropical ocean temperatures of 32° to 37°C except for short-lived cooling by ~5°C during the final Ordovician stage. Evidence for ice sheets spans much of the study interval, but the cooling pulse coincided with a glacial maximum during which ice volumes likely equaled or exceeded those of the last (Pleistocene) glacial maximum. This cooling also coincided with a large perturbation of the carbon cycle and the Late Ordovician mass extinction. arth history is punctuated by glacial episodes that vary widely in their magnitude and duration (1), as well as in their effects on global biodiversity (2). Far more is known

E

1 Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125, USA. 2Department of Earth and Planetary Sciences, Washington University, St. Louis, MO 63130, USA. 3Department of Atmospheric Sciences, University of Washington, Seattle, WA 98195, USA. 4Department of Earth Sciences, University of California, Riverside, CA 92521, USA. 5Department of Earth and Space Sciences and Department of Atmospheric and Oceanic Sciences, Institute of Geophysics and Planetary Physics, University of California, Los Angeles, CA 90095, USA.

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

about the most recent glacial age in the Pleistocene than about older glacial episodes. The Late Ordovician–Early Silurian glaciation of the southern supercontinent of Gondwana (Fig. 1A) is unusual because it occurred during a period when atmospheric partial pressure of CO2 ( pCO2) was generally higher [perhaps 8 to 16 times higher (3)] than today’s pCO2, was short-lived compared to subsequent Gondwanan glaciations (1), and is the only glacial episode that appears to have coincided with a major mass extinction of marine life (4) (Fig. 1B). These observations have led to suggestions that the Late Ordovician–Early Silurian icehouse represents a climate mode distinct from more recent glaciations (5), but fundamental

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22. 23. 24. 25.

26.

27.

28. 29. 30. 31.

stretch-excited reagents under the three polarization angles are related by (I// + 2I⊥) = 3IaM, which provides a stringent check (within T1% in this work) of the consistency of the data. G. Nyman, J. Zhou, B. Zhang, K. Liu, Isr. J. Chem. 47, 1 (2007). R. N. Zare, Ber. Bunsenges. Phys. Chem 86, 422 (1982). R. Altkorn, R. N. Zare, C. H. Greene, Mol. Phys. 55, 1 (1985). R. D. Levine, R. B. Bernstein, Molecular Reaction Dynamics and Chemical Reactivity (Oxford Univ. Press, Oxford, 1987). In an optically aligned (not oriented) CHD3 molecule, the H atom of the aligned C-H bond can point either toward or away from the approaching Cl atom; thus, no distinction of the head-versus-tail dynamics can be made. In a direct reaction, the forward-scattered product is normally associated with large–impact parameter collisions, for which there is a purely kinematic smearing of the alignment effect even for a perfectly aligned reagent (30). R. Martínez, M. González, P. Defazio, C. Petrongolo, J. Chem. Phys. 127, 104302 (2007). J. Zhou, B. Zhang, J. J. Lin, K. Liu, Mol. Phys. 103, 1757 (2005). I. Schetchter, R. D. Levine, J. Chem. Soc. Faraday Trans. II 85, 1059 (1989). We are indebted to S. Yan for earlier attempts of this project and to J. Lam for help with the experiment. This work was supported by National Science Council (NSC-99-2113-M-011-016), Academia Sinica, and the Air Force Office of Scientific Research (AOARD-10-4034).

Downloaded from www.sciencemag.org on February 17, 2011

contrast to the analogous reactions of CHD3 (v1 = 1) with F and O(3P) atoms (9, 10). In retrospect, this implication may not be too surprising. It has been demonstrated (22, 29) that in the ground-state Cl + CHD3 reaction, the product angular distribution essentially mirrors the opacity function (the reaction probability as a function of impact parameters) in a one-to-one correspondent manner [see figures 2 and 5 of (22)]. In accounting for enforcement of such a mirror-like correspondence, a rather weak anisotropic PES in the entrance valley could have been inferred. The structure of the transition state is product-like, and thus the reaction barrier is recessed in the exit valley. Upon vibrational excitation of CHD3(v1 = 1), the elongation of the C-H bond can enlarge the range of attack angles at the reaction barrier, thereby increasing the reaction probability at fixed impact parameters, but the resulting anisotropic interactions may not extend into the entrance valley far enough to appreciably steer the prealigned reagents.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/900/DC1 Figs. S1 to S5 Tables S1 and S2 References 29 October 2010; accepted 13 January 2011 10.1126/science.1199771

questions about the event are still largely unresolved: Estimates of peak ice sheet volume range from ~50 to more than 250 million km3 (6) (Fig. 1A), estimates of its duration range from 35 million years (1) to less than 1 million years (5) (Fig. 1B), and it is unclear how much sea surface temperatures (SSTs) cooled in the tropical environments that hosted much of Late Ordovician biodiversity (7–9). These uncertainties persist because few climate proxies can be reliably applied to Paleozoic rocks. Oxygen isotope ratios (d18O) in wellpreserved marine carbonate and phosphate minerals provide a useful proxy and have been widely applied in Paleozoic climate reconstruction (5, 7, 8) but suffer a fundamental limitation: The d18O value of a mineral is influenced by both the temperature and the isotopic composition of the water reservoir from which it precipitates (d18Owater). Consequently, without independent constraints on one or the other of these variables, interpreting d18O trends in the stratigraphic record poses an underdetermined problem. This issue has been addressed for the Pleistocene last glacial maximum (LGM)—for example, using sediment porewater profiles (10)—but remains largely unresolved for older glaciations. We used carbonate clumped isotope paleothermometry (11, 12) to constrain the precipitation temperatures of a suite of Late Ordovician– Early Silurian carbonates. This approach measures the state of ordering of heavy isotopes (D47) in carbonate minerals and is independent of the

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isotopic composition of water from which the minerals grew. Combination of this approach with conventional carbonate-water oxygen isotope paleothermometry thus provides a means for untangling trends in reservoir composition from those in temperature. To minimize the effects of burial diagenesis, we examined exceptionally fossiliferous and well-studied successions in the U.S. midcontinent and on Anticosti Island, Québec, Canada (13) (Fig. 1A and fig. S1), which have experienced relatively little sedimentary burial. To assay the quality of our proxy measurements, we sampled fossils from several taxonomic groups and surrounding sediments from a broad range of lithotypes and across a large range of preservation states to characterize diagenesis and vital effects (13) (figs. S2 and S3). There is no evidence of a systematic burial overprint on D47 values in our data set. Similar ranges are recorded in the Vauréal Formation and the Jupiter Formation (Fig. 2A) (12) despite ~500 m of intervening strata in the Anticosti Basin (fig. S1). The highest D47 values (lowest temperatures) on Anticosti are not observed in the stratigraphically highest samples but rather in samples from the middle of the stratigraphic column. To confirm that D47 does capture known postdepositional thermal gradients, we sampled micritic carbonates from the Vauréal Formation that are intruded by a Jurassic-aged dike. D47 values immediately adjacent to the dike are the lowest in our entire data set, corresponding to precipitation temperatures exceeding 230°C, but inferred temperatures fall to 39°C within 14 m of the dike-country rock contact (fig. S4). D47 values of skeletal carbonates range from 0.631 to 0.501 (Fig. 2 and table S1), corresponding to a temperature range from 28° to 64°C. This range implies a mixture of plausibly primary and diagenetically altered phases, the latter being typically depleted in both D47 and d18O (fig. S5). Because diagenetic recrystallization of calcite tends to deplete Sr and enrich Mn and Fe (14, 15), we evaluated concentrations of these metals in a large and representative subset of our samples (13) (figs. S6 to S8). Most samples fall within the “well-preserved” compositional range identified by previous studies (fig. S6), but there is a strong relationship between the first principal component (PC1) of trace metal concentrations and D47: High PC1 values (low Sr and high Mn and Fe, fig. S7) are associated with higher precipitation temperatures and in many cases with textural evidence of diagenetic alteration (Fig. 2B). The highest D47 value and lowest PC1 value associated with texturally altered samples are 0.589 and –0.285, respectively. We therefore excluded all samples that fall outside this range from our reconstructions of ocean temperature and chemistry (Fig. 2B). We included samples that were not evaluated for trace metal concentration but that have D47 values higher than 0.589, but similar trends result if these samples are also excluded (fig. S9). Even the best-preserved samples likely

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contain small amounts of dispersed recrystallized phases that cannot easily be avoided given the large sample sizes required for adequately precise measurement of D47. Our temperature reconstructions should therefore be viewed as maximal SST estimates, and we base trend lines on the lowest-temperature samples from each stratigraphic interval. Reconstructed temperatures are nearly indistinguishable from each other and consistent with a narrow range from 32° to 37°C throughout most of the ~20 million years covered by our time series (Fig. 3A). SSTs in this range rarely occur in the modern tropics, but a variety of proxies record similar temperatures during Mesozoic–early Cenozoic greenhouse intervals (16–18), during which atmospheric pCO2 is inferred in some reconstructions to have exceeded five times present atmospheric levels (3). Temperatures below this range (28° to 31°C) occur only in samples from the Laframboise Member of the Ellis Bay Formation on Anticosti Island. This Hirnantian-aged unit (13) records a major drop in sea level (19, 20) and

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a large positive carbon isotope excursion (19); both are recognized globally in other sedimentary successions (4, 20). The best preserved of these successions also record exceptionally enriched d18O values [–2 per mil (‰) to 0‰ Vienna Pee Dee belemnite (VPDB)] during Hirnantian time (8, 20, 21), as do our Laframboise Member samples (Fig. 3B and table S1). d18O values preceding and following the Hirnantian peak are lighter but still enriched relative to the ~ –5‰ (VPDB) baseline values that characterize the beginning and end of the time series (Fig. 3B). Well-preserved brachiopods and trilobites could not be extracted from the Laframboise Member, and hence our data from the Hirnantian maximum are derived exclusively from rugose corals. D47 is not known to be subject to disequilibrium vital effects among modern taxa (11), but such effects cannot be ruled out and there remains uncertainty regarding the possibility of a vital effect on d18O in rugose corals (13). However, our Hirnantian d18O values are similar to those recorded by brachiopods in contemporaneous sections (5, 8, 20, 21), and the Fig. 1. (A) South polar view of a simplified Late Ordovician paleogeographic reconstruction (27), indicating the positions of Late Ordovician to Early Silurian–aged glacial deposits in Gondwana (Gw, tan solid circles and areas) and of the Laurentian (La) localities sampled for this study (red diamonds). Ba indicates Baltica. Two possible reconstructions of the Gondwanan ice sheet (6) are shown: a minimal, discontinuous reconstruction (light blue shading) and a maximal continentspanning reconstruction (dashed blue outline). (B) Hypotheses regarding the duration of the icehouse interval: short and sharp, restricted largely or entirely to the Hirnantian (Hirn.) stage lasting as little as 500,000 years (5), and protracted, with a peak in the Hirnantian interval (1, 28). Marine invertebrate genus diversity (black spindle) (29) declined both at the beginning and at the end of the Hirnantian stage. mya, million years ago.

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Fig. 3. Symbols as in Fig. 2; color indicates provenance: purple, Upper Mississippi Valley; green, Cincinnati Arch; orange, Anticosti Island. Solid symbols indicate samples selected on the basis of both trace metal concentration and ∆47 criteria; open symbols indicate samples selected based only on ∆47 value. Error bars on individual samples (13) are T 1 SE (d18O errors are smaller than symbols) and reflect analytical precision for samples analyzed once and reproducibility for samples analyzed multiple times (table S1). Samples marked by asterisks are the lowesttemperature samples in their respective time intervals and are the basis for trendlines. (A) ∆47derived near-surface ocean temperature trend for the early Katian to late Aeronian interval. (B) d18O (VPDB) trend over the same interval. (C) Relative contributions of temperature and d18Owater to changes in d18O (∆d18O) between successive time intervals. Bars are scaled to the magnitude of ∆d18O, and color proportion is scaled to the relative contribution of temperature change (red) and change in the oxygen isotopic composition of seawater (blue) to ∆d18O. (D) d18Owater (VSMOW) trend. Dotted lines indicate d18Owater value during the Pleistocene LGM (10) and expected d18Owater value for an ice-free world. www.sciencemag.org

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Hirnantian excursion can be defined by using only rugose corals (fig. S10) and thus cannot be explained by systematic differences between rugose corals and other taxa. Our D47 measurements place independent constraints on how much of the temporal variation in d18O can be explained by temperature changes, with the remainder attributable to changes in the isotopic composition of seawater. For much of the study interval, d18O variation is driven almost entirely by changes in d18Owater (Fig. 3C); only during Hirnantian time can changes in temperature account for a substantial proportion of this variation. d18Owater estimates (Fig. 3D) fall between –1‰ Vienna standard mean ocean water (VSMOW), the value expected for an ice-free world (22), and 1‰, the value of LGM seawater (10), for most of the study interval. However, d18Owater estimates exceed 2‰ during the Hirnantian glacial maximum. Assuming (i) the d18O trend reflects changes in mean ocean water, (ii) the d18O of all surface reservoirs combined has been unchanged from the Late Ordovician to the recent (13), and (iii) the d18O of glacial ice was comparable to the LGM, these values imply that continental ice volumes during the Hirnantian maximum substantially exceeded those of the LGM (fig. S11). The mean isotopic composition of Late Ordovician glacial ice cannot be directly measured, but inferred Hirnantian ice volumes exceed those of the LGM for any mean ice value heavier than –60‰, approaching the most depleted values observed in the present day (fig. S11). d18Owater trends suggest multiple episodes of moderate glaciation and deglaciation throughout the mid-late Katian interval, with little evidence of substantial ice sheets before this time (Fig. 3D). The most enriched d18Owater values before the Hirnantian peak come from the sub-Laframboise Ellis Bay Formation, the age of which has been controversial (13). Assigning this unit an early Hirnantian rather than latest Katian age would restrict d18Owater values higher than 1‰ to Hirnantian time (fig. S12), but multiple mid-late Katian samples > 0‰ still indicate the development of substantial pre-Hirnantian ice sheets, at least transiently. Relatively high d18Owater values also occur in latest Hirnantian samples from the lowermost Becscie Formation, which records a sharp rise in sea level and waning of the Hirnantian carbon isotope excursion. These observations reveal that latest Ordovician sea level rise represents only partial deglaciation of Gondwana. d18Owater values consistent with moderate ice sheets persist for several million years, returning to near –1‰ by the end of the Aeronian Stage (Fig. 3D). Our results imply that initial glaciation of Gondwana occurred with little or no cooling of the tropical oceans, that tropical SSTs exceeded the present-day range except during the Hirnantian glacial maximum, and that they warmed rapidly after the Hirnantian maximum despite the persistence of substantial continental ice volumes for several million years. This contrasts with

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Fig. 2. (A) D47 values of all samples examined for this study, keyed to taxonomic group and preservation state, and plotted against stratigraphic position. (B) PC1 of log-transformed Mn, Fe, and Sr concentrations for a large subset (n = 52) of the samples shown in (A). PC1 explains 86% of variation in trace metal composition and receives strong positive loading from Mn and Fe and weak negative loading from Sr. Dashed lines indicate PC1 and D47 cutoffs for inclusion (shaded region) in paleoclimate reconstructions; H, Hirnantian; Rhudd, Rhuddanian; Aer, Aeronian. Ages within stages are interpolated on the basis of stratigraphic position.

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previous work using classical oxygen isotope paleothermometry on conodont apatite from Anticosti and elsewhere (7) that reconstructed temperatures in the modern SST range for much of the Late Ordovician–Early Silurian except for cooling to ~24°C below and above the Laframboise Member (7). These estimates assumed a constant d18Owater of –1.0‰; substituting our d18Owater values from the same units raises inferred temperatures by, on average, 8°C. Recent revision of the phosphate-water oxygen isotope fractionation equation (23) further suggests that all conodontderived temperature estimates should be revised upward, bringing them into the range that we observe for the Late Ordovician–Early Silurian. We cannot rule out the possibility that the trends we observe are influenced by changes in the basin hydrology of the Taconic Foreland, but, if they accurately reflect global trends in the tropical oceans, they imply a nonlinear relationship between tropical ocean temperatures and continental ice volumes (fig. S13A). This contrasts with expectations from climate simulations using a modern continental configuration and from proxy records of the past 60 million years (13) (fig. S13, B and C). Furthermore, coexistence of substantial south polar ice sheets with tropical SSTs regionally in excess of 30°C implies a steeper meridional temperature gradient than during other major glacial episodes (12, 24). Minor glaciations inferred to have occurred under high CO2 conditions in the late Mesozoic–early Cenozoic (16, 25) may have exhibited similar

gradients but were comparatively short-lived. Both of these observations could plausibly be explained by nonlinear changes in the intensity of oceanic meridional overturning circulation (26), similar to those previously invoked to explain changes in the behavior of the Hirnantian carbon cycle (4, 5, 20). Although speculative, some support for this hypothesis is provided by the coincidence of our observed cooling pulse with the globally recognized Hirnantian positive carbon isotope excursion (5, 19, 20). Lastly, by demonstrating that tropical cooling was largely limited to the Hirnantian Stage, our results support hypotheses linking the two-pulsed nature of the Late Ordovician mass extinction to rapid climate changes at the beginning and end of this interval (4, 20). References and Notes 1. L. A. Frakes, J. E. Francis, J. I. Syktus, Climate Modes of the Phanerozoic (Cambridge Univ. Press, Cambridge, 1996). 2. A. Raymond, C. Metz, J. Geol. 112, 655 (2004). 3. R. A. Berner, Geochim. Cosmochim. Acta 70, 5653 (2006). 4. P. M. Sheehan, Annu. Rev. Earth Planet. Sci. 29, 331 (2001). 5. P. J. Brenchley et al., Geology 22, 295 (1994). 6. D. P. Le Heron, J. A. Dowdeswell, J. Geol. Soc. London 166, 277 (2009). 7. J. A. Trotter, I. S. Williams, C. R. Barnes, C. Lécuyer, R. S. Nicoll, Science 321, 550 (2008). 8. J. D. Marshall, P. D. Middleton, J. Geol. Soc. London 147, 1 (1990). 9. A. D. Herrmann, M. E. Patzkowsky, D. Pollard, Palaeogeogr. Palaeoclimatol. Palaeoecol. 206, 59 (2004). 10. D. P. Schrag et al., Quat. Sci. Rev. 21, 331 (2002). 11. P. Ghosh et al., Geochim. Cosmochim. Acta 70, 1439 (2006). 12. R. E. Came et al., Nature 449, 198 (2007).

Hibernation in Black Bears: Independence of Metabolic Suppression from Body Temperature Øivind Tøien,1* John Blake,1 Dale M. Edgar,2† Dennis A. Grahn,3 H. Craig Heller,3 Brian M. Barnes1* Black bears hibernate for 5 to 7 months a year and, during this time, do not eat, drink, urinate, or defecate. We measured metabolic rate and body temperature in hibernating black bears and found that they suppress metabolism to 25% of basal rates while regulating body temperature from 30° to 36°C, in multiday cycles. Heart rates were reduced from 55 to as few as 9 beats per minute, with profound sinus arrhythmia. After returning to normal body temperature and emerging from dens, bears maintained a reduced metabolic rate for up to 3 weeks. The pronounced reduction and delayed recovery of metabolic rate in hibernating bears suggest that the majority of metabolic suppression during hibernation is independent of lowered body temperature. ammalian hibernation is well characterized in species such as marmots, ground squirrels, bats, and dasyurid marsupials (1). These small (<5 kg) hibernators undergo regulated decreases in core body temperature (Tb) to near or below freezing during torpor bouts that last days to weeks (2–5). Torpor is periodically interrupted by arousals to normothermia (35° to 38°C) that usually last for less than one day (6, 7). During torpor, metabolic rates of small

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hibernators decrease to 2 to 5% of basal metabolic rate (BMR) (8–10). However, the relative contributions of temperature-dependent (described by Q10, rate coefficient for a 10°C change in Tb) and temperature-independent mechanisms of metabolic suppression depend on the size of animals and stage of entry into torpor (11). In contrast, the relationships between Tb and metabolism in the large hibernators of the bear family Ursidae have remained unknown because technical lim-

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13. Materials and methods are available as supporting material on Science Online. 14. U. Brand, J. Veizer, J. Sediment. Res. 50, 1219 (1981). 15. G. A. Shields et al., Geochim. Cosmochim. Acta 67, 2005 (2003). 16. A. Bornemann et al., Science 319, 189 (2008). 17. S. Schouten et al., Geology 31, 1069 (2003). 18. P. N. Pearson et al., Nature 413, 481 (2001). 19. A. Desrochers, C. Farley, A. Achab, E. Asselin, J. F. Riva, Palaeogeogr. Palaeoclimatol. Palaeoecol. 296, 248 (2010). 20. P. J. Brenchley et al., Geol. Soc. Am. Bull. 115, 89 (2003). 21. L. Hints et al., Est. J. Earth Sci. 59, 1 (2010). 22. S. M. Savin, Annu. Rev. Earth Planet. Sci. 5, 319 (1977). 23. E. Pucéat et al., Earth Planet. Sci. Lett. 298, 135 (2010). 24. MARGO Project Members, Nat. Geosci. 2, 127 (2009). 25. A. Tripati, J. Backman, H. Elderfield, P. Ferretti, Nature 436, 341 (2005). 26. R. J. Stouffer, S. Manabe, Clim. Dyn. 20, 759 (2003). 27. L. R. M. Cocks, T. H. Torsvik, J. Geol. Soc. London 159, 631 (2002). 28. M. R. Saltzman, S. A. Young, Geology 33, 109 (2005). 29. J. J. Sepkoski Jr., Bull. Am. Paleontol. 363, 560 (2002). 30. We thank T. Raub, M. Rohrssen, and B. Gaines for assistance with field and lab work; D. Boulet and Société des établissements de plein air du Québec (SEPAQ) Anticosti for permission to work in Anticosti National Park; and B. Hunda for supplying samples. This work was funded by an Agouron Institute award to W.W.F. and D.A.F. and NSF Division of Earth Sciences awards to W.W.F. and J.M.E.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200803/DC1 Materials and Methods Figs. S1 to S13 Table S1 References 23 November 2010; accepted 19 January 2011 Published online 27 January 2011; 10.1126/science.1200803

itations have prevented continuous, long-term monitoring in these 30 to 200 kg or larger animals. We used telemetry and respirometry to record Tb, metabolic rates, and heartbeat patterns of black bears, Ursus americanus, through their hibernation and post-hibernation recovery. Black bears were nuisance animals captured in south-central or interior Alaska in late autumn 3 different years and transported to facilities at the Institute of Arctic Biology, University of Alaska Fairbanks. Radio transmitters for Tb and electromyogram (EMG)/electrocardiogram (ECG) were surgically implanted (12), and animals were transferred to outdoor enclosures in an isolated wooded area. Bears hibernated inside 0.8 m3 wooden nest boxes with straw for bedding and equipped with infrared cameras, activity detectors, and telemetry-receiving antennas. Food and water were not provided. Air was continuously collected from the closed hibernacula to record O2 consumption (a measure of metabolic rate). After 1 Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA. 2Department of Psychiatry and Behavioral Sciences, School of Medicine, Stanford University, CA 94305, USA. 3Department of Biological Sciences, Stanford University, CA 94305, USA.

*To whom correspondence should be addressed. E-mail: [email protected] (O.T.); [email protected] (B.M.B.) †Present address: Lilly Research Centre, Eli Lilly, Windlesham, Surrey, GU20 6PH, UK.

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REPORTS

previous work using classical oxygen isotope paleothermometry on conodont apatite from Anticosti and elsewhere (7) that reconstructed temperatures in the modern SST range for much of the Late Ordovician–Early Silurian except for cooling to ~24°C below and above the Laframboise Member (7). These estimates assumed a constant d18Owater of –1.0‰; substituting our d18Owater values from the same units raises inferred temperatures by, on average, 8°C. Recent revision of the phosphate-water oxygen isotope fractionation equation (23) further suggests that all conodontderived temperature estimates should be revised upward, bringing them into the range that we observe for the Late Ordovician–Early Silurian. We cannot rule out the possibility that the trends we observe are influenced by changes in the basin hydrology of the Taconic Foreland, but, if they accurately reflect global trends in the tropical oceans, they imply a nonlinear relationship between tropical ocean temperatures and continental ice volumes (fig. S13A). This contrasts with expectations from climate simulations using a modern continental configuration and from proxy records of the past 60 million years (13) (fig. S13, B and C). Furthermore, coexistence of substantial south polar ice sheets with tropical SSTs regionally in excess of 30°C implies a steeper meridional temperature gradient than during other major glacial episodes (12, 24). Minor glaciations inferred to have occurred under high CO2 conditions in the late Mesozoic–early Cenozoic (16, 25) may have exhibited similar

gradients but were comparatively short-lived. Both of these observations could plausibly be explained by nonlinear changes in the intensity of oceanic meridional overturning circulation (26), similar to those previously invoked to explain changes in the behavior of the Hirnantian carbon cycle (4, 5, 20). Although speculative, some support for this hypothesis is provided by the coincidence of our observed cooling pulse with the globally recognized Hirnantian positive carbon isotope excursion (5, 19, 20). Lastly, by demonstrating that tropical cooling was largely limited to the Hirnantian Stage, our results support hypotheses linking the two-pulsed nature of the Late Ordovician mass extinction to rapid climate changes at the beginning and end of this interval (4, 20). References and Notes 1. L. A. Frakes, J. E. Francis, J. I. Syktus, Climate Modes of the Phanerozoic (Cambridge Univ. Press, Cambridge, 1996). 2. A. Raymond, C. Metz, J. Geol. 112, 655 (2004). 3. R. A. Berner, Geochim. Cosmochim. Acta 70, 5653 (2006). 4. P. M. Sheehan, Annu. Rev. Earth Planet. Sci. 29, 331 (2001). 5. P. J. Brenchley et al., Geology 22, 295 (1994). 6. D. P. Le Heron, J. A. Dowdeswell, J. Geol. Soc. London 166, 277 (2009). 7. J. A. Trotter, I. S. Williams, C. R. Barnes, C. Lécuyer, R. S. Nicoll, Science 321, 550 (2008). 8. J. D. Marshall, P. D. Middleton, J. Geol. Soc. London 147, 1 (1990). 9. A. D. Herrmann, M. E. Patzkowsky, D. Pollard, Palaeogeogr. Palaeoclimatol. Palaeoecol. 206, 59 (2004). 10. D. P. Schrag et al., Quat. Sci. Rev. 21, 331 (2002). 11. P. Ghosh et al., Geochim. Cosmochim. Acta 70, 1439 (2006). 12. R. E. Came et al., Nature 449, 198 (2007).

Hibernation in Black Bears: Independence of Metabolic Suppression from Body Temperature Øivind Tøien,1* John Blake,1 Dale M. Edgar,2† Dennis A. Grahn,3 H. Craig Heller,3 Brian M. Barnes1* Black bears hibernate for 5 to 7 months a year and, during this time, do not eat, drink, urinate, or defecate. We measured metabolic rate and body temperature in hibernating black bears and found that they suppress metabolism to 25% of basal rates while regulating body temperature from 30° to 36°C, in multiday cycles. Heart rates were reduced from 55 to as few as 9 beats per minute, with profound sinus arrhythmia. After returning to normal body temperature and emerging from dens, bears maintained a reduced metabolic rate for up to 3 weeks. The pronounced reduction and delayed recovery of metabolic rate in hibernating bears suggest that the majority of metabolic suppression during hibernation is independent of lowered body temperature. ammalian hibernation is well characterized in species such as marmots, ground squirrels, bats, and dasyurid marsupials (1). These small (<5 kg) hibernators undergo regulated decreases in core body temperature (Tb) to near or below freezing during torpor bouts that last days to weeks (2–5). Torpor is periodically interrupted by arousals to normothermia (35° to 38°C) that usually last for less than one day (6, 7). During torpor, metabolic rates of small

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hibernators decrease to 2 to 5% of basal metabolic rate (BMR) (8–10). However, the relative contributions of temperature-dependent (described by Q10, rate coefficient for a 10°C change in Tb) and temperature-independent mechanisms of metabolic suppression depend on the size of animals and stage of entry into torpor (11). In contrast, the relationships between Tb and metabolism in the large hibernators of the bear family Ursidae have remained unknown because technical lim-

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13. Materials and methods are available as supporting material on Science Online. 14. U. Brand, J. Veizer, J. Sediment. Res. 50, 1219 (1981). 15. G. A. Shields et al., Geochim. Cosmochim. Acta 67, 2005 (2003). 16. A. Bornemann et al., Science 319, 189 (2008). 17. S. Schouten et al., Geology 31, 1069 (2003). 18. P. N. Pearson et al., Nature 413, 481 (2001). 19. A. Desrochers, C. Farley, A. Achab, E. Asselin, J. F. Riva, Palaeogeogr. Palaeoclimatol. Palaeoecol. 296, 248 (2010). 20. P. J. Brenchley et al., Geol. Soc. Am. Bull. 115, 89 (2003). 21. L. Hints et al., Est. J. Earth Sci. 59, 1 (2010). 22. S. M. Savin, Annu. Rev. Earth Planet. Sci. 5, 319 (1977). 23. E. Pucéat et al., Earth Planet. Sci. Lett. 298, 135 (2010). 24. MARGO Project Members, Nat. Geosci. 2, 127 (2009). 25. A. Tripati, J. Backman, H. Elderfield, P. Ferretti, Nature 436, 341 (2005). 26. R. J. Stouffer, S. Manabe, Clim. Dyn. 20, 759 (2003). 27. L. R. M. Cocks, T. H. Torsvik, J. Geol. Soc. London 159, 631 (2002). 28. M. R. Saltzman, S. A. Young, Geology 33, 109 (2005). 29. J. J. Sepkoski Jr., Bull. Am. Paleontol. 363, 560 (2002). 30. We thank T. Raub, M. Rohrssen, and B. Gaines for assistance with field and lab work; D. Boulet and Société des établissements de plein air du Québec (SEPAQ) Anticosti for permission to work in Anticosti National Park; and B. Hunda for supplying samples. This work was funded by an Agouron Institute award to W.W.F. and D.A.F. and NSF Division of Earth Sciences awards to W.W.F. and J.M.E.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1200803/DC1 Materials and Methods Figs. S1 to S13 Table S1 References 23 November 2010; accepted 19 January 2011 Published online 27 January 2011; 10.1126/science.1200803

itations have prevented continuous, long-term monitoring in these 30 to 200 kg or larger animals. We used telemetry and respirometry to record Tb, metabolic rates, and heartbeat patterns of black bears, Ursus americanus, through their hibernation and post-hibernation recovery. Black bears were nuisance animals captured in south-central or interior Alaska in late autumn 3 different years and transported to facilities at the Institute of Arctic Biology, University of Alaska Fairbanks. Radio transmitters for Tb and electromyogram (EMG)/electrocardiogram (ECG) were surgically implanted (12), and animals were transferred to outdoor enclosures in an isolated wooded area. Bears hibernated inside 0.8 m3 wooden nest boxes with straw for bedding and equipped with infrared cameras, activity detectors, and telemetry-receiving antennas. Food and water were not provided. Air was continuously collected from the closed hibernacula to record O2 consumption (a measure of metabolic rate). After 1 Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA. 2Department of Psychiatry and Behavioral Sciences, School of Medicine, Stanford University, CA 94305, USA. 3Department of Biological Sciences, Stanford University, CA 94305, USA.

*To whom correspondence should be addressed. E-mail: [email protected] (O.T.); [email protected] (B.M.B.) †Present address: Lilly Research Centre, Eli Lilly, Windlesham, Surrey, GU20 6PH, UK.

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REPORTS hibernating bears. High and stable Tb patterns occurred during pregnancy in a European brown bear (19). Bears have delayed implantation, and pregnancy starts in late November after denning (20). We suggest that low and fluctuating Tb may not be favorable for embryonic development, and thus, hibernating bears maintain normothermia while pregnant. O2 consumption in hibernating bears varied from minimum levels of 0.06 ml g−1 h−1 sustained for as much as one day to brief peaks of >0.35 ml g−1 h−1 accompanied by movement (Fig. 3A). Tb declined when metabolism and shivering were minimal and increased during intense shivering and heightened metabolism. This varying pattern of endothermic thermoregulation

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can explain the wide range in metabolic rates found in the only previous metabolic study of hibernating bears (18). Predicted BMR for carnivores (21, 22) averages 0.228 ml g−1 h−1. Here, we show BMR (defined as overnight resting and fasting metabolic rate measured 1 month after emergence from hibernation) was 0.276 ml g−1 h−1 (range from 0.267 to 0.285 ml g−1 h−1, n = 3 bears); the mean Tb was 37.8°C. A comparison of the changes in metabolism and corresponding Tb in three nonpregnant bears (Fig. 3B) revealed that during mid-hibernation, when Tb was 32.2°C (30.9° to 33.6°C), minimum metabolic rate (12) was 0.069 (0.056 to 0.086) ml g−1 h−1, or 24.9% of BMR. A reduction to only 0.179 ml g−1 h−1 (64.9% of BMR) would be expected because of

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the spontaneous emergence of bears from their dens in spring, Tb recordings were continued, and minimum metabolism after 24 hours of fasting was determined every four nights for 1 month. Hibernating black bears kept a curled posture (Fig. 1), similar to that previously described (13), that facilitates heat preservation and water economy. Animals changed position twice a day to once every 2 days, when they stood, occasionally groomed, and rearranged bedding material. Tb, which is normally 37° to 38°C, decreased to average levels of 33.0°C (31.7° to 34.0°C, n = 4 bears) in mid hibernation (defined as 21 January to 20 February). Minimum Tb was 30.4°C (29.4° to 32.5°C, n = 4 bears). Before emergence in mid-April, Tb gradually increased over 2 to 4 weeks to 36° to 37°C (Fig. 2). Hibernating bears did not show spontaneous, periodical arousals to normothermic levels of Tb, as do small hibernators. This may be because bear Tb did not decrease below 30°C, a level that may reflect a threshold below which neural deficits, such as loss of neuronal structure (14), begin to occur that require regular returns to high Tb. Hibernating fat-tailed dwarf lemurs that are regularly warmed by the sun to above 30°C also do not show spontaneous arousals (15). Diurnal rhythms in Tb also were not evident in mid-winter; instead, there were unexpected 1.6- to 7.3-day cycles of Tb with 2° to 6°C amplitude. Cycles were shortest during coldest conditions and most regular in the smallest bear (Fig. 2B, top). Tb was higher and more stable in free-ranging black bears studied in Wyoming and Colorado, which could be due to milder ambient temperatures (16). In spring when conditions were warmer in our study, multi-day Tb cycles were also less evident (Fig. 2). Highamplitude multi-day cycles of Tb may be a feature of thermoregulation, when gradients between Tb and environmental temperatures are large. Early studies on hibernating black bears in Alaska or arctic Canada reported Tb of 32° to 35°C (17, 18) and could not reveal any multi-day patterns because of intermittent measurements. The Tb of a female bear remained at normothermic levels through the end of January (Fig. 2C, bottom), when she gave birth to a 243-g cub that died because of a congenital diaphragmatic hernia (12). Afterwards, her Tb became more variable and decreased toward levels of other

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Fig. 3. Body temperature and metabolic rate of hibernating black bears. (A) O2 consumption, EMG activity, Tb, movements, and temperature outside and inside the den of BB97-02F during a 5-day period in January. (B) Minimum O2 direct effects of the 5.5°C decrease in Tb, when a Q10 of 2.2 is assumed (1). When bears emerged from dens in mid-April with Tb of 36.6°C (36.1° to 37.4°C), metabolic rate averaged 0.149 (0.127 to 0.170) ml g−1 h−1 or 52.9% of BMR and stabilized at BMR levels after 2 to 3 weeks (Fig. 3B). Bears began feeding over this period, and their return to BMR may in part involve resumption of a full capacity of the digestive system. In alpine marmots, hibernation is accompanied by a 70% decrease in mass of stomach and intestines, which was reversed in spring (23). Hypothyroidism of hypothalamic origin reported in hibernating black bears (24) may also contribute to metabolic suppression. Decreased metabolism in hibernating bears reduces the need for transport of blood gases and nutrients. Heart rate (HR) of three non-pregnant bears in mid-hibernation decreased from summer resting levels (Fig. 4A) of 55 (44.5 to 63.7) beats per min to 14.4 (8.9 to 20.1) beats per min, which is similar to minima of 8 to 12 beats per min in a captive hibernating black bear reported by Folk (25). Hibernating bears showed a marked variation in inter-beat intervals through the breathing cycle, encompassing a profound sinus arrhythmia (Fig. 4, B and C). Typically, a group of rapid heartbeats occurred as inspiration ended followed by interbeat intervals of 8 to 20 s after expiration. Bursts of shivering were synchronous with rapid heartbeats and breathing (Fig. 4C). At emer-

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consumption and corresponding Tb of three bears during hibernation and during recovery from hibernation. The vertical dashed line indicates the average time of emergence, which varied by T2 days.

Fig. 4. ECG and breathing patterns in summer and hibernating bears. (A) Representative ECG for a bear in summer. (B and C) ECG, breathing (chamber pressure), and EMG amplitude of two hibernating bears, (B) BB99-01M and (C) BB97-02F, showing increased heart rate at inspiration. The open bar marks inspiration, and the filled blue bar marks expiration. The peaks in average EMG amplitude in (C) are shivering bursts, which occurred at the end of inspiration.

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metabolic rate, lack of diurnal Tb patterns, reduced HR, and surviving without feeding or drinking for approximately half a year. However, Tb in hibernating bears is far higher than in small hibernators, which is in part due to the estimated lower levels of thermal conductance in bears (approximately 20%) as compared with ground squirrels [supporting online material (SOM) text and table S1] (10). Whereas smaller hibernators

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References and Notes 1. F. Geiser, Annu. Rev. Physiol. 66, 239 (2004). 2. H. C. Heller, G. W. Colliver, Am. J. Physiol. 227, 583 (1974). 3. G. L. Florant, H. C. Heller, Am. J. Physiol. 232, R203 (1977). 4. B. M. Barnes, Science 244, 1593 (1989).

5. F. Geiser, T. Ruf, Physiol. Zool. 68, 935 (1995). 6. C. P. Lyman, J. S. Willis, A. Malan, L. C. H. Wang, Hibernation and Torpor in Mammals and Birds (Academic Press, New York, 1982). 7. T. N. Lee, B. M. Barnes, C. L. Buck, Ethol. Ecol. Evol. 21, 403 (2009). 8. B. D. Snapp, H. C. Heller, Physiol. Zool. 54, 297 (1981). 9. G. Heldmaier, T. Ruf, J. Comp. Physiol. B 162, 696 (1992). 10. C. L. Buck, B. M. Barnes, Am. J. Physiol. 279, R255 (2000). 11. F. Geiser, J. Comp. Physiol. B 158, 25 (1988). 12. Materials and methods are available as supporting material on Science Online. 13. G. E. Jr, Folk, J. M. Hunt, M. A. Folk, in Bears–Their Biology and Management, C. J. Martinka, K. L. McArthus, Eds. (Bear Biology Association, Tonto Basin, AZ, 1980), pp. 43–47. 14. C. G. von der Ohe, C. C. Garner, C. Darian-Smith, H. C. Heller, J. Neurosci. 27, 84 (2007). 15. K. H. Dausmann, J. Glos, J. U. Ganzhorn, G. Heldmaier, Nature 429, 825 (2004). 16. H. J. Harlow, T. Lohuis, R. C. Anderson-Sprecher, T. D. I. Beck, J. Mammal. 85, 414 (2004). 17. R. J. Hock, 2nd Alaskan Sci. Conf., AAAS 2, 310 (1951). 18. P. D. Watts, C. Cuyler, Acta Physiol. Scand. 134, 149 (1988). 19. R. Hissa, Ann. Zool. Fenn. 34, 267 (1997). 20. R. L. Rausch, Z. Saugetierkd. 26, 65 (1961). 21. V. Hayssen, R. C. Lacy, Comp. Biochem. Physiol. A 81, 741 (1985). 22. B. K. McNab, Comp. Biochem. Physiol. A 151, 5 (2008). 23. I. Hume et al., J. Comp. Physiol. B 172, 197 (2002). 24. F. Azizi, J. E. Mannix, D. Howard, R. A. Nelson, Am. J. Physiol. 237, E227 (1979).

LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia Rik Op den Camp,1 Arend Streng,1 Stéphane De Mita,1* Qingqin Cao,1† Elisa Polone,1,2 Wei Liu,1,3 Jetty S. S. Ammiraju,4 Dave Kudrna, Rod Wing,4 Andreas Untergasser,1‡ Ton Bisseling,1,5§ René Geurts1 Rhizobium–root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor–like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis. he rhizobial nodule symbiosis is widespread in the legume family (Fabaceae). Although this nitrogen-fixing symbiosis provides the plant with a major advantage, it is in principle restricted to a single family, and it is a major challenge for future agriculture to transfer this symbiosis to nonlegumes (1). The genus Parasponia could provide a key to this, because it encompasses the only nonlegume species that acquired also the rhizobium symbiosis (2, 3), where “rhizobium” refers to all species and genera that form nodules on legumes. Parasponia comprises several tropical tree species and belongs to Celtidaceae (4). Celtidaceae (order Rosales) and Fabaceae (order Fabales) are only remotely related.

T

Further, not a single species phylogenetically positioned between Parasponia and Fabaceae is able to establish such rhizobium symbiosis. Hence, in all probability the common ancestor of present Parasponia species gained the rhizobium-nodule symbiosis independent from legumes. Therefore, a legume-Parasponia comparison provides a key to identifying genetic constraints underlying this symbiosis. In this study, we focused on parallel evolution of the recognition of the rhizobial signal that starts the symbiotic interaction, the Nod factor. Parasponia makes lateral rootlike nodules that are associated with cell divisions in the root cortex (5). Rhizobium enters the Parasponia root intercellularly and becomes imbedded in a dense

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25. G. E. Jr, Folk, in Mammalian Hibernation, vol. III, K. Fisher, A. R. Dawe, C. P. Lyman, Schönbaum, F. E. Jr. South, Eds. (Oliver & Boyd, Edinburgh, Scotland, 1967), pp. 75–85. 26. O. L. Nelson, C. T. Robbins, J. Comp. Physiol. B 180, 465 (2010). 27. H. J. Harlow, T. Lohuis, T. D. I. Beck, P. A. Iaizzo, Nature 409, 997 (2001). 28. V. B. Fedorov et al., Physiol. Genomics 37, 108 (2009). 29. S. W. Donahue, M. R. Vaughan, L. M. Demers, H. J. Donahue, J. Exp. Biol. 206, 4233 (2003). 30. C. Shao et al., Mol. Cell. Proteomics 9, 313 (2010). 31. This work was supported by U.S. Army Medical Research and Materiel Command grant 05178001; NSF grants 9819540, 0076039, and 0732755; NIH HD-00973; and gift funds to Stanford University, American Heart Association #0020626Z, and the Fulbright Program. We thank the Alaska Department of Fish and Game for providing bears, D. Ritter for technical assistance, and J. Kenagy and J. Duman for comments.

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show long torpor bouts interrupted by regular arousal episodes, black bears in Alaska exhibit distinct cyclic non-diurnal Tb patterns. Bear metabolism is reduced by 53% from BMR, even when Tb has returned to normothermic levels. These observations expand the phenotype of mammalian hibernation that occurs in diverse animals over body mass ranges from 0.005 to 200 kg. Insights into how hibernating bears achieve and cope with these reductions in energy need and Tb, as well as conservation of muscle (27, 28) and bone mass (29) despite prolonged seasonal inactivity and disuse, could lead to the development of novel clinical therapies. Current molecular and genetic approaches (28, 30) in combination with better physiological knowledge can increase our understanding of the regulation of hibernation in small and large hibernators and their evolution.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/906/DC1 Materials and Methods SOM Text Table S1 References 22 October 2010; accepted 13 January 2011 10.1126/science.1199435

matrix. Rhizobium obtains an intracellular lifestyle when it reaches a nodule primordium. There, cortical cells are infected via threadlike structures that remain connected to the plasma membrane. These so-called fixation threads branch, fill up the cells, and provide a niche to rhizobia to fix nitrogen (5). This is illustrated by the expression, in these threads, of the rhizobium nifH gene that encodes one of the subunits of nitrogenase (fig. S1). In contrast, rhizobia enter most legume roots via root hair–based intracellular infection threads, and the bacteria are released in nodule cells as membrane-surrounded nitrogen-fixing organelle-like structures (symbiosomes) that harbor a single or only a few bacteria. Legume nodules are considered to be genuine organs with a unique ontogeny (6). The fact that the Rhizobium symbiosis is very common in 65-million-year-old Fabaceae led to the conclusion that the symbiotic 1 Department of Plant Sciences, Laboratory of Molecular Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands. 2Department of Agricultural Biotechnologies, Universitá di Padova, Viale dell’Universitá 16, 35020 Legnaro (Padova), Italy. 3Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 4University of Arizona, Plant Sciences Department, 303 Forbes Building, Tucson, AZ 85721–0036, USA. 5College of Science, King Saud University, Post Office Box 2455, Riyadh 11451, Saudi Arabia.

*Present address: Institut de Recherche pour le Développement Montpellier, 911 Avenue, Agropolis BP 64501, 34394 Montpellier, cedex 5, France. †Present address: Department of Biotechnology, Beijing University of Agriculture, No. 7 Beinong Road, Huilongguan Changping District, Beijing, People’s Republic of China. ‡Present address: Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. §To whom correspondence should be addressed. E-mail: [email protected]

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References and Notes 1. F. Geiser, Annu. Rev. Physiol. 66, 239 (2004). 2. H. C. Heller, G. W. Colliver, Am. J. Physiol. 227, 583 (1974). 3. G. L. Florant, H. C. Heller, Am. J. Physiol. 232, R203 (1977). 4. B. M. Barnes, Science 244, 1593 (1989).

5. F. Geiser, T. Ruf, Physiol. Zool. 68, 935 (1995). 6. C. P. Lyman, J. S. Willis, A. Malan, L. C. H. Wang, Hibernation and Torpor in Mammals and Birds (Academic Press, New York, 1982). 7. T. N. Lee, B. M. Barnes, C. L. Buck, Ethol. Ecol. Evol. 21, 403 (2009). 8. B. D. Snapp, H. C. Heller, Physiol. Zool. 54, 297 (1981). 9. G. Heldmaier, T. Ruf, J. Comp. Physiol. B 162, 696 (1992). 10. C. L. Buck, B. M. Barnes, Am. J. Physiol. 279, R255 (2000). 11. F. Geiser, J. Comp. Physiol. B 158, 25 (1988). 12. Materials and methods are available as supporting material on Science Online. 13. G. E. Jr, Folk, J. M. Hunt, M. A. Folk, in Bears–Their Biology and Management, C. J. Martinka, K. L. McArthus, Eds. (Bear Biology Association, Tonto Basin, AZ, 1980), pp. 43–47. 14. C. G. von der Ohe, C. C. Garner, C. Darian-Smith, H. C. Heller, J. Neurosci. 27, 84 (2007). 15. K. H. Dausmann, J. Glos, J. U. Ganzhorn, G. Heldmaier, Nature 429, 825 (2004). 16. H. J. Harlow, T. Lohuis, R. C. Anderson-Sprecher, T. D. I. Beck, J. Mammal. 85, 414 (2004). 17. R. J. Hock, 2nd Alaskan Sci. Conf., AAAS 2, 310 (1951). 18. P. D. Watts, C. Cuyler, Acta Physiol. Scand. 134, 149 (1988). 19. R. Hissa, Ann. Zool. Fenn. 34, 267 (1997). 20. R. L. Rausch, Z. Saugetierkd. 26, 65 (1961). 21. V. Hayssen, R. C. Lacy, Comp. Biochem. Physiol. A 81, 741 (1985). 22. B. K. McNab, Comp. Biochem. Physiol. A 151, 5 (2008). 23. I. Hume et al., J. Comp. Physiol. B 172, 197 (2002). 24. F. Azizi, J. E. Mannix, D. Howard, R. A. Nelson, Am. J. Physiol. 237, E227 (1979).

LysM-Type Mycorrhizal Receptor Recruited for Rhizobium Symbiosis in Nonlegume Parasponia Rik Op den Camp,1 Arend Streng,1 Stéphane De Mita,1* Qingqin Cao,1† Elisa Polone,1,2 Wei Liu,1,3 Jetty S. S. Ammiraju,4 Dave Kudrna, Rod Wing,4 Andreas Untergasser,1‡ Ton Bisseling,1,5§ René Geurts1 Rhizobium–root nodule symbiosis is generally considered to be unique for legumes. However, there is one exception, and that is Parasponia. In this nonlegume, the rhizobial nodule symbiosis evolved independently and is, as in legumes, induced by rhizobium Nod factors. We used Parasponia andersonii to identify genetic constraints underlying evolution of Nod factor signaling. Part of the signaling cascade, downstream of Nod factor perception, has been recruited from the more-ancient arbuscular endomycorrhizal symbiosis. However, legume Nod factor receptors that activate this common signaling pathway are not essential for arbuscular endomycorrhizae. Here, we show that in Parasponia a single Nod factor–like receptor is indispensable for both symbiotic interactions. Therefore, we conclude that the Nod factor perception mechanism also is recruited from the widespread endomycorrhizal symbiosis. he rhizobial nodule symbiosis is widespread in the legume family (Fabaceae). Although this nitrogen-fixing symbiosis provides the plant with a major advantage, it is in principle restricted to a single family, and it is a major challenge for future agriculture to transfer this symbiosis to nonlegumes (1). The genus Parasponia could provide a key to this, because it encompasses the only nonlegume species that acquired also the rhizobium symbiosis (2, 3), where “rhizobium” refers to all species and genera that form nodules on legumes. Parasponia comprises several tropical tree species and belongs to Celtidaceae (4). Celtidaceae (order Rosales) and Fabaceae (order Fabales) are only remotely related.

T

Further, not a single species phylogenetically positioned between Parasponia and Fabaceae is able to establish such rhizobium symbiosis. Hence, in all probability the common ancestor of present Parasponia species gained the rhizobium-nodule symbiosis independent from legumes. Therefore, a legume-Parasponia comparison provides a key to identifying genetic constraints underlying this symbiosis. In this study, we focused on parallel evolution of the recognition of the rhizobial signal that starts the symbiotic interaction, the Nod factor. Parasponia makes lateral rootlike nodules that are associated with cell divisions in the root cortex (5). Rhizobium enters the Parasponia root intercellularly and becomes imbedded in a dense

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25. G. E. Jr, Folk, in Mammalian Hibernation, vol. III, K. Fisher, A. R. Dawe, C. P. Lyman, Schönbaum, F. E. Jr. South, Eds. (Oliver & Boyd, Edinburgh, Scotland, 1967), pp. 75–85. 26. O. L. Nelson, C. T. Robbins, J. Comp. Physiol. B 180, 465 (2010). 27. H. J. Harlow, T. Lohuis, T. D. I. Beck, P. A. Iaizzo, Nature 409, 997 (2001). 28. V. B. Fedorov et al., Physiol. Genomics 37, 108 (2009). 29. S. W. Donahue, M. R. Vaughan, L. M. Demers, H. J. Donahue, J. Exp. Biol. 206, 4233 (2003). 30. C. Shao et al., Mol. Cell. Proteomics 9, 313 (2010). 31. This work was supported by U.S. Army Medical Research and Materiel Command grant 05178001; NSF grants 9819540, 0076039, and 0732755; NIH HD-00973; and gift funds to Stanford University, American Heart Association #0020626Z, and the Fulbright Program. We thank the Alaska Department of Fish and Game for providing bears, D. Ritter for technical assistance, and J. Kenagy and J. Duman for comments.

Downloaded from www.sciencemag.org on February 17, 2011

show long torpor bouts interrupted by regular arousal episodes, black bears in Alaska exhibit distinct cyclic non-diurnal Tb patterns. Bear metabolism is reduced by 53% from BMR, even when Tb has returned to normothermic levels. These observations expand the phenotype of mammalian hibernation that occurs in diverse animals over body mass ranges from 0.005 to 200 kg. Insights into how hibernating bears achieve and cope with these reductions in energy need and Tb, as well as conservation of muscle (27, 28) and bone mass (29) despite prolonged seasonal inactivity and disuse, could lead to the development of novel clinical therapies. Current molecular and genetic approaches (28, 30) in combination with better physiological knowledge can increase our understanding of the regulation of hibernation in small and large hibernators and their evolution.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/906/DC1 Materials and Methods SOM Text Table S1 References 22 October 2010; accepted 13 January 2011 10.1126/science.1199435

matrix. Rhizobium obtains an intracellular lifestyle when it reaches a nodule primordium. There, cortical cells are infected via threadlike structures that remain connected to the plasma membrane. These so-called fixation threads branch, fill up the cells, and provide a niche to rhizobia to fix nitrogen (5). This is illustrated by the expression, in these threads, of the rhizobium nifH gene that encodes one of the subunits of nitrogenase (fig. S1). In contrast, rhizobia enter most legume roots via root hair–based intracellular infection threads, and the bacteria are released in nodule cells as membrane-surrounded nitrogen-fixing organelle-like structures (symbiosomes) that harbor a single or only a few bacteria. Legume nodules are considered to be genuine organs with a unique ontogeny (6). The fact that the Rhizobium symbiosis is very common in 65-million-year-old Fabaceae led to the conclusion that the symbiotic 1 Department of Plant Sciences, Laboratory of Molecular Biology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands. 2Department of Agricultural Biotechnologies, Universitá di Padova, Viale dell’Universitá 16, 35020 Legnaro (Padova), Italy. 3Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 4University of Arizona, Plant Sciences Department, 303 Forbes Building, Tucson, AZ 85721–0036, USA. 5College of Science, King Saud University, Post Office Box 2455, Riyadh 11451, Saudi Arabia.

*Present address: Institut de Recherche pour le Développement Montpellier, 911 Avenue, Agropolis BP 64501, 34394 Montpellier, cedex 5, France. †Present address: Department of Biotechnology, Beijing University of Agriculture, No. 7 Beinong Road, Huilongguan Changping District, Beijing, People’s Republic of China. ‡Present address: Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. §To whom correspondence should be addressed. E-mail: [email protected]

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interaction emerged as early as 60 million years ago (7). In contrast, the lateral rootlike nodule structure and more primitive rhizobium infections in Parasponia (5), together with the very close relation with the nonnodulating genus Trema (2–4), strongly suggest that Parasponia gained the Rhizobium symbiosis more recently than legumes. A key step in rhizobium symbiosis is the recognition by the host of bacterial Nod factors, which are specific lipochito-oligosaccharides. This holds for (almost) all nodulated legumes but also for Parasponia (8). This implies that a nonlegume species evolved independently from legumes, a Nod factor perception mechanism. In legumes, Nod factors are perceived by specific LysM receptor kinases that coevolved with the Nod factor structure of their host-specific rhizobium species (9–12). Legume Nod factor receptors activate a common signaling cascade that is shared with and recruited from the more common and far more ancient arbuscular mycorrhizal symbiosis (13, 14). This common signaling pathway comprises an additional plasma membrane receptor kinase, several components in the nuclear envelope including a cation ion channel and subunits of nuclear pores, and a nuclear-localized calcium/calmodulin-dependent kinase (CCaMK) (13, 14). Rhizobium- and mycorrhizae-induced signaling diverge downstream of CCaMK, possibly because of a different nature of the induced calcium spiking (14, 15). Because legume Nod factor receptors are not essential for mycorrhization, it is generally assumed that mycorrhizal symbiosis is controlled by other receptors specific for mycorrhizal signals (i.e., Myc factor). Such Myc factor receptors, like Nod factor receptors, are presumed to activate the common symbiotic signaling pathway (13–17). Two scenarios can be envisioned for how Nod factor receptors could have evolved. The complete mycorrhizal signaling pathway, including the Myc receptor, has been recruited by legumes, resulting in a common signaling pathway. In such a case, present Nod factor receptors have emerged upon gene duplication events and subsequently neofunctionalized during coevolution with specific rhizobium species. In this scenario, Myc receptors would be close homologs of known Nod factor receptors, as was argued previously (16, 17). However, such a scenario also implies that early in rhizobium symbiosis evolution a single receptor fulfilled a dual function, namely in mycorrhization as well as in Rhizobium symbiosis. A second scenario is that only the common signaling pathway devoid of a fungal-specific Myc receptor was recruited, and a novel receptor obtained the ability to activate this common signaling pathway upon Nod factor recognition. We favor the first hypothesis, because it is more simple and finds support in the fact that the chitooligosaccharide backbone of Nod factors is a “fungal” characteristic; chitin is a major component in fungal cell walls. The occurrence of Nod factor signaling in Parasponia provides a possibility to investigate this hypothesis.

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First, we confirmed and extended the idea that Parasponia-rhizobium symbiosis is induced by Nod factors. To this end, we used Parasponia andersonii, a species that can be nodulated by the broad host strain Sinorhizobium sp. NGR234 (18). A mutant of Sinorhizobium sp. NGR234 (NGR234DnodABC) that does not produce Nod factors was unable to trigger nodule formation or to infect roots of P. andersonii plantlets (0 of 30), whereas wild-type NGR234 does form nodules on ~40% of the plantlets [12 of 30; 8 weeks post inoculation (wpi)], similar as reported previously (5). Furthermore, root cortical cell divisions could be induced by local application of Nod factors (16 of 19; fig. S2). Next, we obtained evidence that, also in P. andersonii, the common symbiotic pathway is recruited to facilitate rhizobium symbiosis. A dominant active form of

Medicago truncatula MtCCaMK was introduced in P. andersonii roots (19). In legumes, CCaMK is a key element in the common symbiotic pathway, and dominant active forms of this kinase result in spontaneous nodulation in absence of rhizobia (20, 21). In P. andersonii, we also observed spontaneous formation of nodule-like structures (6 of 30, Fig. 1), indicating that activation of the common signaling pathway is sufficient to induce nodule organogenesis. These data suggest that in P. andersonii the common signaling pathway is activated upon Nod factor perception. In legumes, two different Nod factor receptor types are involved. One of these, MtLYK3/LjNFR1 in M. truncatula/Lotus japonicus, has several paralogous genes that resulted from recent duplication events (9, 11, 16, 17, 22, 23). In contrast, the second Nod factor receptor (MtNFP/

Fig. 1. P. andersonii spontaneous nodule-like structure triggered by dominant active MtCCaMK. Scale bars indicate 50 mm. (A) Nodule-like structure on atransgenic P.andersonii root (selected on the basis of red fluorescence resulting from DsRED1 expression). Scale bar indicates 0.5 mm. (B) Longitudinal section of spontaneous nodule-like structure. Nodule-like structure originates from cortical and pericycle cell layers and has a rudimentary stele (s), reflecting the lateral rootlike origin of P. andersonii nodules.

Fig. 2. Maximum-likelihood phylogeny of MtNFP/LjNFR5-like genes in the Rosid I (Fabidae) clade. P. andersonii (Pa), apple (Md), and castor bean (Rc) contain only a single gene, whereas in poplar (Pt), cucumber (Cs), and legumes (Gm/Lj/Mt) lineage-specific duplications have occurred. In legumes, LjNFR5, MtNFP, GmNFR5a, and GmNFR5b are Rhizobium Nod factor receptors. Branch lengths are proportional to the number of amino acid substitutions per site. Branch support was obtained from 1000 bootstrap repetitions. LjLYS16 and the closest MtNFP/LjNFR5 homolog in Oryza sativa (OsLYR1) were used as outgroups.

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REPORTS

REPORTS Also a substantial level of microsynteny in paralogous regions as well as a low level of nucleotide substitutions in paralogous gene pairs supports the recent nature of the duplication (17, 23). To determine whether this duplication predates the Fabaceae, we searched for MtNFP/LjNFR5-like sequences in a collection of cDNA clones from the basal legume Chamaecrista fasciculata (25). We identified a single clone (named CfNFP1) that is phylogenetically ancestral to the duplication observed in M. truncatula and L. japonicus (fig. S4). Therefore, we conclude that the duplication of MtNFP/LjNFR5 in the legume lineage was not essential to gain symbiosis with Rhizobium. Reverse transcription PCR studies revealed that PaNFP is expressed in roots (fig. S5). To study whether PaNFP has a symbiotic function, we performed RNA interference (RNAi) knockdown experiments (19). P. andersonii roots transformed with the empty vector (control roots) could be nodulated effectively with Sinorhizobium sp. NGR234 (Fig. 3A; 11 out of 30 plants formed nodules, and in total 55 nodules were formed 8 wpi). Transgenic P. andersonii roots that express a PaNFP RNAi construct have markedly reduced PaNFP expression levels (often below detection level, and, in cases where it is detected, it is ≥50% reduced; fig. S5). Inoculation of such RNAi roots with Sinorhizobium sp. NGR234 resulted only in a few nodules (PaNFP RNAi roots had 13 nodules on 30 plants, 8 wpi), and these were much smaller than nodules on control roots (Fig. 3, A and D). Sectioning of NFP RNAi nodules showed that they harbored rhizobia intercellularly, but fixation thread formation was com-

Fig. 3. Rhizobium nodulation and mycorrhization on P. andersonii control (A to C) and PaNFP RNAi knockdown (D to F) roots. (A) Control nodule. Scale bar, 1.0 mm. (B) Rhizobium fixation threads in control nodule. Scale bar, 10 mm. (C) Arbuscle in inner root cortical cell of (slightly squashed) control roots. Scale bar, 50 mm. (D) PaNFP RNAi nodule. Scale bar, 1.0 mm. (E) Aborted fixation threads in PaNFP RNAi nodule. Scale bar, 10 mm. (F) Aborted intracellular infection of Glomus intraradices in PaNFP RNAi root. Scale bar, 50 mm. www.sciencemag.org

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pletely blocked in all nodules investigated (n = 10) (Fig. 3, B and E). This demonstrated that PaNFP is involved in nodule formation and is essential for the switch to an intracellular lifestyle of rhizobia. Also in legumes, MtNFP/LjNFR5 is essential for nodule formation as well as intracellular accommodation of rhizobia (11, 12). On the basis of these results, we conclude that P. andersonii has recruited a gene orthologous to the MtNFP/LjNFR5 Nod factor receptor in legumes to control rhizobium symbiosis. This points to constraints in evolution of Nod factor perception mechanisms. As hypothesized above, a Nod factor receptor could have been recruited from the mycorrhizal signaling pathway. Because P. andersonii has only a single MtNFP/LjNFR5like gene, we determined whether PaNFP is also essential for endomycorrhization. PaNFP RNAi knockdown and control roots were inoculated with Glomus intraradices. This experiment showed that both are equally well infected by fungal hyphae. However, arbuscle formation is blocked in PaNFP RNAi roots, whereas in control roots arbuscules were effectively formed (Fig. 3, C and F, and fig. S6). PaNFP therefore is also essential for successful intracellular infection during arbuscle formation by mycorrhizal fungi. We conclude that in P. andersonii a single MtNFP/LjNFR5-like receptor, PaNFP, fulfills a dual symbiotic function and controls the intracellular life style of both arbuscular mycorrhizae fungi and rhizobium. Our findings in P. andersonii provide strong support for the hypothesis that during evolution a Myc factor receptor, as part of the common signaling cascade, was recruited to serve as Nod factor receptor in the rhizobial-plant symbiosis. Because in P. andersonii PaNFP fulfills a dual function, we suggest that only a few adaptions, if any at all, will have occurred to enable perception of a new ligand, rhizobium Nod factors. Also this result suggests that the Myc factor will have structural characteristics similar to those of Nod factors. In most legumes, MtNFP/LjNFR5 underwent at least one round of gene duplication (Fig. 2). However, our data suggest that this duplication occurred within the Papilionoideae subfamily of the Fabaceae (e.g., Medicago, Lotus, and Glycine), because CfNFP of Chamaecrista, as part of the basal Caesalpinioideae subfamily, is ancestral to the duplication events (fig. S4). Therefore it is likely that in Chamaecrista mycorrhization and Rhizobium symbiosis are controlled by just a single receptor, CfNFP. In more recent legumes like M. truncatula and L. japonicus, a duplication of this receptor has occurred, and only one of these has evolved as a Nod factor receptor. It seems very probable that the second copy functions as a Myc factor receptor. The bacterial genera collectively named rhizobium that evolved the ability to establish a nodule symbiosis, in general, acquired this by horizontal transfer of nod genes (26). This event allowed them to produce fungal-like molecules, namely Nod factors, by which they could use the ancient mechanism by which endomycorrhizal

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LjNFR5) has only one paralog in M. truncatula and L. japonicus (11, 23), and a putative orthologous gene is absent in Arabidopsis, a species that is unable to establish mycorrhizal symbiosis (11, 16, 17). Interestingly, in M. truncatula this paralog, MtLYR1, is transcriptionally up-regulated during mycorrhization (24). Therefore, we focused on the putative MtNFP/LjNFR5 orthologous gene in P. andersonii. To clone P. andersonii homologs a bacterial artificial chromosome (BAC) library was constructed and screened with MtNFP as probe. All eight positive BACs came from a single locus and shared the region containing one MtNFP/LjNFR5-like LysM receptor that we named PaNFP (Parasponia andersonii NOD FACTOR PERCEPTION). Southern blotting as well as sequencing of P. andersonii MtNFP/LjNFR5– like sequences generated by polymerase chain reaction (PCR) using degenerated primers and genomic DNA, as well as nodule and root cDNA, confirmed that P. andersonii has a single NFPlike gene. Next we searched for MtNFP/LjNFR5like genes in available genome sequences of other Fabidae (Rosid I) species (19). Apple (Malus x domestica), a close relative of P. andersonii, also has only a single MtNFP/LjNFR5-like gene that we named MdLYR1 [Malus x domestica LYKRELATED1 (11)]. Subsequent phylogenetic analysis revealed that PaNFP and MdLYR1 are close homologs of legume MtNFP/LjNFR5 and MtLYR1/ LjLYS11 (Fig. 2). On the basis of this result, we conclude that, in contrast to legumes, P. andersonii contains only a single MtNFP/LjNFR5-like gene. The legume-specific nature of the gene duplications is supported by the presence of two conserved deletions in the legume genes (fig. S3).

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References and Notes 1. V. Gewin, Nature 466, 552 (2010). 2. M. J. Trinick, Nature 244, 459 (1973). 3. A. D. L. Akkermans, S. Abdulkadir, M. J. Trinick, Nature 274, 190 (1978). 4. K. J. Sytsma et al., Am. J. Bot. 89, 1531 (2002). 5. G. Webster, P. R. Poulton, E. Cocking, M. R. Davey, J. Exp. Bot. 46, 1131 (1995).

6. K. Pawlowski, T. Bisseling, Plant Cell 8, 1899 (1996). 7. J. Sprent, J. Exp. Bot. 59, 1081 (2008). 8. D. J. Marvel, J. G. Torry, F. M. Ausubel, Proc. Natl. Acad. Sci. U.S.A. 84, 1319 (1987). 9. E. Limpens et al., Science 302, 630 (2003); 10.1126/science.1090074. 10. S. Radutoiu et al., Nature 425, 585 (2003). 11. J. F. Arrighi et al., Plant Physiol. 142, 265 (2006). 12. S. Radutoiu et al., EMBO J. 26, 392 (2007). 13. K. Markmann, M. Parniske, Trends Plant Sci. 14, 77 (2009). 14. G. E. D. Oldroyd, M. J. Harrison, U. Paszkowski, Science 324, 753 (2009). 15. S. Kosuta et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9823 (2008). 16. X. C. Zhang et al., Plant Physiol. 144, 623 (2007). 17. X. C. Zhang, S. B. Cannon, G. Stacey, BMC Evol. Biol. 9, 623 (2009). 18. S. G. Pueppke, W. J. Broughton, Mol. Plant Microbe Interact. 12, 293 (1999). 19. Materials and methods are available as supporting material on Science Online. 20. C. Gleason et al., Nature 441, 1149 (2006). 21. L. Tirichine et al., Nature 441, 1153 (2006).

The Antiproliferative Action of Progesterone in Uterine Epithelium Is Mediated by Hand2 Quanxi Li,1 Athilakshmi Kannan,1 Francesco J. DeMayo,2 John P. Lydon,2 Paul S. Cooke,1 Hiroyuki Yamagishi,3 Deepak Srivastava,4 Milan K. Bagchi,5* Indrani C. Bagchi1* During pregnancy, progesterone inhibits the growth-promoting actions of estrogen in the uterus. However, the mechanism for this is not clear. The attenuation of estrogen-mediated proliferation of the uterine epithelium by progesterone is a prerequisite for successful implantation. Our study reveals that progesterone-induced expression of the basic helix-loop-helix transcription factor Hand2 in the uterine stroma suppresses the production of several fibroblast growth factors (FGFs) that act as paracrine mediators of mitogenic effects of estrogen on the epithelium. In mouse uteri lacking Hand2, continued induction of these FGFs in the stroma maintains epithelial proliferation and stimulates estrogen-induced pathways, resulting in impaired implantation. Thus, Hand2 is a critical regulator of the uterine stromal-epithelial communication that directs proper steroid regulation conducive for the establishment of pregnancy. sequential and timely interplay of the steroid hormones 17b-estradiol (E) and progesterone (P) regulates critical uterine functions during the reproductive cycle and pregnancy (1–3). Whereas E drives uterine epithelial proliferation in cycling females, P counteracts E-induced endometrial hyperplasia. In mice, preovulatory ovarian E stimulates uterine epithelial growth and proliferation on days 1 and 2 of pregnancy (1). However, starting on day 3, P produced by the corpora lutea terminates E-mediated

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1 Department of Comparative Biosciences, University of Illinois Urbana/Champaign, Urbana, IL 61820, USA. 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. 3Keio University School of Medicine, Tokyo 160-8582, Japan. 4The Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94158, USA. 5Department of Molecular and Integrative Physiology, University of Illinois Urbana/Champaign, Urbana, IL 61820, USA.

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

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epithelial proliferation. In response to P, epithelial cells exit from the cell cycle and enter a differentiation pathway to acquire the receptive state that supports embryo implantation on day 4 of pregnancy (4–6). To identify the P-regulated pathways that underlie the implantation process, we had previously examined alterations in mouse uterine mRNA expression profiles in the periimplantation period in response to RU-486 (mifepristone), a well-characterized progesterone receptor (PR) antagonist (7). Our results identified Hand2, a critical regulator of morphogenesis in a variety of tissues (8, 9), as a potential PRregulated gene. Real-time polymerase chain reaction (PCR) confirmed that the expression of Hand2 mRNA was greatly reduced in the uteri of RU-486–treated mice (10) (fig. S1A). The expression of Hand2 protein, localized exclusively in the uterine stroma, was also abolished after RU-486 treatment (fig. S1B), which indicated that PR controls Hand2 expression in the mouse uterus during early pregnancy.

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22. H. Zhu, B. K. Riely, N. J. Burns, J. M. Ane, Genetics 172, 2491 (2006). 23. G. V. Lohmann et al., Mol. Plant Microbe Interact. 23, 510 (2010). 24. S. K. Gomez et al., BMC Plant Biol. 9, 10 (2009). 25. S. B. Cannon et al., PLoS One 5, e11630 (2010). 26. L. Moulin, A. Munive, B. Dreyfus, C. Boivin-Masson, Nature 411, 948 (2001). 27. We thank W. Broughton for NGR234; E. James, P. Hadobas, and T. J. Higgens for Parasponia seeds; P. van Dijk for flow cytometry; and J. Talag and W. Golser for BAC library construction. This research is funded by the Dutch Science Organization (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) (VIDI 864.06.007).

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1198181/DC1 Materials and Methods Figs. S1 to S16 References 23 September 2010; accepted 13 December 2010 Published online 23 December 2010; 10.1126/science.1198181

To further confirm P regulation of Hand2, ovaries were removed from nonpregnant mice, and then these animals were injected with either vehicle or P. We observed intense nuclear expression of Hand2 protein in uterine stromal cells after P treatment. Similar treatment of PR-null females showed no induction of Hand2 protein (Fig. 1A). These results established that P induces Hand2 expression in the uterine stroma. Consistent with its regulation by P, Hand2 expression was observed in the stromal cells underlying the luminal epithelium on days 3 and 4 of pregnancy (Fig. 1B). To investigate the function of Hand2 in the uterus, we created a conditional knockout of this gene in the adult uterine tissue. Crossing of mice harboring the “floxed” Hand2 gene (Hand2 f/f ) with PR-Cre mice (in which Cre recombinase was inserted into the PR gene) generated Hand2d/d mice in which the Hand2 gene is deleted selectively in cells expressing PR. As shown in fig. S2, Hand2 expression was successfully abrogated in uteri of Hand2d/d mice. A breeding study demonstrated that Hand2d/d females are infertile (table S1). An analysis of the ovulation and fertilization in Hand2f/f and Hand2d/d females revealed no significant difference in either the number or the morphology of the embryos recovered from their uteri (fig. S3, A and B). The serum levels of P and E were comparable in Hand2f/f and Hand2d/d females on day 4 of pregnancy, which indicated normal ovarian function (fig. S3, C and D). We next examined embryo attachment to the uterine epithelium by using the blue dye assay, which assesses increased vascular permeability at implantation sites. Hand2f/f mice displayed distinct blue bands, indicative of implantation sites on day 5 of pregnancy (fig. S4). In contrast, none of the Hand2d/d females showed any sign of implantation. Implanted embryos with decidual swellings were also absent in Hand2d/d uteri on days 6 and 7 of pregnancy. Histological analysis of Hand2f/f females on day 5 of pregnancy

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fungi establish an intracellular life style and turned these rhizobia from free-living bacteria into nitrogen-fixing endosymbionts. However, although the endomycorrhizal symbiosis is widespread in the plant kingdom only very few plant lineages, namely legumes and Parasponia, have recruited this mechanism for the rhizobial nodule symbiosis. Studies on the constraints underlying this evolutionary event in Parasponia can provide insight into whether and how this nitrogen-fixing symbiosis can be transferred to other nonlegumes.

REPORTS

References and Notes 1. V. Gewin, Nature 466, 552 (2010). 2. M. J. Trinick, Nature 244, 459 (1973). 3. A. D. L. Akkermans, S. Abdulkadir, M. J. Trinick, Nature 274, 190 (1978). 4. K. J. Sytsma et al., Am. J. Bot. 89, 1531 (2002). 5. G. Webster, P. R. Poulton, E. Cocking, M. R. Davey, J. Exp. Bot. 46, 1131 (1995).

6. K. Pawlowski, T. Bisseling, Plant Cell 8, 1899 (1996). 7. J. Sprent, J. Exp. Bot. 59, 1081 (2008). 8. D. J. Marvel, J. G. Torry, F. M. Ausubel, Proc. Natl. Acad. Sci. U.S.A. 84, 1319 (1987). 9. E. Limpens et al., Science 302, 630 (2003); 10.1126/science.1090074. 10. S. Radutoiu et al., Nature 425, 585 (2003). 11. J. F. Arrighi et al., Plant Physiol. 142, 265 (2006). 12. S. Radutoiu et al., EMBO J. 26, 392 (2007). 13. K. Markmann, M. Parniske, Trends Plant Sci. 14, 77 (2009). 14. G. E. D. Oldroyd, M. J. Harrison, U. Paszkowski, Science 324, 753 (2009). 15. S. Kosuta et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9823 (2008). 16. X. C. Zhang et al., Plant Physiol. 144, 623 (2007). 17. X. C. Zhang, S. B. Cannon, G. Stacey, BMC Evol. Biol. 9, 623 (2009). 18. S. G. Pueppke, W. J. Broughton, Mol. Plant Microbe Interact. 12, 293 (1999). 19. Materials and methods are available as supporting material on Science Online. 20. C. Gleason et al., Nature 441, 1149 (2006). 21. L. Tirichine et al., Nature 441, 1153 (2006).

The Antiproliferative Action of Progesterone in Uterine Epithelium Is Mediated by Hand2 Quanxi Li,1 Athilakshmi Kannan,1 Francesco J. DeMayo,2 John P. Lydon,2 Paul S. Cooke,1 Hiroyuki Yamagishi,3 Deepak Srivastava,4 Milan K. Bagchi,5* Indrani C. Bagchi1* During pregnancy, progesterone inhibits the growth-promoting actions of estrogen in the uterus. However, the mechanism for this is not clear. The attenuation of estrogen-mediated proliferation of the uterine epithelium by progesterone is a prerequisite for successful implantation. Our study reveals that progesterone-induced expression of the basic helix-loop-helix transcription factor Hand2 in the uterine stroma suppresses the production of several fibroblast growth factors (FGFs) that act as paracrine mediators of mitogenic effects of estrogen on the epithelium. In mouse uteri lacking Hand2, continued induction of these FGFs in the stroma maintains epithelial proliferation and stimulates estrogen-induced pathways, resulting in impaired implantation. Thus, Hand2 is a critical regulator of the uterine stromal-epithelial communication that directs proper steroid regulation conducive for the establishment of pregnancy. sequential and timely interplay of the steroid hormones 17b-estradiol (E) and progesterone (P) regulates critical uterine functions during the reproductive cycle and pregnancy (1–3). Whereas E drives uterine epithelial proliferation in cycling females, P counteracts E-induced endometrial hyperplasia. In mice, preovulatory ovarian E stimulates uterine epithelial growth and proliferation on days 1 and 2 of pregnancy (1). However, starting on day 3, P produced by the corpora lutea terminates E-mediated

A

1 Department of Comparative Biosciences, University of Illinois Urbana/Champaign, Urbana, IL 61820, USA. 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. 3Keio University School of Medicine, Tokyo 160-8582, Japan. 4The Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94158, USA. 5Department of Molecular and Integrative Physiology, University of Illinois Urbana/Champaign, Urbana, IL 61820, USA.

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

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epithelial proliferation. In response to P, epithelial cells exit from the cell cycle and enter a differentiation pathway to acquire the receptive state that supports embryo implantation on day 4 of pregnancy (4–6). To identify the P-regulated pathways that underlie the implantation process, we had previously examined alterations in mouse uterine mRNA expression profiles in the periimplantation period in response to RU-486 (mifepristone), a well-characterized progesterone receptor (PR) antagonist (7). Our results identified Hand2, a critical regulator of morphogenesis in a variety of tissues (8, 9), as a potential PRregulated gene. Real-time polymerase chain reaction (PCR) confirmed that the expression of Hand2 mRNA was greatly reduced in the uteri of RU-486–treated mice (10) (fig. S1A). The expression of Hand2 protein, localized exclusively in the uterine stroma, was also abolished after RU-486 treatment (fig. S1B), which indicated that PR controls Hand2 expression in the mouse uterus during early pregnancy.

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22. H. Zhu, B. K. Riely, N. J. Burns, J. M. Ane, Genetics 172, 2491 (2006). 23. G. V. Lohmann et al., Mol. Plant Microbe Interact. 23, 510 (2010). 24. S. K. Gomez et al., BMC Plant Biol. 9, 10 (2009). 25. S. B. Cannon et al., PLoS One 5, e11630 (2010). 26. L. Moulin, A. Munive, B. Dreyfus, C. Boivin-Masson, Nature 411, 948 (2001). 27. We thank W. Broughton for NGR234; E. James, P. Hadobas, and T. J. Higgens for Parasponia seeds; P. van Dijk for flow cytometry; and J. Talag and W. Golser for BAC library construction. This research is funded by the Dutch Science Organization (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) (VIDI 864.06.007).

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1198181/DC1 Materials and Methods Figs. S1 to S16 References 23 September 2010; accepted 13 December 2010 Published online 23 December 2010; 10.1126/science.1198181

To further confirm P regulation of Hand2, ovaries were removed from nonpregnant mice, and then these animals were injected with either vehicle or P. We observed intense nuclear expression of Hand2 protein in uterine stromal cells after P treatment. Similar treatment of PR-null females showed no induction of Hand2 protein (Fig. 1A). These results established that P induces Hand2 expression in the uterine stroma. Consistent with its regulation by P, Hand2 expression was observed in the stromal cells underlying the luminal epithelium on days 3 and 4 of pregnancy (Fig. 1B). To investigate the function of Hand2 in the uterus, we created a conditional knockout of this gene in the adult uterine tissue. Crossing of mice harboring the “floxed” Hand2 gene (Hand2 f/f ) with PR-Cre mice (in which Cre recombinase was inserted into the PR gene) generated Hand2d/d mice in which the Hand2 gene is deleted selectively in cells expressing PR. As shown in fig. S2, Hand2 expression was successfully abrogated in uteri of Hand2d/d mice. A breeding study demonstrated that Hand2d/d females are infertile (table S1). An analysis of the ovulation and fertilization in Hand2f/f and Hand2d/d females revealed no significant difference in either the number or the morphology of the embryos recovered from their uteri (fig. S3, A and B). The serum levels of P and E were comparable in Hand2f/f and Hand2d/d females on day 4 of pregnancy, which indicated normal ovarian function (fig. S3, C and D). We next examined embryo attachment to the uterine epithelium by using the blue dye assay, which assesses increased vascular permeability at implantation sites. Hand2f/f mice displayed distinct blue bands, indicative of implantation sites on day 5 of pregnancy (fig. S4). In contrast, none of the Hand2d/d females showed any sign of implantation. Implanted embryos with decidual swellings were also absent in Hand2d/d uteri on days 6 and 7 of pregnancy. Histological analysis of Hand2f/f females on day 5 of pregnancy

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fungi establish an intracellular life style and turned these rhizobia from free-living bacteria into nitrogen-fixing endosymbionts. However, although the endomycorrhizal symbiosis is widespread in the plant kingdom only very few plant lineages, namely legumes and Parasponia, have recruited this mechanism for the rhizobial nodule symbiosis. Studies on the constraints underlying this evolutionary event in Parasponia can provide insight into whether and how this nitrogen-fixing symbiosis can be transferred to other nonlegumes.

REPORTS the uteri of Hand2d/d mice. However, the expression of leukemia inhibitory factor (Lif ), a glandular factor that regulates uterine receptivity (2), was significantly reduced in Hand2d/d uteri (fig. S8). Down-regulation of Muc-1 in the luminal epithelium is indicative of a receptive uterus (12). In contrast, persistent Muc-1 expression impairs acquisition of uterine receptivity and embryo implantation. On days 4 and 5 of gestation, a marked reduction in Muc-1 level was seen in uterine epithelia of Hand2f/f mice, consistent with the attainment of receptive status (fig. S9). However, Muc1 expression persisted in uteri of Hand2d/d mice at the time of implantation. Thus, elevated epithelial ER signaling led to increased expression of Muc-1 and corresponded to disrupted uterine receptivity and implantation failure in Hand2d/d mice. In normal pregnant uteri, the receptive state is also marked by a cessation in epithelial cell proliferation before implantation (1, 2). As expected, in Hand2f/f mice, Ki-67, a cell proliferation marker, was undetectable in the uterine epithelium as it attains receptive status on day 4 of pregnancy (Fig. 2B, a). Hand2d/d uteri, on the other hand, exhibited robust Ki-67 expression in the luminal epithelium, which indicated sustained epithelial cell proliferation in the absence of Hand2 (Fig. 2B, b). The persistent proliferative state of uterine epithelium in the Hand2d/d mice raised the possibility that stromal expression of Hand2 mediates P action that opposes E-mediated epithelial proliferation. Administration of E to ovariectomized Hand2f/f and Hand2d/d mice led to robust uterine epithelial proliferation (Fig. 2C, a and b). Treatment with P alone induced proliferation exclusively in the uterine stromal cells of

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both genotypes (Fig. 2C, c and d). In Hand2f/f mice pretreated with P, administration of E showed no proliferative activity in the epithelium, which suggested a complete blockade of E-dependent proliferation by P. Under similar treatment conditions, Hand2d/d uteri exhibited a marked E-induced epithelial proliferation, indicating an absence of antiproliferative effects of P on the uterine epithelium (Fig. 2C, e and f). These results established Hand2 as a critical mediator of the actions of P in the stroma that inhibit E-dependent epithelial proliferation. To identify downstream target(s) of Hand2 in the uterus, we performed gene expression profiling of uterine stromal cells isolated from Hand2f/f and Hand2d/d mice on day 4 of pregnancy. This study revealed elevated expression of mRNAs corresponding to several members of the fibroblast growth factor family (FGFs)—namely, Fgf1, Fgf2, Fgf9, and Fgf18—in uterine stroma of Hand2d/d mice. Real-time PCR confirmed the induction of Fgf1, Fgf9, Fgf2, and Fgf18 mRNAs in uterine stromal cells of Hand2d/d mice (Fig. 3A). The expression of Hbegf mRNA, encoding the heparin-binding epidermal growth factor (HB-EGF), was also increased in the mutant uteri (fig. S10). In contrast, the expression of mRNAs of several other growth factor genes was either unaffected or slightly reduced in the Hand2-null uteri (fig. S10). We also observed that the uterine expression of Fgf2, Fgf9, and Fgf18 progressively declined with the rise of Hand2 expression as pregnancy advanced from day 1 to day 4 (fig. S11). FGFs exert their paracrine responses through the cell surface FGF receptors (FGFRs) and a

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showed, as expected, a close contact of embryonic trophectoderm with uterine luminal epithelium (Fig. 1C, a and b). In contrast, in Hand2d/d uteri, blastocysts remained unattached in the lumen (Fig. 1C, c and d). These results suggested that, in the absence of Hand2 expression in the stroma, the luminal epithelium fails to acquire competency for embryo implantation. In mice, the window of uterine receptivity coincides with the P-mediated down-regulation of ER activity in uterine luminal epithelium (5, 6). The levels of PR and estrogen receptor a (ERa) proteins in the luminal epithelium or stroma of Hand2 d/d uteri were comparable to those of Hand2 f/f controls (fig. S5). An examination of the phosphorylation of ERa at serine 118, indicative of its transcriptionally active state (11), revealed a sharp reduction of this modification in the luminal epithelial cells of Hand2 f/f uteri on days 3 and 4 of pregnancy (fig. S6, a to d). In contrast, an increase in ERa phosphorylation was evident on these days in luminal epithelium of Hand2 d/d uteri (fig. S6, e to h). Consistent with this increase in ER transcriptional activity, expression of mRNAs corresponding to mucin 1 (Muc-1) and lactoferrin (Lf ), well-characterized E-responsive genes in uterine epithelium (12), was significantly elevated in the Hand2-null uterus on day 4 of pregnancy (Fig. 2A). In contrast, the expression of Ihh (13), Alox15 (7), and Irg1 (7), which are known P-responsive genes in uterine epithelium, remained unaltered in Hand2d/d uteri (fig. S7). In addition, the mRNA levels of Hoxa10, a P-regulated stromal factor (3), and Nr2f2, a downstream target of Ihh in the uterine stroma (13), were unaffected in

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Fig. 1. P-regulated expression of Hand2 in the uterus is critical for implantation. (A) Immunohistochemistry (IHC) of Hand2 protein in the uterine sections of ovariectomized WT mice treated with vehicle (a) or P (b) and PR-null mice treated with P (c). (d) Sections treated with nonimmune immunoglobulin G (IgG). (B) Uterine sections obtained from mice on days 2 to 4 (D2 to D4) of pregnancy were subjected to IHC using an antibody against Hand2. Magnification, 20×. (C) Hematoxylin-andeosin staining of uterine sections obtained from Hand2f/f (a and b) and d/d Hand2 (c and d) mice on day 5 (n = 6) of pregnancy. (b) and (d) Magnified images of (a) and (c), respectively. Wide and narrow arrows point to embryo and luminal epithelium. L and S represent luminal epithelium and stroma, respectively.

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Fig. 2. Enhanced ERa activity and proliferation in the luminal epithelium of Hand2d/d uteri. (A) Real-time PCR was performed to monitor the expression of Muc1 and Ltf in the uteri of day 4 pregnant Hand2f/f and Hand2d/d mice, *P < 0.001. (B) IHC of Ki-67 in Hand2f/f (a) and Hand2d/d (b) uteri on day 4 of pregnancy, 20×. (c) Uterine sections from Hand2d/d mice treated with nonimmune IgG, 40×. (C) IHC of Ki-67 in the uterine sections of ovariectomized Hand2f/f and Hand2d/d mice treated with E for 1 day (a) and (b), P for 3 days (c) and (d), or 2 days of P treatment followed by P and E (e) and (f).

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Fig. 3. Enhanced FGFR signaling in the luminal epithelium of Hand2d/d uteri. (A) Relative level of mRNA expression of FGF family of growth factors in the uterine stroma of Hand2f/f and Hand2d/d mice on day 4 of pregnancy, * P < 0.001. The levels of p-FRS2 (B) and p-ERK1/2 (C) were examined in the uterine sections (n = 3) of both genotypes by IHC. (a and b) Uterine sections obtained from day 4 pregnant mice. (c and d) Uterine sections from ovariectomized mice treated with 2 days of P followed by P and E.

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Fig. 4. The inhibitor PD173074 (A) or PD184352 (B) was administered to one uterine horn of Hand2d/d mice on day 3 of pregnancy (n = 5). The other horn served as vehicle-treated control. Uterine horns were collected on the morning of day 4, and sections were subjected to IHC to detect p-FRS2, p-ERK1/2, and Ki-67. (C) IHC of pERa and Muc-1 in uterine sections of Hand2d/d mice treated with PD173074 or PD184352.

FRS2 was undetectable in uterine epithelium of ovariectomized Hand2f/f mice in which P effectively blocks E-mediated proliferation (Fig. 3B, c). However, the epithelial cells of Hand2null uteri, which showed proliferative activity under similar hormone treatment conditions, exhibited a marked elevation in the level of phosphoFRS2, reflecting the activation of FGFR signaling in these cells (Fig. 3B, d). We also monitored the activated phosphorylated states of ErbB1 and ErbB4, the primary receptors mediating the actions of Hbegf (15). The activated forms of these receptors were undetectable in uterine epithelia of Hand2f/f and Hand2d/d mice on day 4 of gestation (fig. S13), which indicated that the increased HBEGF produced by the stroma of Hand2-null uteri did not act directly on the epithelial cells. We next investigated whether the ERK1/2 and/or PI3K/Akt signaling pathways were activated downstream of FGFR in the epithelia of Hand2-ablated uteri. As shown in Fig. 3C, an increase in the level of phospho-ERK1/2 was seen in uterine epithelium of Hand2-null mice on day 4 of pregnancy (Fig. 3C, b) and also in response to E treatment in the presence of P (Fig. 3C, d). In

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contrast, the phospho-AKT levels were undetectable or low and remained unaltered in the uterine epithelia of these mice (fig. S14), which suggested that the ERK1/2 pathway, but not the PI3K/Akt pathway, is the key downstream mediator of enhanced FGF signaling in Hand2-null uteri. To examine whether the elevated mitogenic activity in the luminal epithelium of Hand2d/d uteri is induced by FGF and ERK1/2 signaling, we administered PD173074 [a FGFR-specific inhibitor (16)] or vehicle into uterine horns of Hand2d/d mice at the time of implantation. The epithelia of vehicle-treated horn showed prominent expression of p-FRS2 and p-ERK1/2 (Fig. 4A, a and c). However, the levels of both p-FRS2 and p-ERK1/2 were reduced in the epithelia of PD173074-treated horn on day 4 of pregnancy (Fig. 4A, b and d). We also observed a marked decline in the proliferative activity of Hand2-null uterine epithelia, as indicated by decreased Ki-67 staining (Fig. 4A, e and f). In parallel experiments, administration of PD184352, an inhibitor of the ERK1/2 pathway (17), to uterine horns of Hand2d/d mice suppressed the level of pERK1/2 (Fig. 4B, a and b), as well as luminal epithelial

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docking protein complex (14). FGF-stimulation of FGFRs induces phosphorylation of specific tyrosine residues in a critical docking protein, FGFR substrate2 (FRS2), which guides the assembly of distinct multiprotein complexes, leading to activation of either extracellular signal–regulated kinase (ERK) 1 and 2 (ERK1/2) or phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling cascades (14). We determined the expression of FGFRs 1 and 2 in uteri on days 1 and 4 of pregnancy and found that these receptors localized to the epithelium (fig. S12A). The levels of mRNAs corresponding to Fgfr 1 to 3 were comparable in uteri of Hand2f/f and Hand2d/d mice on day 4 of pregnancy (fig. S12B). The activation of the FGFR signaling pathway in uteri of Hand2f/f and Hand2d/d mice was monitored by examining the tyrosine phosphorylation status of FRS2. Whereas only low levels of phospho-FRS2 were seen in the uterine epithelium of Hand2f/f mice on day 4 of pregnancy (Fig. 3B, a), a marked increase in its level was observed in the epithelium of Hand2-null uteri, indicating that FGF signaling is activated in the absence of Hand2 (Fig. 3B, b). Phospho-

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REPORTS cation remained unknown. Our study has delineated a pathway in which Hand2 operates downstream of P to regulate the production of FGFs, mitogenic paracrine signals that originate in the stroma and act on the FGFR(s) in epithelium to control its E responsiveness (fig. S15). The antiproliferative action of P in uterine epithelium is of clinical significance, because the breakdown of this action underpins E-dependent endometrial cancer (19). Hand2, therefore, is an important factor to be considered for hormone therapy to block the proliferative actions of E in the endometrium.

References and Notes 1. C. A. Finn, L. Martin, J. Reprod. Fertil. 39, 195 (1974). 2. D. D. Carson et al., Dev. Biol. 223, 217 (2000). 3. C. Y. Ramathal, I. C. Bagchi, R. N. Taylor, M. K. Bagchi, Semin. Reprod. Med. 28, 17 (2010). 4. L. Martin, R. M. Das, C. A. Finn, J. Endocrinol. 57, 549 (1973). 5. I. C. Bagchi, Y. P. Cheon, Q. Li, M. K. Bagchi, Front. Biosci. 8, s852 (2003). 6. H. Pan, Y. Deng, J. W. Pollard, Proc. Natl. Acad. Sci. U.S.A. 103, 14021 (2006). 7. I. C. Bagchi et al., Semin. Reprod. Med. 23, 38 (2005). 8. D. Srivastava et al., Nat. Genet. 16, 154 (1997). 9. A. B. Firulli, Gene 312, 27 (2003).

Distinct Properties of the XY Pseudoautosomal Region Crucial for Male Meiosis Liisa Kauppi,1 Marco Barchi,2,3 Frédéric Baudat,2* Peter J. Romanienko,2 Scott Keeney,1,4† Maria Jasin2† Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11b isoform–specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination. eiotic recombination, initiated by programmed double-strand breaks (DSBs), promotes homologous chromosome (homolog) pairing during prophase I (1). A subset of DSBs matures into crossovers that physically

M

1 Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 2Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 3Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, 00133 Rome, Italy. 4Howard Hughes Medical Institute, Memorial SloanKettering Cancer Center, New York, NY 10065, USA.

*Present address: Institute of Human Genetics, CNRS, 34090 Montpellier, France. †To whom correspondence should be addressed. E-mail: [email protected] (M.J.); [email protected] (S.K.)

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connect homologs so that they orient properly on the first meiotic spindle. Because sex chromosome recombination and pairing are restricted to the PAR (2), at least one DSB must form within this small region, and the homologous PAR must be located and engaged in recombination to lead to a crossover. Accordingly, the PAR in males exhibits high crossover frequency (2, 3), but sex chromosomes also missegregate more frequently than autosomes (4). Nevertheless, X-Y nondisjunction is rare, which suggests that there are mechanisms that ensure successful X-Y recombination. X-Y pairing is more challenging than autosomal pairing, as it cannot be mediated by multiple DNA interactions along the length of the chromosomes. We used fluorescence in situ hy-

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10. Materials and methods are available as supporting material on Science Online. 11. S. Kato et al., Science 270, 1491 (1995). 12. G. A. Surveyor et al., Endocrinology 136, 3639 (1995). 13. K. Y. Lee et al., Nat. Genet. 38, 1204 (2006). 14. V. P. Eswarakumar, I. Lax, J. Schlessinger, Cytokine Growth Factor Rev. 16, 139 (2005). 15. R. Iwamoto, E. Mekada, Cytokine Growth Factor Rev. 11, 335 (2000). 16. M. Koziczak, T. Holbro, N. E. Hynes, Oncogene 23, 3501 (2004). 17. D. B. Solit et al., Nature 439, 358 (2006). 18. T. Kurita et al., Endocrinology 139, 4708 (1998). 19. J. J. Kim, E. Chapman-Davis, Semin. Reprod. Med. 28, 81 (2010). 20. We thank M. Laws for genotyping and Y. Li for immunohistochemistry. This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, through U54HD055787 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. The Gene Expression Omnibus (GEO) microarray accession number is GSE25881.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/912/DC1 Materials and Methods Figs. S1 to S15 Table S1 References 7 September 2010; accepted 15 December 2010 10.1126/science.1197454

bridization (FISH) (5) to compare timing of meiotic X-Y and autosomal pairing in mice (Fig. 1). At leptonema, when DSBs begin to form and only short chromosome axis segments are present, PAR and autosomal FISH probes were mostly unpaired. By early to mid-zygonema, when axes elongate and homologs become juxtaposed, distal ends of chr 18 and 19 were paired in ~50% of nuclei; by late zygonema, these regions were paired in nearly all nuclei (Fig. 1B and fig. S1). In contrast, the X and Y PARs were rarely paired before pachynema (Fig. 1B); hence, X-Y pairing is delayed compared with that of autosomes. DSBs precede and are required for efficient homolog pairing in mouse meiosis (6, 7). Nucleuswide (“global”) foci of DSB markers RAD51/ DMC1 peak in number at early to mid-zygonema (Fig. 2A) (8, 9). Because stable X-Y pairing occurs late, we asked whether PAR DSB kinetics is also delayed (Fig. 2B and fig. S2). More than half of cells had no RAD51/DMC1 focus in the PAR before late zygonema (Fig. 2C), distinct from global patterns. Only when global foci were already declining did the majority of cells (~70%) display PAR foci (Fig. 2C and fig. S2i). We interpret the lack of PAR foci to indicate that DSBs have not yet formed. Thus, we propose that PAR DSB formation and/or turnover are under distinct temporal control. We cannot exclude the alternative possibility that PAR DSBs have formed but are cytologically undetectable, for example, because RAD51/DMC1 have not yet been loaded onto DSB ends or because foci have already turned over. In either case, DSB dynamics and/or processing differs on the PAR. Most sites marked by PAR RAD51/DMC1 foci appeared incapable of mediating stable pairing before early pachynema (~70% of late zygotene

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proliferation (Fig. 4B, c and d). Collectively these results are consistent with the hypothesis that increased FGF production by the Hand2-null uterine stroma stimulates epithelial proliferation by activating the FGFR-ERK1/2 pathway. The ERK1/2-dependent phosphorylation of epithelial ERa at Ser118 is critical for the transcriptional activation of ERa (11). Administration of either PD173074 (Fig. 4C, a to d) or PD184352 (Fig. 4C, e to h) to Hand2-null uterine horns blocked the phosphorylation of epithelial ERa at Ser118 and the expression of Muc-1. This result supported our view that elevated signaling by FGFR-ERK1/2 pathway in Hand2d/d uteri is responsible for phosphorylation and activation of ERa in epithelial cells, which promotes persistent expression of Muc-1 and which in turn creates a barrier that prevents embryo attachment. Earlier studies using tissue recombinants prepared with uterine epithelium and stroma isolated from neonatal wild-type and PR-null mice indicated that the stromal PR plays an obligatory role in mediating the inhibitory actions of P on E-induced epithelial cell proliferation (18). However, the mechanism of this stromal-epithelial communi-

REPORTS cation remained unknown. Our study has delineated a pathway in which Hand2 operates downstream of P to regulate the production of FGFs, mitogenic paracrine signals that originate in the stroma and act on the FGFR(s) in epithelium to control its E responsiveness (fig. S15). The antiproliferative action of P in uterine epithelium is of clinical significance, because the breakdown of this action underpins E-dependent endometrial cancer (19). Hand2, therefore, is an important factor to be considered for hormone therapy to block the proliferative actions of E in the endometrium.

References and Notes 1. C. A. Finn, L. Martin, J. Reprod. Fertil. 39, 195 (1974). 2. D. D. Carson et al., Dev. Biol. 223, 217 (2000). 3. C. Y. Ramathal, I. C. Bagchi, R. N. Taylor, M. K. Bagchi, Semin. Reprod. Med. 28, 17 (2010). 4. L. Martin, R. M. Das, C. A. Finn, J. Endocrinol. 57, 549 (1973). 5. I. C. Bagchi, Y. P. Cheon, Q. Li, M. K. Bagchi, Front. Biosci. 8, s852 (2003). 6. H. Pan, Y. Deng, J. W. Pollard, Proc. Natl. Acad. Sci. U.S.A. 103, 14021 (2006). 7. I. C. Bagchi et al., Semin. Reprod. Med. 23, 38 (2005). 8. D. Srivastava et al., Nat. Genet. 16, 154 (1997). 9. A. B. Firulli, Gene 312, 27 (2003).

Distinct Properties of the XY Pseudoautosomal Region Crucial for Male Meiosis Liisa Kauppi,1 Marco Barchi,2,3 Frédéric Baudat,2* Peter J. Romanienko,2 Scott Keeney,1,4† Maria Jasin2† Meiosis requires that each chromosome find its homologous partner and undergo at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11b isoform–specific transgenic mice revealed that late RAD51/DMC1 foci in the PAR are genetically distinct from both early PAR foci and global foci and that late PAR foci promote efficient X-Y pairing, recombination, and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination. eiotic recombination, initiated by programmed double-strand breaks (DSBs), promotes homologous chromosome (homolog) pairing during prophase I (1). A subset of DSBs matures into crossovers that physically

M

1 Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 2Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. 3Department of Public Health and Cell Biology, Section of Anatomy, University of Rome Tor Vergata, 00133 Rome, Italy. 4Howard Hughes Medical Institute, Memorial SloanKettering Cancer Center, New York, NY 10065, USA.

*Present address: Institute of Human Genetics, CNRS, 34090 Montpellier, France. †To whom correspondence should be addressed. E-mail: [email protected] (M.J.); [email protected] (S.K.)

916

connect homologs so that they orient properly on the first meiotic spindle. Because sex chromosome recombination and pairing are restricted to the PAR (2), at least one DSB must form within this small region, and the homologous PAR must be located and engaged in recombination to lead to a crossover. Accordingly, the PAR in males exhibits high crossover frequency (2, 3), but sex chromosomes also missegregate more frequently than autosomes (4). Nevertheless, X-Y nondisjunction is rare, which suggests that there are mechanisms that ensure successful X-Y recombination. X-Y pairing is more challenging than autosomal pairing, as it cannot be mediated by multiple DNA interactions along the length of the chromosomes. We used fluorescence in situ hy-

18 FEBRUARY 2011

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10. Materials and methods are available as supporting material on Science Online. 11. S. Kato et al., Science 270, 1491 (1995). 12. G. A. Surveyor et al., Endocrinology 136, 3639 (1995). 13. K. Y. Lee et al., Nat. Genet. 38, 1204 (2006). 14. V. P. Eswarakumar, I. Lax, J. Schlessinger, Cytokine Growth Factor Rev. 16, 139 (2005). 15. R. Iwamoto, E. Mekada, Cytokine Growth Factor Rev. 11, 335 (2000). 16. M. Koziczak, T. Holbro, N. E. Hynes, Oncogene 23, 3501 (2004). 17. D. B. Solit et al., Nature 439, 358 (2006). 18. T. Kurita et al., Endocrinology 139, 4708 (1998). 19. J. J. Kim, E. Chapman-Davis, Semin. Reprod. Med. 28, 81 (2010). 20. We thank M. Laws for genotyping and Y. Li for immunohistochemistry. This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, through U54HD055787 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. The Gene Expression Omnibus (GEO) microarray accession number is GSE25881.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/912/DC1 Materials and Methods Figs. S1 to S15 Table S1 References 7 September 2010; accepted 15 December 2010 10.1126/science.1197454

bridization (FISH) (5) to compare timing of meiotic X-Y and autosomal pairing in mice (Fig. 1). At leptonema, when DSBs begin to form and only short chromosome axis segments are present, PAR and autosomal FISH probes were mostly unpaired. By early to mid-zygonema, when axes elongate and homologs become juxtaposed, distal ends of chr 18 and 19 were paired in ~50% of nuclei; by late zygonema, these regions were paired in nearly all nuclei (Fig. 1B and fig. S1). In contrast, the X and Y PARs were rarely paired before pachynema (Fig. 1B); hence, X-Y pairing is delayed compared with that of autosomes. DSBs precede and are required for efficient homolog pairing in mouse meiosis (6, 7). Nucleuswide (“global”) foci of DSB markers RAD51/ DMC1 peak in number at early to mid-zygonema (Fig. 2A) (8, 9). Because stable X-Y pairing occurs late, we asked whether PAR DSB kinetics is also delayed (Fig. 2B and fig. S2). More than half of cells had no RAD51/DMC1 focus in the PAR before late zygonema (Fig. 2C), distinct from global patterns. Only when global foci were already declining did the majority of cells (~70%) display PAR foci (Fig. 2C and fig. S2i). We interpret the lack of PAR foci to indicate that DSBs have not yet formed. Thus, we propose that PAR DSB formation and/or turnover are under distinct temporal control. We cannot exclude the alternative possibility that PAR DSBs have formed but are cytologically undetectable, for example, because RAD51/DMC1 have not yet been loaded onto DSB ends or because foci have already turned over. In either case, DSB dynamics and/or processing differs on the PAR. Most sites marked by PAR RAD51/DMC1 foci appeared incapable of mediating stable pairing before early pachynema (~70% of late zygotene

www.sciencemag.org

Downloaded from www.sciencemag.org on February 17, 2011

proliferation (Fig. 4B, c and d). Collectively these results are consistent with the hypothesis that increased FGF production by the Hand2-null uterine stroma stimulates epithelial proliferation by activating the FGFR-ERK1/2 pathway. The ERK1/2-dependent phosphorylation of epithelial ERa at Ser118 is critical for the transcriptional activation of ERa (11). Administration of either PD173074 (Fig. 4C, a to d) or PD184352 (Fig. 4C, e to h) to Hand2-null uterine horns blocked the phosphorylation of epithelial ERa at Ser118 and the expression of Muc-1. This result supported our view that elevated signaling by FGFR-ERK1/2 pathway in Hand2d/d uteri is responsible for phosphorylation and activation of ERa in epithelial cells, which promotes persistent expression of Muc-1 and which in turn creates a barrier that prevents embryo attachment. Earlier studies using tissue recombinants prepared with uterine epithelium and stroma isolated from neonatal wild-type and PR-null mice indicated that the stromal PR plays an obligatory role in mediating the inhibitory actions of P on E-induced epithelial cell proliferation (18). However, the mechanism of this stromal-epithelial communi-

REPORTS

Fig. 2. Distinct temporal and structural properties of the PAR. (A) Nucleuswide RAD51/DMC1 foci in spermatocytes (bars show means T SD). (B) Assay for PAR DSB formation. IF against RAD51/DMC1 and SYCP3 (i) and FISH (ii) with probes shown in Fig. 1Ai on a leptotene spermatocyte nucleus. Scale bar, 10 mm. (iii) Magnified views of Y and X PARs from frames in (i) (5) and an overlay of the PAR FISH signal with SYCP3 (right), here with a RAD51/DMC1 www.sciencemag.org

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Fig. 1. Late PAR pairing during male meiosis. (A) FISH assay for pairing. (i and ii) Example of immunofluorescence (IF) and two sequential rounds of FISH on a late zygotene spermatocyte nucleus. Nuclei stained with an antibody against axis protein SYCP3 were subjected first to PAR FISH (i), then to distal chr 18 and distal chr 19 FISH (ii). Scale bar, 10 mm. (B) Nuclei (%) with unpaired and paired (≤2 mm apart) FISH signals. Chromosome synapsis status was also recorded at sites of paired signals.

nuclei had foci, but <20% showed PAR pairing) (Figs. 1B and 2C). The number of PAR foci per cell also increased over time. In leptonema and early to mid-zygonema, most cells with a PAR RAD51/DMC1 focus had only one (typically on X), whereas by late zygonema, two PAR foci were often present (both X and Y) (Fig. 2C). Foci on both PARs could represent two independent DSBs. If so, then having more than one X-Y recombination interaction may stabilize pairing, similar to multiple interactions that stabilize pairing of autosomes (10). Alternatively, foci on both PARs could represent the two, separated ends of a single DSB (11, 12)—with one focus marking the broken PAR and the second focus marking the other PAR (fig. S3A). In this “ends-apart” model, nuclei that have two PAR foci are those in which the X and Y PARs have successfully engaged each other. However, we found that most such nuclei showed no evidence of a preferential X-Y spatial relationship (fig. S3B), and most PAR pairing occurred abruptly at the zygonema-topachynema transition, i.e., after the stage when many cells displayed two PAR foci (compare Figs. 1B and 2C). Sex body formation (13) may facilitate this sudden completion of X-Y pairing by providing homology-independent X-Y proximity that simplifies the homology search. The haploid mouse genome averages fewer than one DSB per 10 Mb (Fig. 2A), whereas the

focus only on the X PAR. (C) Nuclei (%) with one or two PAR RAD51/DMC1 foci. (D) Axis/loop segments as a determinant of DSB potential [after (15)]. Only one homolog is shown. DNA organized on a longer axis into more and smaller loops (i) has more DSB potential than if the same DNA is organized on a shorter axis into fewer and larger loops (ii). (E) Examples of chromatin extension (gray brackets in insets); see also Table 1. Scale bar, 5 mm. VOL 331

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REPORTS chr 18 and 19 (regions with relatively frequent crossing-over) (19), DNA content was 10 to 13 Mb per mm and correlated well with axis length, i.e., the distal ~10% of DNA occupied ~10% total axis length (Table 1A). The ≥10-fold difference between PAR and autosome axes is of the magnitude expected for a region that experiences more than 10 times as many DSBs. Axes of nonPAR portions of the X and Y had a DNA content more like autosomes (≥14 Mb per mm) (fig. S4). Long PAR axes predict short chromatin loops. As a proxy for loop size, we measured FISH signal extension from axes for probes in the PAR and autosomal subtelomeric regions (Fig. 2E and Table 1B). PAR FISH signals were substantially more compact at all stages (about one-third to one-seventh as extended), consistent with smaller

loops. Thus, chromosome structure could be one factor that facilitates high-frequency DSB formation in the PAR. The distinct temporal and structural features outlined above raised the possibility that mechanisms ensuring efficient PAR recombination and pairing may be under different genetic control from autosomes. Characterization of a variant of SPO11, the evolutionarily conserved meiotic DSB catalyst (1), validated this hypothesis (Fig. 3). Two major mRNA splicing isoforms in mice and humans are Spo11a and Spo11b (7, 20–22) (Fig. 3Ai and fig. S5). Spo11b is expressed early in meiosis, when most DSBs are formed, whereas Spo11a is expressed later (7, 20, 23) (Fig. 3Aii and fig. S6A). Thus, SPO11b is likely responsible for most DSB formation.

Fig. 3. Genetic control of PAR recombination and pairing. (A) Spo11 splice variants (see also fig. S5). (i) Genomic organization and splicing. Spo11b includes exon 2, Spo11a excludes it. Y, catalytic tyrosine. (ii and iii) Reverse transcription polymerase chain reaction from flowsorted meiocyte populations of adult mice. –RT, no reverse transcription; L/Z, leptonema/zygonema; P/D, pachynema/diplonema; S, spermatids. (iv) SPO11 protein levels in adult testis extracts. Asterisk, a lower-mobility protein likely originating from the knockout allele (fig. S6D). (B) IF of SYCP1 and SYCP3 on pachytene nuclei (i) and of SYCP3 plus whole-chromosome FISH of early metaphase I spermatocyte nuclei (ii) from mice of the indicated genotypes. Inset in (i), schematic of X and Y chromosomes. Scale bars, 10 mm. (iii) Quantification of X-Y association; 57 to 65 nuclei scored per genotype. (C) Terminaldeoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL)– stained testis sections; apoptotic cells stain brown. Elongating spermatids (arrows) are rare in Spo11bonly mice. Inset shows a lagging chromosome (arrowhead) in a TUNEL-positive cell. (D) RAD51/DMC1 focus counts in spermatocytes from control and Spo11b-only mice (bars show means T SD). (E) Nuclei (%) with PAR RAD51/DMC1 foci in mice of the indicated genotypes; for each genotype, 41 to 55 nuclei were scored per stage. *P ≤ 0.0002 (two-tailed Mann-Whitney test); n.s., not significant (P = 0.09).

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<1 Mb PAR (14) undergoes one or two DSBs (Fig. 2C), which is 10 to 20 times the genome average. We speculated that distinct higher-order chromosome structure could render the PAR more conducive to DSB formation. Meiotic recombination is proposed to occur within DNA segments residing in chromatin loops that become transiently tethered to chromosome axes (15). Loop density per micrometer of axis is constant (16) and produces an inverse relation between loop size and axis length (17). DNA arranged into smaller loops may have higher DSB potential (Fig. 2D) (18); indeed, autosomal crossover frequency in male mice correlates with axis length (19). We found that PAR axes were disproportionately long relative to DNA length and incorporated ~1 Mb per mm of axis (Table 1A). At the distal ends of

REPORTS Table 1. Chromosome axis lengths and chromatin extension in PARs and distal ends of chr 18 and 19. Axis lengths are means T SD and FISH signal extensions are means T SD, for the number of observations in parentheses. Probe size is the size of the bacterial artificial chromosome (5). (A) DNA content versus chromosome axis lengths (late zygonema) Chromosome Y X 18 19

Total size (Mb)

Probe-distal region (Mb)

95 167 91 61

0.7 0.7 8 6

Probe-distal axis

Total chromosome axis (mm) 4.2 12.7 6.1 5.1

T T T T

0.7 2.8 0.9 0.5

Length (mm)

(17) (13) (11) (11)

0.7 0.8 0.6 0.6

T T T T

0.2 0.2 0.1 0.1

DNA content (Mb/mm)

(20) (23) (12) (10)

1 1 13 10

(B) Length of chromatin extension from axes

Y PAR X PAR Distal chr 18 Distal chr 19

FISH signal extension (mm)

Probe size (kb)

Early to mid-zygonema

Leptonema 0.5 0.6 2.2 2.3

146 207 182

T T T T

0.2 0.4 0.8 0.9

(25) (23) (26) (18)

0.6 0.7 3.2 3.6

T T T T

0.3 0.5 1.5 2.0

(21) (17) (31)* (33)*

Late zygonema 0.6 0.6 4.5 5.3

T T T T

0.3 0.5 1.9 3.0

(21) (20) (35)* (40)*

Pachynema

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Locus

1.2 T 0.5 (23)* 5.4 T 2.2 (21)* 5.9 T 2.3 (23)*

*Some or all measurements are from paired FISH signals.

We generated transgenic mice expressing Spo11bB cDNA (fig. S5) from a meiosis-specific promoter (24) (fig. S6B). Tg(Xmr-Spo11bB) transcript expression overlapped with Spo11b mRNA appearance in wild type (Fig. 3Aii and fig. S6, A and C). In testis extracts of Spo11–/– Tg (Xmr-Spo11bB)+/+ (hereafter, “Spo11b-only”) mice, SPO11bB protein approximated the total level of SPO11 in wild type (Fig. 3Aiii). The transgene did not cause obvious meiotic phenotypes in mice heterozygous at the endogenous Spo11 locus [i.e., Spo11+/– Tg(Xmr-Spo11bB)+/+], and these mice were used as controls. The profound meiotic defects of Spo11–/– mice [no recombination, failure of homolog pairing and synapsis, and infertility (6, 7, 25)] were mostly rescued by Tg (Xmr-Spo11bB) in both sexes: Autosomal homologous pairing, synapsis, and MLH1 focus formation (a crossover marker) appeared normal (Fig. 3Bi and fig. S7A). Moreover, ovaries of Spo11b-only mice contained abundant primordial follicles (fig. S7B), and Spo11b-only females were fully fertile with normal litter sizes. Thus SPO11bB supports autosomal crossing-over, pairing, and synapsis, and (in females) full meiotic progression and accurate chromosome segregation. Male meiosis was not fully rescued, however. Although sex bodies formed (fig. S7C), the X and Y failed to pair and synapse in ~70% of spermatocytes (Fig. 3B). Spo11b-only testis sections showed numerous apoptotic metaphase I cells (Fig. 3C), many with a lagging chromosome (Fig. 3C, inset, and fig. S7D), consistent with spindle checkpoint-induced apoptosis (9, 26, 27), triggered by the failure of nonrecombinant X and Y to orient properly on the metaphase I spindle. Few postmeiotic cells were formed, and testis sizes were reduced (Fig. 3C and fig. S7, D and E), so that although some Spo11b-only males produced offspring, most were infertile.

Nucleus-wide numbers and timing of RAD51/ DMC1 foci were indistinguishable between Spo11bonly and control males (Fig. 3D and fig. S7F), which indicated that the X-Y pairing defect cannot be attributed to reduced global DSB levels. Similarly, the frequency of PAR RAD51/ DMC1 foci in leptonema was not affected (Fig. 3E). In contrast, the percentage of late zygotene nuclei with a PAR focus was reduced in Spo11b-only males, consistent with a defect in a late-forming DSB population (PAR-specific, or possibly including a small subset of autosomal DSBs). About 70% of late zygotene nuclei lacked PAR foci (Fig. 3E), which was similar to the percentage of cells with X-Y pairing failure (Fig. 3Biii). Thus, the few PAR foci that form early in both wild-type and Spo11b-only males seem to persist until late zygonema (fig. S4, discussion), at which time recombination-mediated X-Y pairing occurs. We propose that a lack of late PAR DSBs is the cause of infertility in Spo11b-only males. In females, two fully homologous X chromosomes make PAR recombination dispensable. Spo11a is the only splice variant missing from Spo11b-only mice that is known to be developmentally regulated, and its expression in wild type correlates with the timing of late PAR DSBs as inferred from the appearance of RAD51/DMC1 foci. It is thus possible that SPO11a, by itself or in combination with SPO11b, is needed for DSB formation in late zygonema. In this scenario, lateforming PAR DSBs are genetically separable from both global DSBs and early-forming PAR DSBs, and the surge of late-forming PAR DSBs is crucial for efficient X-Y pairing and fertility. PAR recombination occasionally fails in humans, as evidenced by paternally inherited sex chromosome aneuploidies [e.g., Klinefelter’s or Turner syndromes (28)]. Because Spo11 isoforms are conserved, we speculate that variation in Spo11

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splicing patterns may be a human X-Y nondisjunction susceptibility trait. References and Notes 1. F. Cole, S. Keeney, M. Jasin, Genes Dev. 24, 1201 (2010). 2. F. Rouyer et al., Nature 319, 291 (1986). 3. P. Soriano et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7218 (1987). 4. Q. Shi et al., Am. J. Med. Genet. 99, 34 (2001). 5. Materials and methods are available as supporting material on Science Online. 6. F. Baudat, K. Manova, J. P. Yuen, M. Jasin, S. Keeney, Mol. Cell 6, 989 (2000). 7. P. J. Romanienko, R. D. Camerini-Otero, Mol. Cell 6, 975 (2000). 8. X. Ding et al., Dev. Cell 12, 863 (2007). 9. M. Barchi et al., PLoS Genet. 4, e1000076 (2008). 10. B. M. Weiner, N. Kleckner, Cell 77, 977 (1994). 11. N. Hunter, in Molecular Genetics of Recombination, A. Aguilera, Rothstein, R., Eds. (Springer-Verlag, Heidelberg, 2007), vol. 17, pp. 381–442. 12. A. Storlazzi et al., Cell 141, 94 (2010). 13. P. S. Burgoyne, S. K. Mahadevaiah, J. M. Turner, Nat. Rev. Genet. 10, 207 (2009). 14. J. Perry, S. Palmer, A. Gabriel, A. Ashworth, Genome Res. 11, 1826 (2001). 15. Y. Blat, R. U. Protacio, N. Hunter, N. Kleckner, Cell 111, 791 (2002). 16. D. Zickler, N. Kleckner, Annu. Rev. Genet. 33, 603 (1999). 17. E. Revenkova et al., Nat. Cell Biol. 6, 555 (2004). 18. N. Kleckner, A. Storlazzi, D. Zickler, Trends Genet. 19, 623 (2003). 19. L. Froenicke, L. K. Anderson, J. Wienberg, T. Ashley, Am. J. Hum. Genet. 71, 1353 (2002). 20. P. J. Romanienko, R. D. Camerini-Otero, Genomics 61, 156 (1999). 21. S. Keeney et al., Genomics 61, 170 (1999). 22. M. A. Bellani, K. A. Boateng, D. McLeod, R. D. Camerini-Otero, Mol. Cell. Biol. 30, 4391 (2010). 23. M. J. Neale, J. Pan, S. Keeney, Nature 436, 1053 (2005). 24. P. J. Romanienko, dissertation, Cornell University Ithaca, NY (1997). 25. M. Di Giacomo et al., Proc. Natl. Acad. Sci. U.S.A. 102, 737 (2005). 26. S. Eaker, J. Cobb, A. Pyle, M. A. Handel, Dev. Biol. 249, 85 (2002).

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REPORTS American-Italian Cancer Foundation (AICF) (M.B.); and the Charles H. Revson Foundation (F.B.). We thank M. Leversha [Memorial Sloan-Kettering Cancer Center (MSKCC)], P. Bois (Scripps Florida), and K. Manova (MSKCC) for valuable advice and protocols. We are grateful to Keeney and Jasin lab members, especially I. Roig, E. de Boer, and F. Cole, and to N. Hunter (University of California, Davis) for insightful comments.

Classic Selective Sweeps Were Rare in Recent Human Evolution Ryan D. Hernandez,1* Joanna L. Kelley,1 Eyal Elyashiv,2 S. Cord Melton,1 Adam Auton,3 Gilean McVean,3,4 1000 Genomes Project, Guy Sella,2† Molly Przeworski1,5,6†‡ Efforts to identify the genetic basis of human adaptations from polymorphism data have sought footprints of “classic selective sweeps” (in which a beneficial mutation arises and rapidly fixes in the population). Yet it remains unknown whether this form of natural selection was common in our evolution. We examined the evidence for classic sweeps in resequencing data from 179 human genomes. As expected under a recurrent-sweep model, we found that diversity levels decrease near exons and conserved noncoding regions. In contrast to expectation, however, the trough in diversity around human-specific amino acid substitutions is no more pronounced than around synonymous substitutions. Moreover, relative to the genome background, amino acid and putative regulatory sites are not significantly enriched in alleles that are highly differentiated between populations. These findings indicate that classic sweeps were not a dominant mode of human adaptation over the past ~250,000 years. umans have experienced myriad adaptations since the common ancestor with chimpanzees and more recently have adapted to a wide range of environments. Efforts to infer the molecular basis of these adaptations from polymorphism data have largely been guided by the “classic selective sweep” model, in which a new, strongly beneficial mutation increases in frequency to fixation in the population [reviewed in (1, 2)]. In this scenario, the allele ascends rapidly enough in frequency for there to be little opportunity for recombination to uncouple it from its genetic background, such that its rise sweeps out variation at linked sites, reducing linked neutral diversity in the population and distorting allele frequencies and patterns of linkage disequilibrium (3). In humans, the effects of sweeps are expected to persist for approximately 10,000 generations or about 250,000 years (4).

H

1 Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA. 2Department of Ecology, Evolution and Behavior, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel. 3Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. 4 Department of Statistics, University of Oxford, Oxford OX1 3TG, UK. 5Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA. 6Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA.

*Present address: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143, USA. †These authors contributed equally to this work. ‡To whom correspondence should be addressed. E-mail: [email protected]

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Identifying the footprint of a sweep against a noisy genomic background is challenging, because patterns of genetic variation reflect the effects of multiple modes of natural selection as well as of demographic history, mutation, and recombination. To date, applications of statistical tests based on the sweep model have led to the identification of more than 2000 genes as potential targets of positive selection in the human genome (2) and to the suggestion that diversity patterns in ~10% of the human genome have been affected by linkage to recent sweeps [e.g., (5)]. The list of functionally characterized cases of genetic adaptations is short, however, and the false discovery rate of selection scans is potentially high (6). Thus, it remains unknown whether the well-documented cases are typical of human adaptations, or whether they represent rare instances where the genetic architecture of the adaptation was conducive to classic sweeps (7, 8), with most adaptations occurring by other modes (e.g., polygenic selection and selection on standing variation). Two main lines of evidence have been advanced in support of the hypothesis that classic selective sweeps were common. First, regions of low recombination, in which a single sweep should have a larger span, exhibit lower diversity (after correcting for variation in mutation rates) relative to regions of high recombination (9–11). Regions of low recombination also show greater differentiation between populations (12), as expected from local adaptation or, for some parameters, from the fixation of globally advantageous alleles (13).

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Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/916/DC1 SOM Text Materials and Methods Figs. S1 to S7 References 28 July 2010; accepted 21 December 2010 10.1126/science.1195774

Second, under the sensible assumption that amino acid and conserved noncoding sites are enriched among targets of adaptation, one would expect that the signal of selection would be most clearly visible at or around such sites [e.g., (10, 14)]. Consistent with this expectation, diversity levels decrease with the number of human-specific substitutions at amino acid or conserved noncoding sites (in 200- to 600-kb windows) (10), and genic regions show an enrichment of alleles that are highly differentiated between populations relative to nongenic regions (15, 16). These patterns are informative but are only indirectly related to theoretical predictions. Moreover, some—possibly all— of these patterns may instead result from purifying selection acting on deleterious mutations at linked sites (“background selection”) (9–11, 16–18). To evaluate the importance of classic sweeps in shaping human diversity, we analyzed resequencing data for 179 human genomes from four populations, collected as part of the low-coverage pilot for the 1000 Genomes Project (19). These data overcome ascertainment biases arising in the study of genotyping data, with ~99% power to detect variants with a population frequency above 10% for 86% of the euchromatic genome (19). We examined the extent to which selection affects diversity levels at linked sites by calculating the average diversity as a function of genetic distance from the nearest exons, collating all exons across the genome (fig. S1). To estimate neutral diversity levels, we focused only on nonconserved noncoding and fourfold degenerate sites (11). To correct for systematic variation in the mutation rate, we divided diversity by human– rhesus macaque divergence [to which the contribution of ancestral polymorphism is minor (11)]. Our estimate of relative diversity appears little affected by the low fold coverage of individuals or variation in sequencing depth (fig. S2, C to E). Scaled diversity levels are lowest near exons (Fig. 1A and fig. S3), recovering half the drop by 0.03 to 0.04 cM, depending on the population, and 80% by 0.07 to 0.1 cM [see (20)]. Given that diversity is scaled by divergence, the trough in scaled diversity around exons does not reflect systematic variation in mutation rates as a function of the distance from exons, strong purifying selection on the sites themselves (which would decrease both diversity and divergence), or weak selection near exons (which should inflate, not decrease, diversity levels divided by divergence). Rather, the trough provides evidence for the effects of directional selection at linked sites, extending over 100 kb.

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27. T. Odorisio, T. A. Rodriguez, E. P. Evans, A. R. Clarke, P. S. Burgoyne, Nat. Genet. 18, 257 (1998). 28. H. Hall, P. Hunt, T. Hassold, Curr. Opin. Genet. Dev. 16, 323 (2006). 29. This work was supported by NIH grant R01 HD040916 (M.J. and S.K); International Grants in Cancer Research (AIRC) [My First AIRC Grant (MFAG) grant 4765], Italian Ministry for Education, University and Research (MIUR), the Lalor Foundation, and the

REPORTS American-Italian Cancer Foundation (AICF) (M.B.); and the Charles H. Revson Foundation (F.B.). We thank M. Leversha [Memorial Sloan-Kettering Cancer Center (MSKCC)], P. Bois (Scripps Florida), and K. Manova (MSKCC) for valuable advice and protocols. We are grateful to Keeney and Jasin lab members, especially I. Roig, E. de Boer, and F. Cole, and to N. Hunter (University of California, Davis) for insightful comments.

Classic Selective Sweeps Were Rare in Recent Human Evolution Ryan D. Hernandez,1* Joanna L. Kelley,1 Eyal Elyashiv,2 S. Cord Melton,1 Adam Auton,3 Gilean McVean,3,4 1000 Genomes Project, Guy Sella,2† Molly Przeworski1,5,6†‡ Efforts to identify the genetic basis of human adaptations from polymorphism data have sought footprints of “classic selective sweeps” (in which a beneficial mutation arises and rapidly fixes in the population). Yet it remains unknown whether this form of natural selection was common in our evolution. We examined the evidence for classic sweeps in resequencing data from 179 human genomes. As expected under a recurrent-sweep model, we found that diversity levels decrease near exons and conserved noncoding regions. In contrast to expectation, however, the trough in diversity around human-specific amino acid substitutions is no more pronounced than around synonymous substitutions. Moreover, relative to the genome background, amino acid and putative regulatory sites are not significantly enriched in alleles that are highly differentiated between populations. These findings indicate that classic sweeps were not a dominant mode of human adaptation over the past ~250,000 years. umans have experienced myriad adaptations since the common ancestor with chimpanzees and more recently have adapted to a wide range of environments. Efforts to infer the molecular basis of these adaptations from polymorphism data have largely been guided by the “classic selective sweep” model, in which a new, strongly beneficial mutation increases in frequency to fixation in the population [reviewed in (1, 2)]. In this scenario, the allele ascends rapidly enough in frequency for there to be little opportunity for recombination to uncouple it from its genetic background, such that its rise sweeps out variation at linked sites, reducing linked neutral diversity in the population and distorting allele frequencies and patterns of linkage disequilibrium (3). In humans, the effects of sweeps are expected to persist for approximately 10,000 generations or about 250,000 years (4).

H

1 Department of Human Genetics, University of Chicago, Chicago, IL 60637, USA. 2Department of Ecology, Evolution and Behavior, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel. 3Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. 4 Department of Statistics, University of Oxford, Oxford OX1 3TG, UK. 5Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA. 6Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637, USA.

*Present address: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143, USA. †These authors contributed equally to this work. ‡To whom correspondence should be addressed. E-mail: [email protected]

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Identifying the footprint of a sweep against a noisy genomic background is challenging, because patterns of genetic variation reflect the effects of multiple modes of natural selection as well as of demographic history, mutation, and recombination. To date, applications of statistical tests based on the sweep model have led to the identification of more than 2000 genes as potential targets of positive selection in the human genome (2) and to the suggestion that diversity patterns in ~10% of the human genome have been affected by linkage to recent sweeps [e.g., (5)]. The list of functionally characterized cases of genetic adaptations is short, however, and the false discovery rate of selection scans is potentially high (6). Thus, it remains unknown whether the well-documented cases are typical of human adaptations, or whether they represent rare instances where the genetic architecture of the adaptation was conducive to classic sweeps (7, 8), with most adaptations occurring by other modes (e.g., polygenic selection and selection on standing variation). Two main lines of evidence have been advanced in support of the hypothesis that classic selective sweeps were common. First, regions of low recombination, in which a single sweep should have a larger span, exhibit lower diversity (after correcting for variation in mutation rates) relative to regions of high recombination (9–11). Regions of low recombination also show greater differentiation between populations (12), as expected from local adaptation or, for some parameters, from the fixation of globally advantageous alleles (13).

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Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/916/DC1 SOM Text Materials and Methods Figs. S1 to S7 References 28 July 2010; accepted 21 December 2010 10.1126/science.1195774

Second, under the sensible assumption that amino acid and conserved noncoding sites are enriched among targets of adaptation, one would expect that the signal of selection would be most clearly visible at or around such sites [e.g., (10, 14)]. Consistent with this expectation, diversity levels decrease with the number of human-specific substitutions at amino acid or conserved noncoding sites (in 200- to 600-kb windows) (10), and genic regions show an enrichment of alleles that are highly differentiated between populations relative to nongenic regions (15, 16). These patterns are informative but are only indirectly related to theoretical predictions. Moreover, some—possibly all— of these patterns may instead result from purifying selection acting on deleterious mutations at linked sites (“background selection”) (9–11, 16–18). To evaluate the importance of classic sweeps in shaping human diversity, we analyzed resequencing data for 179 human genomes from four populations, collected as part of the low-coverage pilot for the 1000 Genomes Project (19). These data overcome ascertainment biases arising in the study of genotyping data, with ~99% power to detect variants with a population frequency above 10% for 86% of the euchromatic genome (19). We examined the extent to which selection affects diversity levels at linked sites by calculating the average diversity as a function of genetic distance from the nearest exons, collating all exons across the genome (fig. S1). To estimate neutral diversity levels, we focused only on nonconserved noncoding and fourfold degenerate sites (11). To correct for systematic variation in the mutation rate, we divided diversity by human– rhesus macaque divergence [to which the contribution of ancestral polymorphism is minor (11)]. Our estimate of relative diversity appears little affected by the low fold coverage of individuals or variation in sequencing depth (fig. S2, C to E). Scaled diversity levels are lowest near exons (Fig. 1A and fig. S3), recovering half the drop by 0.03 to 0.04 cM, depending on the population, and 80% by 0.07 to 0.1 cM [see (20)]. Given that diversity is scaled by divergence, the trough in scaled diversity around exons does not reflect systematic variation in mutation rates as a function of the distance from exons, strong purifying selection on the sites themselves (which would decrease both diversity and divergence), or weak selection near exons (which should inflate, not decrease, diversity levels divided by divergence). Rather, the trough provides evidence for the effects of directional selection at linked sites, extending over 100 kb.

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27. T. Odorisio, T. A. Rodriguez, E. P. Evans, A. R. Clarke, P. S. Burgoyne, Nat. Genet. 18, 257 (1998). 28. H. Hall, P. Hunt, T. Hassold, Curr. Opin. Genet. Dev. 16, 323 (2006). 29. This work was supported by NIH grant R01 HD040916 (M.J. and S.K); International Grants in Cancer Research (AIRC) [My First AIRC Grant (MFAG) grant 4765], Italian Ministry for Education, University and Research (MIUR), the Lalor Foundation, and the

REPORTS ratio of X to autosome scaled diversity near exons than farther away, potentially confounding demographic analysis (21) (fig. S4). A similar effect is seen around conserved noncoding regions (CNCs), but the trough is more dif-

Fig. 1. (A to D) Diversity levels divided by human–rhesus macaque divergence (at nonconserved, noncoding sites) as a function of genetic distance from exons [(A) and (C)] and from conserved noncoding regions [(B) and (D)]. Autosome data are shown in (A) and (B); X chromosome data are shown in (C) and (D). Populations are YRI (green), CEU (orange), and CHB+JPT (purple). Shown are LOESS curves obtained for a span of 0.1 and a bin size of 1.2 × 10−5 cM. Above each figure is a histogram of the number of kilobases in each bin (plotted on a log scale). See (20) for alternative versions. Fig. 2. CEU diversity levels divided by human–rhesus macaquedivergencearound human-specific substitutions, across autosomes. In the main plot, LOESS curves have a span of 0.2 and a bin size of 1.2 × 10−5 cM; the inset has a span of 0.05 to show added detail near the substitutions. The light blue shaded area represents the central 95th percentile of diversity estimates obtained from 100 bootstrap simulations. For alternative versions of this figure, including the same plot for YRI and CHB+JPT, as well as the X chromosome, see fig. S5. www.sciencemag.org

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fuse (Fig. 1, B and D, and fig. S3). Because CNCs tend to be linked to exons (the median distance of a CNC to the nearest exon is 0.08 cM), the trough around CNCs could be a by-product of the effects of selection on exons (see below); alternatively, it could reflect less widespread selection on mutations in CNCs relative to exons (11, 22). If the trough in scaled diversity results from classic sweeps at linked sites, it should be deepest around those changes most likely to have functional consequences (i.e., within exons, around amino acid substitutions). We tested this prediction by considering the average scaled diversity around human-specific amino acid fixations and, as a control for other evolutionary forces, around synonymous substitutions. Our rationale was as follows: Human and chimpanzee species split approximately 5 million years ago [e.g., (23)], such that, assuming a constant rate of substitution, about 5% of human-specific substitutions could have left a detectable sweep in their wake (i.e., have occurred in the past 250,000 years). Thus, if a substantial fraction of amino acid changes are the result of classic sweeps, average diversity should be decreased around amino acid substitutions as compared to synonymous substitutions. In the fly Drosophila simulans, diversity levels are indeed significantly lower and suggest that ~13% of amino acid substitutions involved classic sweeps (24). In contrast, human diversity levels around amino acid substitutions are not lower than around synonymous substitutions (Fig. 2; P = 0.90 for a window of size 0.02 cM around the focal substitution). This conclusion is robust to alternative approaches for inferring substitutions or estimating divergence and to the choice of genetic map (fig. S5). The similar troughs indicate either that amino acid and synonymous mutations (including fourfold degenerate mutations; Fig. 2) experienced recurrent classic selective sweeps of similar intensities and rates or, more plausibly, that few amino acid substitutions resulted from classic sweeps. Simulations suggest that even if only 10% of human-specific amino acid substitutions were strongly favored or if 25% of amino acid fixations were favored with weak effects, there should be a significant decrease in the diversity levels relative to what would be expected if all fixations were neutral (Fig. 3A) (20). These simulations mimic the data structure but do not fully capture the clustering of substitutions in the genome (fig. S6). Because amino acid substitutions are more clustered with one another than with synonymous substitutions (fig. S6A), this omission is conservative, leading to an underestimate of the power to detect the effects of classic sweeps (fig. S7) (20). Thus, our finding strongly constrains the maximal fraction of protein changes that could have resulted from classic sweeps in the past 250,000 years. The troughs in diversity around both synonymous and amino acid fixations could instead be due to strong purifying selection at linked sites. Indeed, we found that under a model of back-

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This pattern is even more pronounced on the X chomosome, where the trough is deeper and wider, recovering diversity over twice the genetic distance (Fig. 1C and fig. S3). The greater footprint of linked selection on the X leads to a smaller

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tions, enrichments of highly differentiated alleles at nonsynonymous sites, at 5′ and 3′ UTRs, and within 1 kb upstream of the TSS are either not significant or only marginally significant when tested against the genomic background (Fig. 4, C to F, and fig. S10). This finding reflects the small numbers of cases of highly differentiated alleles (fig. S11) and underscores how few local adaptations resulted from the extreme changes in allele frequencies among populations expected from classic sweeps. In particular, there are only four fixed amino acid differences between YRI and CEU, suggesting a rate of classic sweeps far below 10% since the two populations split [see (20)]. Moreover, the cases of extreme differentiation could also arise under an alternative model

to the classic sweep, in which the beneficial allele was not a new mutation but was already segregating in the population. Although tests based on the frequency spectrum or the decay of linkage disequilibrium have low power to detect this mode of selection [e.g., (6, 27)], measures of differentiation should have substantial power so long as there was little or no gene flow between the populations and the allele was at low frequency when first favored (28) (as is likely to be the case for both neutral and previously deleterious alleles). Intriguingly, the alleles with the largest differences in frequency between populations, which should be most enriched for targets of selection (6), often segregate in both populations (fig. S12) and tend to lie on a shorter haplotype

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ground selection (17), the expected troughs are of similar depths to the observed ones, with lower diversity predicted around fourfold degenerate substitutions than around amino acid substitutions, as observed (Fig. 3B) (20). Interestingly, even though the model assumes extremely weak purifying selection in CNCs, it predicts a trough around them as well (fig. S8), indicating that the observed decrease in diversity around CNCs may primarily reflect selection on linked exons. The prevalence of classic sweeps can also be evaluated by considering the genetic differentiation among the three population samples, whose ancestors occupied a range of environments. The two Eurasian populations [a population of European ancestry (CEU) and the combined Chinese and Japanese (CHB+JPT)] are thought to have split from the Yoruba (YRI) more than 100,000 years ago, and CEU and CHB+JPT are thought to have split about 23,000 years ago [e.g., (25)]. Given this time frame, local adaptation involving classic sweeps would have led to fixed differences or extreme differences in allele frequencies between YRI and CEU/CHB+JPT (and possibly between CEU and CHB+JPT) at targets of selection [see (16)]. Consistent with this expectation, there is an enrichment of highly differentiated single-nucleotide polymorphisms (SNPs) between population pairs in genic regions relative to nongenic regions (Fig. 4A) (15, 16). However, the enrichments can also be explained by a 10 to 15% decrease in the effective population size near exons due to background selection (i.e., a trough similar to that in Fig. 1A) (16). In turn, CNCs are not significantly enriched for highly differentiated SNPs relative to nonconserved noncoding regions (Fig. 4B and fig. S9B). Although the enrichment of genic SNPs could in principle result from purifying selection alone, there are well-documented examples of adaptations among the most highly differentiated SNPs, notably in genes involved in pigmentation or infectious disease susceptibility (19)—in other words, there are at least a handful of loci that conform to the sweep model. To ask whether these cases represent the tip of the iceberg of sweeps yet to be discovered, we tested for an enrichment of highly differentiated alleles that cause amino acid changes or that lie in putative regulatory regions, at which the highest fraction of changes would be expected, a priori, to have phenotypic consequences and hence to be possible targets of sweeps. Because CNCs may be unusual regulatory elements, we also considered SNPs in untranslated regions (UTRs) or 1 kb upstream of the transcription start site (TSS), annotations in which more than 10% of substitutions were estimated to be beneficial (22) and which are most strongly enriched for expression quantitative trait loci in cell lines (26). These annotations all fall within genic regions, so a test of enrichment against the genomic background is confounded by background selection or other modes of selection that increase population differentiation at genic sites. Nonetheless, in comparisons among the three human popula-

Fig. 3. (A) The power to detect a decrease in diversity levels around amino acid substitutions due to classic sweeps. The upper panels show the power at a given genetic distance from the substitution, for the three sets of selection parameters [see (20)]; the lower panels show the diversity patterns expected around amino acid substitutions for four sets of selection parameters, as well as for a model of purely neutral fixations, after LOESS smoothing (with a span of 0.2). For each set of parameters, the shaded area represents the central 95th percentile obtained from 100 bootstrap simulations. The depth of the trough reflects the fraction of substitutions that were beneficial; its width reflects the typical strength of selection (24). (B) Relative diversity levels around nonsynonymous, synonymous, and fourfold degenerate synonymous substitutions predicted under a model of background selection [see (20)]. The symbol B denotes the predicted diversity level relative to what is expected with no effects of background selection (i.e., under strict neutrality), taking into account variation in mutation rates (11). OBS refers to the observed value of average scaled diversity (i.e., diversity divided by human–rhesus macaque divergence). For the expected diversity around exons and CNCs, as well as predictions for the X chromosome, see fig. S8. The inset has a span of 0.05 to show added detail near the substitutions.

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REPORTS neutral sites, for example, suggest that 10 to 15% [and possibly as many as 40% (29)] of amino acid differences between humans and chimpanzees were adaptive [e.g., (30)], as were 5% of substitutions in conserved noncoding regions (22, 29) and ~20% in UTRs (22). Given the paucity of classic sweeps revealed by our findings, an excess of functional divergence would point to the importance of other modes of adaptation. One way to categorize modes of adaptation is in terms of their effect on the allele frequencies at sites that affect the beneficial phenotype. In this view, classic sweeps bring new alleles to fixation; selection on standing variation or on multiple beneficial alleles brings rare or intermediate frequency alleles to fixation; and other forms of adaptation, such as selection on

polygenic traits, increase or decrease allele frequencies to a lesser extent. Such changes in allele frequencies can decrease variation at closely linked sites—to a lesser extent than in a full sweep—and might therefore contribute to a reduction in diversity near functional elements (31) as well as to excess divergence. Alternatives to classic sweeps are likely for parameters applicable to human populations (7, 32); in particular, many phenotypes of interest are quantitative and plausibly result from selection at many loci of small effect (8). An important implication is that in the search for targets of human adaptation, a change in focus is warranted. To date, selection scans have relied almost entirely on the sweep model, either explicitly (by considering strict neutrality as the null hypothesis and a classic sweep as the alternative) or implicitly (by ranking regions by a statistic thought to be sensitive to classic sweeps and focusing on the tails of the empirical distribution). It appears that few adaptations in humans took the form that these approaches are designed to detect, such that low-hanging fruits accessible by existing approaches may be largely depleted. Conversely, the more common modes of adaptation likely remain undetected. Thus, to dissect the genetic basis of human adaptations and assess what fraction of the genome was affected by positive selection, we need new tests to detect other modes of selection, such as comparisons between closely related populations that have adapted to drastically different environments [e.g., (33)] or methods that consider loci that contribute to the same phenotype jointly [e.g., (34)]. Moreover, if alleles that contribute to recent adaptations are often polymorphic within a population, genome-wide association studies should be highly informative.

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than expected from a classic sweep (16), consistent with selection on standing variation rather than ongoing classic sweeps. In summary, patterns of diversity around genic substitutions and of highly differentiated alleles are inconsistent with the expectation for frequent classic sweeps, but could result, at least in part, from background selection. Thus, although some substitutions in proteins and regulatory positions undoubtedly involved classic sweeps, they were too infrequent within the past 250,000 years to have had discernible effects on genomic diversity. This conclusion does not imply that humans have experienced few phenotypic adaptations, or that adaptations have not shaped genomic patterns of diversity. Comparisons of diversity and divergence levels at putatively functional versus

References and Notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Fig. 4. (A) Enrichment of highly differentiated SNPs in genic relative to nongenic sites. Shown is the CEUYRI comparison (other population comparisons are in fig. S9A). The total number of SNPs considered in each pairwise comparison is provided within each panel. Central 90% and 98% confidence intervals are shown with gray and black dashed lines, respectively; they were obtained by bootstrapping 200-kb regions 1000 times (16). (B) Enrichment of highly differentiated SNPs in conserved noncoding positions relative to nonconserved, noncoding positions (20) for the CEU-YRI comparison. Other population comparisons are in fig. S9B. (C to F) Enrichment of specific genic annotations relative to the genomic background for the CEU-YRI comparison, with central 90% and 98% confidence intervals shown as gray and black dashed lines, respectively. Note that an enrichment of 0 corresponds to no SNPs with that level of differentiation, and the confidence interval is not estimated in this case. Other population comparisons are shown in fig. S10. For the numbers of each bin, see fig. S11. Enrichments calculated on the folded frequency spectrum are shown in fig. S13B. For a comparison of synonymous and nonsynonymous SNPs in an alternative resequencing data set, see fig. S14. www.sciencemag.org

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11. 12. 13. 14. 15. 16. 17. 18. 19.

P. C. Sabeti et al., Science 312, 1614 (2006). J. M. Akey, Genome Res. 19, 711 (2009). J. Maynard Smith, J. Haigh, Genet. Res. 23, 23 (1974). M. Przeworski, Genetics 160, 1179 (2002). S. H. Williamson et al., PLoS Genet. 3, e90 (2007). K. M. Teshima, G. Coop, M. Przeworski, Genome Res. 16, 702 (2006). J. Hermisson, P. S. Pennings, Genetics 169, 2335 (2005). J. K. Pritchard, J. K. Pickrell, G. Coop, Curr. Biol. 20, R208 (2010). I. Hellmann et al., Genome Res. 18, 1020 (2008). J. J. Cai, J. M. Macpherson, G. Sella, D. A. Petrov, PLoS Genet. 5, e1000336 (2009). G. McVicker, D. Gordon, C. Davis, P. Green, PLoS Genet. 5, e1000471 (2009). A. Keinan, D. Reich, PLoS Genet. 6, e1000886 (2010). E. Santiago, A. Caballero, Genetics 169, 475 (2005). P. C. Sabeti et al., International HapMap Consortium, Nature 449, 913 (2007). L. B. Barreiro, G. Laval, H. Quach, E. Patin, L. Quintana-Murci, Nat. Genet. 40, 340 (2008). G. Coop et al., PLoS Genet. 5, e1000500 (2009). B. Charlesworth, M. T. Morgan, D. Charlesworth, Genetics 134, 1289 (1993). B. Charlesworth, M. Nordborg, D. Charlesworth, Genet. Res. 70, 155 (1997). R. M. Durbin et al., 1000 Genomes Project Consortium, Nature 467, 1061 (2010).

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See supporting material on Science Online. M. F. Hammer et al., Nat. Genet. 42, 830 (2010). D. G. Torgerson et al., PLoS Genet. 5, e1000592 (2009). N. Patterson, D. J. Richter, S. Gnerre, E. S. Lander, D. Reich, Nature 441, 1103 (2006). S. Sattath, E. Elyashiv, O. Kolodny, Y. Rinott, G. Sella, PLoS Genet. pgen.1001302 (2010). R. N. Gutenkunst, R. D. Hernandez, S. H. Williamson, C. D. Bustamante, PLoS Genet. 5, e1000695 (2009). J. B. Veyrieras et al., PLoS Genet. 4, e1000214 (2008). M. Przeworski, G. Coop, J. D. Wall, Evolution 59, 2312 (2005). H. Innan, Y. Kim, Genetics 179, 1713 (2008).

29. A. Eyre-Walker, P. D. Keightley, Mol. Biol. Evol. 26, 2097 (2009). 30. A. R. Boyko et al., PLoS Genet. 4, e1000083 (2008). 31. E. Santiago, A. Caballero, Genetics 139, 1013 (1995). 32. P. L. Ralph, G. Coop, Genetics 186, 647 (2010). 33. G. H. Perry et al., Nat. Genet. 39, 1256 (2007). 34. H. A. Orr, Genetics 149, 2099 (1998). 35. We thank G. Coop, T. Long, G. McVicker, J. Pickrell, J. Pritchard, and K. Thornton for helpful discussions, and G. Coop, A. Di Rienzo, and J. Pritchard for comments. Supported by an NSF minority postdoctoral fellowship (R.D.H.), National Research Service Award postdoctoral fellowship GM087069 (J.L.K.), Wellcome Trust grant

Early Tagging of Cortical Networks Is Required for the Formation of Enduring Associative Memory Edith Lesburguères, Oliviero L. Gobbo, Stéphanie Alaux-Cantin, Anne Hambucken, Pierre Trifilieff, Bruno Bontempi* Although formation and stabilization of long-lasting associative memories are thought to require time-dependent coordinated hippocampal-cortical interactions, the underlying mechanisms remain unclear. Here, we present evidence that neurons in the rat cortex must undergo a “tagging process” upon encoding to ensure the progressive hippocampal-driven rewiring of cortical networks that support remote memory storage. This process was AMPA- and N-methyl-D-aspartate receptor–dependent, information-specific, and capable of modulating remote memory persistence by affecting the temporal dynamics of hippocampal-cortical interactions. Post-learning reinforcement of the tagging process via time-limited epigenetic modifications resulted in improved remote memory retrieval. Thus, early tagging of cortical networks is a crucial neurobiological process for remote memory formation whose functional properties fit the requirements imposed by the extended time scale of systems-level memory consolidation. emories for facts and events are not acquired in their definitive form but undergo a gradual process of stabilization over time (1–3). According to the so-called standard theory of systems-level memory consolidation, the hippocampus (HPC) is believed to integrate, in the form of an anatomical index, information transmitted from distributed cortical networks that support the various features of a whole experience (4). Upon encoding, the HPC rapidly fuses these different features into a coherent memory trace. Consolidation of this new memory trace at the cortical level would then occur slowly via repeated and coordinated reactivation of hippocampal-cortical networks in order to progressively increase the strength and stability of cortical-cortical connections that represent the original experience. Over days to weeks as memories mature, the role of the HPC would gradually diminish, presumably leaving cortical areas to become capable of sustaining permanent memories and mediating their retrieval inde-

M

Institut des Maladies Neurodégénératives, CNRS UMR 5293, Universités Bordeaux 1 et 2, Avenue des Facultés, 33405 Talence, France. *To whom correspondence should be addressed. E-mail: [email protected]

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pendently (4, 5). Using brain imaging, we have provided evidence supporting the time-limited role of the HPC as a consolidation organizing device of remote memory in the cortex (5, 6). Yet the nature and dynamics of plasticity phenomena as well as the neuronal constraints within hippocampal-cortical networks responsible for the formation of remote memories have remained elusive. To pinpoint the post-learning mechanisms underlying the hippocampal-cortical dialogue during the course of systems-level memory consolidation, we used the social transmission of food preference (STFP) paradigm, which involves an ethologically based form of associative olfactory memory (7). In this task, rats learn, within only one single interaction session of 30 min, about the safety of potential food sources by sampling the odor of those sources on the breath of littermates (8). After establishing the necessary role played by the HPC in acquisition of associative olfactory memory (9) (fig. S1), we trained independent groups of rats and tested them for memory retrieval either 1 day (recent memory) or 30 days (remote memory) later. Interaction of experimental observer rats with a demonstrator rat fed with cumin reversed the innate preference typically expressed by

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WT086084MA (G.M.), Israeli Science Foundation grant 1492/ 10 and NIH grant GM083228 (G.S.), and NIH grants GM20373 and GM72861 (M.P.). M.P. is a Howard Hughes Medical Institute Early Career Scientist.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/920/DC1 Materials and Methods Figs. S1 to S16 References 12 October 2010; accepted 6 January 2011 10.1126/science.1198878

control observer rats (food preference), which interacted with a demonstrator rat fed with plain food (fig. S2A). The acquired memory for cumin was robust and long-lasting, which makes the STFP task particularly suitable to studying the processes underlying remote memory formation. Cellular imaging of the activity-dependent gene c-fos (6) coupled to region-specific pharmacological inactivation of HPC by using tetrodotoxin or the AMPA receptor antagonist 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) revealed a transitory role of this region in remote memory storage of associative olfactory information (figs. S2, B to D, and S3). At the cortical level, we focused our analyses on the orbitofrontal cortex (OFC) because of its privileged role in processing the relevance of associative olfactory information (fig. S2E) (10). The observed hippocampal disengagement was associated with a concomitant increase in Fos immunoreactivity in the OFC (fig. S2B). Accordingly, inactivation of the OFC selectively impaired remote memory retrieval (figs. S2, C and D, and S3). Although a broad cortical network is likely to be involved in the processing of remote associative olfactory memory, our results identify the OFC as a critical node within this network. Concurring with this, we found neuronal networks of the OFC to undergo time-dependent morphological changes at pre- and postsynaptic sites (fig. S4). To determine whether such a progressive synaptic remodeling in the OFC during remote memory storage is driven by the HPC, we chronically inactivated the dorsal HPC during two critical post-learning periods: an early (from day 0 to day 12) and a late (from day 15 to day 27) time window (Fig. 1A). This reversible pharmacological approach enabled us to target selectively post-learning consolidation mechanisms without interfering with retrieval processes (fig. S3). Remote memory retrieval examined at day 30 was impaired when hippocampal activity was silenced during the early, but not the late, postlearning period (Fig. 1B and fig. S5). This early hippocampal dysfunction also completely abolished the late development of dendritic spine growth on OFC neurons (Fig. 1B). Delaying intrahippocampal infusion of CNQX by 1 day so as to prevent any confounding interference with cellular consolidation mechanisms (1, 2, 5) that were triggered immediately upon encoding yielded similar results (fig. S6). Therefore, these findings

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20. 21. 22. 23.

REPORTS

24. 25. 26. 27. 28.

See supporting material on Science Online. M. F. Hammer et al., Nat. Genet. 42, 830 (2010). D. G. Torgerson et al., PLoS Genet. 5, e1000592 (2009). N. Patterson, D. J. Richter, S. Gnerre, E. S. Lander, D. Reich, Nature 441, 1103 (2006). S. Sattath, E. Elyashiv, O. Kolodny, Y. Rinott, G. Sella, PLoS Genet. pgen.1001302 (2010). R. N. Gutenkunst, R. D. Hernandez, S. H. Williamson, C. D. Bustamante, PLoS Genet. 5, e1000695 (2009). J. B. Veyrieras et al., PLoS Genet. 4, e1000214 (2008). M. Przeworski, G. Coop, J. D. Wall, Evolution 59, 2312 (2005). H. Innan, Y. Kim, Genetics 179, 1713 (2008).

29. A. Eyre-Walker, P. D. Keightley, Mol. Biol. Evol. 26, 2097 (2009). 30. A. R. Boyko et al., PLoS Genet. 4, e1000083 (2008). 31. E. Santiago, A. Caballero, Genetics 139, 1013 (1995). 32. P. L. Ralph, G. Coop, Genetics 186, 647 (2010). 33. G. H. Perry et al., Nat. Genet. 39, 1256 (2007). 34. H. A. Orr, Genetics 149, 2099 (1998). 35. We thank G. Coop, T. Long, G. McVicker, J. Pickrell, J. Pritchard, and K. Thornton for helpful discussions, and G. Coop, A. Di Rienzo, and J. Pritchard for comments. Supported by an NSF minority postdoctoral fellowship (R.D.H.), National Research Service Award postdoctoral fellowship GM087069 (J.L.K.), Wellcome Trust grant

Early Tagging of Cortical Networks Is Required for the Formation of Enduring Associative Memory Edith Lesburguères, Oliviero L. Gobbo, Stéphanie Alaux-Cantin, Anne Hambucken, Pierre Trifilieff, Bruno Bontempi* Although formation and stabilization of long-lasting associative memories are thought to require time-dependent coordinated hippocampal-cortical interactions, the underlying mechanisms remain unclear. Here, we present evidence that neurons in the rat cortex must undergo a “tagging process” upon encoding to ensure the progressive hippocampal-driven rewiring of cortical networks that support remote memory storage. This process was AMPA- and N-methyl-D-aspartate receptor–dependent, information-specific, and capable of modulating remote memory persistence by affecting the temporal dynamics of hippocampal-cortical interactions. Post-learning reinforcement of the tagging process via time-limited epigenetic modifications resulted in improved remote memory retrieval. Thus, early tagging of cortical networks is a crucial neurobiological process for remote memory formation whose functional properties fit the requirements imposed by the extended time scale of systems-level memory consolidation. emories for facts and events are not acquired in their definitive form but undergo a gradual process of stabilization over time (1–3). According to the so-called standard theory of systems-level memory consolidation, the hippocampus (HPC) is believed to integrate, in the form of an anatomical index, information transmitted from distributed cortical networks that support the various features of a whole experience (4). Upon encoding, the HPC rapidly fuses these different features into a coherent memory trace. Consolidation of this new memory trace at the cortical level would then occur slowly via repeated and coordinated reactivation of hippocampal-cortical networks in order to progressively increase the strength and stability of cortical-cortical connections that represent the original experience. Over days to weeks as memories mature, the role of the HPC would gradually diminish, presumably leaving cortical areas to become capable of sustaining permanent memories and mediating their retrieval inde-

M

Institut des Maladies Neurodégénératives, CNRS UMR 5293, Universités Bordeaux 1 et 2, Avenue des Facultés, 33405 Talence, France. *To whom correspondence should be addressed. E-mail: [email protected]

924

pendently (4, 5). Using brain imaging, we have provided evidence supporting the time-limited role of the HPC as a consolidation organizing device of remote memory in the cortex (5, 6). Yet the nature and dynamics of plasticity phenomena as well as the neuronal constraints within hippocampal-cortical networks responsible for the formation of remote memories have remained elusive. To pinpoint the post-learning mechanisms underlying the hippocampal-cortical dialogue during the course of systems-level memory consolidation, we used the social transmission of food preference (STFP) paradigm, which involves an ethologically based form of associative olfactory memory (7). In this task, rats learn, within only one single interaction session of 30 min, about the safety of potential food sources by sampling the odor of those sources on the breath of littermates (8). After establishing the necessary role played by the HPC in acquisition of associative olfactory memory (9) (fig. S1), we trained independent groups of rats and tested them for memory retrieval either 1 day (recent memory) or 30 days (remote memory) later. Interaction of experimental observer rats with a demonstrator rat fed with cumin reversed the innate preference typically expressed by

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WT086084MA (G.M.), Israeli Science Foundation grant 1492/ 10 and NIH grant GM083228 (G.S.), and NIH grants GM20373 and GM72861 (M.P.). M.P. is a Howard Hughes Medical Institute Early Career Scientist.

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/920/DC1 Materials and Methods Figs. S1 to S16 References 12 October 2010; accepted 6 January 2011 10.1126/science.1198878

control observer rats (food preference), which interacted with a demonstrator rat fed with plain food (fig. S2A). The acquired memory for cumin was robust and long-lasting, which makes the STFP task particularly suitable to studying the processes underlying remote memory formation. Cellular imaging of the activity-dependent gene c-fos (6) coupled to region-specific pharmacological inactivation of HPC by using tetrodotoxin or the AMPA receptor antagonist 6-cyano-7nitroquinoxaline-2,3-dione (CNQX) revealed a transitory role of this region in remote memory storage of associative olfactory information (figs. S2, B to D, and S3). At the cortical level, we focused our analyses on the orbitofrontal cortex (OFC) because of its privileged role in processing the relevance of associative olfactory information (fig. S2E) (10). The observed hippocampal disengagement was associated with a concomitant increase in Fos immunoreactivity in the OFC (fig. S2B). Accordingly, inactivation of the OFC selectively impaired remote memory retrieval (figs. S2, C and D, and S3). Although a broad cortical network is likely to be involved in the processing of remote associative olfactory memory, our results identify the OFC as a critical node within this network. Concurring with this, we found neuronal networks of the OFC to undergo time-dependent morphological changes at pre- and postsynaptic sites (fig. S4). To determine whether such a progressive synaptic remodeling in the OFC during remote memory storage is driven by the HPC, we chronically inactivated the dorsal HPC during two critical post-learning periods: an early (from day 0 to day 12) and a late (from day 15 to day 27) time window (Fig. 1A). This reversible pharmacological approach enabled us to target selectively post-learning consolidation mechanisms without interfering with retrieval processes (fig. S3). Remote memory retrieval examined at day 30 was impaired when hippocampal activity was silenced during the early, but not the late, postlearning period (Fig. 1B and fig. S5). This early hippocampal dysfunction also completely abolished the late development of dendritic spine growth on OFC neurons (Fig. 1B). Delaying intrahippocampal infusion of CNQX by 1 day so as to prevent any confounding interference with cellular consolidation mechanisms (1, 2, 5) that were triggered immediately upon encoding yielded similar results (fig. S6). Therefore, these findings

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Fig. 1. Conjoint functional participation of HPC and OFC in the early, but not late, post-acquisition period is required for systems-level consolidation of remote associative olfactory memory. (A) Experimental design depicting region-specific pharmacological inactivation of HPC or OFC with CNQX during either the early or late post-acquisition periods after social interaction. Control rats received artificial cerebrospinal fluid (aCSF) as vehicle. (B) Early, but not late, hippocampal inactivation impaired remote memory retrieval at day 30 (left, treatment × delay interaction, F1,31 = 4.74; P < 0.05) and prevented the associated increase in spine density along basal dendrites of OFC neurons that is typically observed in aCSF rats (right, treatment × delay interaction, F1,15 = 5.56; P < 0.04). This interaction was close to reaching significance for apical dendrites ( F1,15 = 3.65; P < 0.07). (C) Both early and late inactivation of OFC impaired remote memory retrieval (left, main treatment effect F1,36 = 15.26, P < 0.001) and prevented structural plasticity in the OFC compared with aCSF rats (right, main treatment effect, apical, F1,16 = 17.82, P < 0.01; basal, F1,16 = 11.25, P < 0.01). The dotted line represents performance or cortical spine density in food preference controls. *P < 0.05 versus aCSF, n = 5 to 12 rats per group. www.sciencemag.org

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in the cortex (13, 14). How then does the HPC manage to activate the proper experience-relevant set of cortical neurons during the course of systemslevel consolidation? Insights can be gained from a repertoire of cellular and molecular mechanisms that enables activated synapses to be “tagged” within the few hours that follow an event (15, 16)—a time course compatible with cellular consolidation (2, 5). To examine the relevance of the synaptic tagging framework to the process of systems-level memory consolidation that operates over much longer time windows, we carried out four complementary experiments (SOM text). Building on the hippocampal indexing theory (14), we hypothesized that tagged synapses should contribute to the formation of a hippocampal index that in turn would act as a coincidence regenerator for activating and strengthening over time the relevant set of distributed cortical cell assemblies (17). Thus, we predicted that hippocampal and cortical networks should be tagged simultaneously at the time of encoding, albeit possibly through different mechanisms. Because the HPC is crucial for acquisition of the STFP task, interfering with hippocampal function during encoding will inevitably result in memory retrieval impairment, which is a deleterious effect observed in most hippocampal-dependent tasks (fig. S1) (5). To circumvent this confounding factor, we focused on the OFC and first examined whether its inactivation immediately before social interaction interfered with memory retrieval tested 7 days later. In accordance with previous findings (18), memory retrieval at this early time point was unaffected, therefore indicating that the OFC is not necessary for acquisition of the STFP task (Fig. 2A and SOM text). Reliance only on the olfactory component of the STFP paradigm was insufficient to explain this lack of effect (fig. S8). In sharp contrast, a similar inactivation resulted in impaired memory retrieval probed at a longer time point (day 30) (Fig. 2A) and completely abolished the later occurrence of dendritic spine growth on OFC neurons (Fig. 2B). Thus, impairing the tagging of cortical networks at the time of encoding most likely interfered with the post-acquisition hippocampal-cortical dialogue, preventing the HPC to activate the adequate pattern of cortical neurons and synapses recruited during the initial learning episode. This finding identifies early cortical tagging as a potentially critical process responsible for the progressive embedding of enduring memories within cortical networks. However, the validity of such a neurobiological process with respect to synaptic plasticity and remote memory formation rests in demonstrating additional key features, that is, (i) Nmethyl-D-aspartate (NMDA) receptor dependency, (ii) information specificity, and (iii) modulation of memory persistence. As for AMPA receptors (Fig. 2A), selectively blocking NMDA receptors in the OFC led to a robust impairment in remote memory retrieval at day 30 (Fig. 2C). If cortical tagging is information-specific, then disrupting

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post-learning period (Fig. 1C)—a time window reported above as being no longer hippocampaldependent. Thus despite hippocampal disengagement, additional cortical-cortical interactions appear to be required to ensure stability of cortical networks. Alternatively, disrupting cortical activity during the late phase of the consolidation process may have interfered with neuronal mechanisms underlying memory maintenance (11, 12). To rule out the possibility that the observed memory disruption was due to a nonspecific impairment in hippocampal or OFC function, we established that the same chronically inactivated animals could relearn and consolidate when tested 1 day (HPC groups) or 30 days (OFC groups) after a novel interaction with a demonstrator rat fed with a different flavor (fig. S7). Hippocampal-cortical projections are topographically organized, which makes the HPC ideally suited for organizing structural changes

point to the crucial, but time-limited, involvement of the HPC in driving at least in part wiring plasticity in the OFC [supporting online material (SOM) text]. Because the establishment of cortical memory is critically dependent on coordinated hippocampalcortical interactions (1–5), we predicted that cortical disruption during the early post-learning period should preclude cortical processing of hippocampal inputs and therefore lead to memory degradation or erasure. We thus applied to the OFC the same experimental design as for the HPC (Fig. 1A). Silencing neuronal activity during the early post-learning period impaired remote memory retrieval and structural plasticity, indicating that early cortical activity is required for subsequent maturation and stabilization of the memory trace (Fig. 1C and figs. S5 and S6). More unexpected was the finding of similar alterations after OFC inactivation during the late

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REPORTS trieval for the first one (Fig. 2D). Therefore, cortical tagging appears as highly specific for a given memory trace. This property could contribute to minimizing interference with the processing of other information that is already engaged in the consolidation process and needs to be interleaved with existing cortical knowledge or mental schemas (19). We next tested the ability of the cortical tagging process to modulate memory persistence by manipulating its efficacy. Because multiple cell assemblies are thought to act in concert during

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Fig. 2. Early tagging of OFC neurons is a prerequisite for the formation of remote associative olfactory memory. (A) Intra-OFC infusion of CNQX before social interaction (day 0) impaired remote memory formation assessed at day 30 while sparing retrieval of recent memory probed at day 7 (treatment × delay interaction, F1,31 = 5.67; P < 0.05). (B) OFC inactivation upon encoding prevented the late development of structural plasticity seen in this region in aCSF controls. OFC inactivation reduced cortical spine density along both apical (left, t9 = 2.66; P < 0.05) and basal (right, t9 = 2.31; P < 0.05) dendrites and resulted in a lower proportion of neurons exhibiting intermediate and high spine density (bottom). (C) Injecting AP-5 into OFC before social interaction resulted in impaired remote memory retrieval at day 30 (F1,14 = 17.39; P < 0.001). (D) Intra-OFC infusion of CNQX at the time of the second interaction (cumin) impaired selectively remote memory for this second flavor while sparing remote memory for the first one (cocoa) (treatment × flavor pair, F1,11 = 13.62; P < 0.01). (Bottom left) Schematic of OFC neurons presumably tagged

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upon encoding of the two olfactory associative informations. Blue, tagged neurons during cocoa interaction; crossed green, neurons whose tagging was prevented by CNQX during cumin interaction. (E and F) Although extensive OFC inactivation impaired memory retrieval at both day 15 and 30 (main treatment effect, F2,37 = 12.0; P < 0.01), partial inactivation affected remote memory only at day 30 (main treatment × delay interaction, F2,38 = 3.42; P < 0.05). (G and H) Extent of OFC inactivation upon encoding differentially affected the post-acquisition kinetics of hippocampal and cortical activation assessed by means of Fos counts relative to controls (dotted line indicates 100%). Persistence of hippocampal activity at day 15 after partial OFC inactivation [(H) top, main treatment, F1,24 = 13.32; P < 0.01] was not observed after extensive inactivation [(G) top, F1,22 = 0.87; P > 0.30, not significant]. Both types of OFC inactivation prevented the functional recruitment of this region seen over time in aCSF controls (main treatment, extensive, F1,22 = 30.65, P < 0.0001; partial, F1,24 = 12.79, P < 0.01). *P < 0.05 versus aCSF; n = 5 to 10 rats per group. SCIENCE

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memory processing, we reasoned that the threedimensional extent (partial versus extensive) of the OFC inactivation should differentially affect the number of interconnected neurons tagged upon encoding and potentially alter the efficacy of hippocampal-cortical interactions in the guidance of remote memory formation. To assess the time course of memory persistence, independent groups of rats were tested at 7, 15, and 30 days after social interaction (Fig. 2, E and F). Extensive OFC inactivation upon encoding impaired memory retrieval regardless of the two

Fos counts (% control)

this process should predominantly prevent the formation and stabilization of one given memory trace but enable systems-level consolidation of another one. We submitted rats to two consecutive social interactions by using two different flavors and inactivated the OFC only before the second interaction, thus ensuring the occurrence of tagging of cortical cell assemblies coding for the first olfactory information (Fig. 2D). Remote memory was probed 30 days after each interaction. OFC inactivation selectively impaired retrieval for the second flavor while sparing re-

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extracellular-signal regular kinase (MAPK/ERK) cascade in the OFC immediately before social interaction. Downstream of MAPK/ERK, we also targeted the nuclear mitogen- and stress-activated protein kinase 1 (MSK1), which acts as a main regulating gateway of histone H3 modifications (23). Blocking activation of these targets upon acquisition prevented the associated increase in histone H3 acetylation (fig. S10), impaired remote memory assessed 30 days later (Fig. 3B), and abolished the late development of cortical structural plasticity (fig. S11). These effects are consistent with that observed after blockade of NMDA receptors whose activity is coupled with the activation of the MAPK cascade (Fig. 2C and fig. S11). Although confirming the important role played by the MAPK/ERK pathway in regulating gene expression associated with remote memory processing (24), these results identify MSK1 as one important nuclear mediator of the cortical tagging process. Because the observed STFPinduced acetylation in the OFC was transitory but pharmacologically modulable (Fig. 3C and figs. S10 and S12), we investigated whether maintenance of a higher level of acetylation would translate into a potentially easier-to-retrieve remote memory. Starting immediately upon completion of the social interaction, we performed repeated intracortical infusions of the histone deacetylase inhibitors sodium butyrate (NaB) or Trichostatin A during the early post-acquisition period. This resulted in improved remote mem-

4h

ory retrieval probed 30 days later (Fig. 3D and fig. S12) and in a higher level of expression of synaptophysin in the OFC (fig. S13). Administering NaB during the late post-acquisition period was ineffective (Fig. 3D). Although cortical tags have to be set upon encoding and persist thereafter, these findings indicate that it may be possible to facilitate remote memory formation and retrieval by potentiating, in a time-dependent manner, the epigenetic mechanisms that underlie their formation and subsequent regulation (SOM text). Efficacy of this type of pharmacological manipulation matched the hippocampal-dependent phase of the memory consolidation process. Thus, cortical tags may act as the scaffolding to support the progressive hippocampal-driven embedding of permanent memories into cortical networks via “weight” (changes in the efficacy of synaptic transmission between existing synapses) and wiring plasticity (25), potentially making these memories more resistant over time when the initial scaffolding is reinforced (SOM text). Replay of encoding-related activity during phases of sleep has emerged as a core mechanism for driving structural changes within hippocampalcortical neuronal networks (26 ). In this context, our findings identify early tagging of cortical networks as a crucial process for the formation of enduring memories and suggest that synaptic tags contribute actively, although not exclusively, to the development but also the maintenance of this form of plasticity in the cortex (SOM text) (11, 12, 27). The identity of synaptic tags needs to be further explored, although potential candidates have started to emerge (28, 29). Given the complex nature of memory traces that typically integrate the various modalities of an event, it is likely that a plethora of synaptic molecules act in concert to capture the various patterns of synaptic activity elicited upon encoding. These tags may then be recruited differentially during the course of systems-level consolidation depending on the status of existing knowledge in the cortex (2, 19).

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longer retention delays, indicating a rapid decay of the associative memory (Fig. 2E). In contrast, partial silencing of OFC activity was deleterious only at the 30-day interval (Fig. 2F). Manipulating cortical tagging affected the temporal dynamics of hippocampal-cortical interactions. The preserved memory at day 15 after partial OFC inactivation was indeed associated with an unusual maintenance of hippocampal activation at this time point, possibly to compensate for the absence of gradual recruitment of the OFC normally seen in control animals over the 30-day period (Fig. 2, G and H, and SOM text). Thus, despite the potential of the HPC to act as a temporary memory buffer, the alteration of cortical tagging led to an unstable memory trace that was more prone to degradation. This indicates that synaptic tags may serve as an early and persistent signature of activity in the cortex that is necessary to ensure the progressive rewiring of cortical networks that support remote memory storage. Because chromatin remodeling events are crucial enduring regulatory mechanisms involved in gene transcription, we examined whether the setting of synaptic tags triggered specific signaling cascades leading to histone-tail acetylation in the OFC, a modification that modulates memory function (20–22). Increased acetylation of histone H3 in this cortical region was memoryspecific (Fig. 3A and fig. S9). We next blocked upstream the mitogen-activated protein kinase/

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Fig. 3. The tagging process in the OFC triggers specific signaling cascades involved in the regulation of chromatin remodeling. (A) Experimental (EXP) rats showed increased histone H3 acetylation at lysine 9 in the OFC compared with food preference (FP) controls 1 hour after social interaction (F1,10 = 19.72; P < 0.01). (B) Intra-OFC infusion of the MAPK/ERK kinase inhibitor U0126 or the MSK1 inhibitor H89 before social interaction impaired remote memory retrieval assessed 30 days later (F2,18 = 4.03; P < 0.05). (C) Intra-OFC infusion of the histone deactelylase inhibitor NaB immediately upon completion of social interaction increased the level of histone H3 acetylation assessed 4 hours after acquisition compared with aCSF rats ( F1,9 = 77.62; P < 0.0001). (D) Experimental design used to investigate the effects of maintaining in the OFC the level of histone H3 acetylation during the early or late STFP post-acquisition periods on remote memory retrieval at day 30 (left). Intra-OFC infusion of NaB improved remote memory only when delivered during the early, but not late, post-acquisition period (right, treatment × delay interaction, F1,35 = 4.27; P < 0.05). *P < 0.05 versus FP; *P < 0.05 versus aCSF; n = 5 to 12 rats per group. www.sciencemag.org

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References and Notes 1. Y. Dudai, Annu. Rev. Psychol. 55, 51 (2004). 2. S. H. Wang, R. G. Morris, Annu. Rev. Psychol. 61, 49, C1 (2010). 3. L. R. Squire, P. J. Bayley, Curr. Opin. Neurobiol. 17, 185 (2007). 4. L. R. Squire, P. Alvarez, Curr. Opin. Neurobiol. 5, 169 (1995). 5. P. W. Frankland, B. Bontempi, Nat. Rev. Neurosci. 6, 119 (2005). 6. T. Maviel, T. P. Durkin, F. Menzaghi, B. Bontempi, Science 305, 96 (2004). 7. B. G. Galef Jr., E. E. Whiskin, Learn. Behav. 31, 160 (2003). 8. Materials and methods are available as supporting material on Science Online. 9. P. Alvarez, P. A. Lipton, R. Melrose, H. Eichenbaum, Learn. Mem. 8, 79 (2001). 10. G. Schoenbaum, M. Roesch, Neuron 47, 633 (2005). 11. J. I. Rossato, L. R. Bevilaqua, I. Izquierdo, J. H. Medina, M. Cammarota, Science 325, 1017 (2009). 12. R. Shema, T. C. Sacktor, Y. Dudai, Science 317, 951 (2007). 13. H. Eichenbaum, Neuron 44, 109 (2004). 14. T. J. Teyler, J. W. Rudy, Hippocampus 17, 1158 (2007).

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REPORTS 24. G. M. Thomas, R. L. Huganir, Nat. Rev. Neurosci. 5, 173 (2004). 25. D. B. Chklovskii, B. W. Mel, K. Svoboda, Nature 431, 782 (2004). 26. S. Diekelmann, J. Born, Nat. Rev. Neurosci. 11, 114 (2010). 27. C. A. Miller et al., Nat. Neurosci. 13, 664 (2010). 28. K. C. Martin, K. S. Kosik, Nat. Rev. Neurosci. 3, 813 (2002). 29. D. Okada, F. Ozawa, K. Inokuchi, Science 324, 904 (2009). 30. We thank T. Durkin for comments on earlier drafts of the manuscript and T. Maviel, L. Restivo, M. Ammassari-Teule, C. Saugier, L. Decorte, and A. Faugère for their experimental

Microtubule Stabilization Reduces Scarring and Causes Axon Regeneration After Spinal Cord Injury Farida Hellal,1 Andres Hurtado,2 Jörg Ruschel,1 Kevin C. Flynn,1 Claudia J. Laskowski,1 Martina Umlauf,1 Lukas C. Kapitein,3,4 Dinara Strikis,5 Vance Lemmon,5 John Bixby,5 Casper C. Hoogenraad,3,4 Frank Bradke1* Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor–b signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow. icrotubule dynamics regulate key processes during scarring, including cell proliferation, migration, and differentiation as well as intracellular trafficking and secretion of extracellular matrix (ECM) molecules (1, 2). Moreover, moderate microtubule stabilization prevents axonal retraction and swelling of the axon tip after central nervous system (CNS) injury (3) and stimulates axon growth of cultured neurons (4), enabling them to overcome the growth inhibitory effect of CNS myelin (3). We hypothesized that moderate microtubule stabilization with Taxol, an approved drug, would facilitate axonal regeneration after spinal cord injury (SCI) by decreasing scar formation and enhancing intrinsic axonal growth.

M

1 Axonal Growth and Regeneration Group, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany. 2International Center for Spinal Cord Injury, Hugo W. Moser Research Institute at Kennedy Krieger, Department of Neurology, Johns Hopkins University, 707 North Broadway, Suite 523, Baltimore, MD 21205, USA. 3Department of Neuroscience, Erasmus Medical Center, Post Office Box 2040, 3000 CA Rotterdam, Netherlands. 4Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. 5The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 1095 Northwest 14th Terrace, Miami, FL 33136, USA.

*To whom correspondence should be addressed: fbradke@ neuro.mpg.de

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We first examined whether Taxol treatment reduced scarring after SCI. Adult rats underwent a dorsal hemisection at the eighth thoracic spinal

contribution or technical advice. This work was supported by the CNRS, Fondation Simone et Cino del Duca (B.B. and O.L.G.), Fédération pour la Recherche sur le Cerveau (B.B.), and the Agence Nationale de la Recherche (ANR grant 09-MNPS-005-01 to B.B.).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/924/DC1 Materials and Methods SOM Text Figs. S1 to S13 References 6 August 2010; accepted 16 December 2010 10.1126/science.1196164

cord level; Taxol was continuously delivered at the injury site using an intrathecal catheter connected to an osmotic minipump. Seven days after injury, the lesion of vehicle-treated animals was filled with laminin, fibronectin, and collagen IV, hallmarks of a fibrotic scar (Fig. 1, A and B, and fig. S1), which is a major impediment for axon regeneration (5–7). In contrast, in the lesion of animals treated with Taxol at 256 ng/day, a much lower dose than used for chemotherapy (8), laminin, fibronectin, and collagen IV were strongly reduced (Fig. 1, C and D, and fig. S1). At this Taxol concentration, astrocytes surrounded the injury as in vehicle controls (Fig. 1, B, C, and E); the injury size was equivalent in both groups (Fig. 1F), which suggests that astrocytes isolated the lesion from undamaged CNS tissue (9). Phosphohistone-H3 immunostaining and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) showed that the numbers of proliferating and apoptotic cells were similar between Taxol- and vehicle-treated animals (figs. S2 and S3). Thus, at low doses, Taxol reduced fibrotic scarring by

Fig. 1. Taxol decreases scarring induced by spinal cord injury. (A) Representation of lesioned spinal cord (box). (B and C) Midsagittal sections of lesion site from rats treated with (B) vehicle or (C) Taxol (256 ng/day) 7 days after injury. Scale bars, 300 mm. (D) Taxol significantly decreases fibrotic scarring (expressed as percentage of vehicle control; n = 12 rats per group; **P = 0.002; two-tailed t test) without affecting glial compaction (28 days after injury) (E) or injury size (F) (n = 7 to 10 rats per group; P = 0.951; two-tailed t test). Data expressed as mean T SEM. GFAP, glial fibrillary acidic protein.

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15. U. Frey, R. G. Morris, Trends Neurosci. 21, 181 (1998). 16. A. Barco, M. Lopez de Armentia, J. M. Alarcon, Neurosci. Biobehav. Rev. 32, 831 (2008). 17. G. M. Wittenberg, J. Z. Tsien, Trends Neurosci. 25, 501 (2002). 18. C. A. Smith, B. S. East, P. J. Colombo, Behav. Brain Res. 208, 243 (2010). 19. D. Tse et al., Science 316, 76 (2007). 20. T. L. Roth, J. D. Sweatt, Curr. Opin. Neurobiol. 19, 336 (2009). 21. C. G. Vecsey et al., J. Neurosci. 27, 6128 (2007). 22. M. W. Swank, J. D. Sweatt, J. Neurosci. 21, 3383 (2001). 23. W. B. Chwang, J. S. Arthur, A. Schumacher, J. D. Sweatt, J. Neurosci. 27, 12732 (2007).

REPORTS 24. G. M. Thomas, R. L. Huganir, Nat. Rev. Neurosci. 5, 173 (2004). 25. D. B. Chklovskii, B. W. Mel, K. Svoboda, Nature 431, 782 (2004). 26. S. Diekelmann, J. Born, Nat. Rev. Neurosci. 11, 114 (2010). 27. C. A. Miller et al., Nat. Neurosci. 13, 664 (2010). 28. K. C. Martin, K. S. Kosik, Nat. Rev. Neurosci. 3, 813 (2002). 29. D. Okada, F. Ozawa, K. Inokuchi, Science 324, 904 (2009). 30. We thank T. Durkin for comments on earlier drafts of the manuscript and T. Maviel, L. Restivo, M. Ammassari-Teule, C. Saugier, L. Decorte, and A. Faugère for their experimental

Microtubule Stabilization Reduces Scarring and Causes Axon Regeneration After Spinal Cord Injury Farida Hellal,1 Andres Hurtado,2 Jörg Ruschel,1 Kevin C. Flynn,1 Claudia J. Laskowski,1 Martina Umlauf,1 Lukas C. Kapitein,3,4 Dinara Strikis,5 Vance Lemmon,5 John Bixby,5 Casper C. Hoogenraad,3,4 Frank Bradke1* Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. Here, moderate microtubule stabilization decreased scar formation after spinal cord injury in rodents through various cellular mechanisms, including dampening of transforming growth factor–b signaling. It prevented accumulation of chondroitin sulfate proteoglycans and rendered the lesion site permissive for axon regeneration of growth-competent sensory neurons. Microtubule stabilization also promoted growth of central nervous system axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow. icrotubule dynamics regulate key processes during scarring, including cell proliferation, migration, and differentiation as well as intracellular trafficking and secretion of extracellular matrix (ECM) molecules (1, 2). Moreover, moderate microtubule stabilization prevents axonal retraction and swelling of the axon tip after central nervous system (CNS) injury (3) and stimulates axon growth of cultured neurons (4), enabling them to overcome the growth inhibitory effect of CNS myelin (3). We hypothesized that moderate microtubule stabilization with Taxol, an approved drug, would facilitate axonal regeneration after spinal cord injury (SCI) by decreasing scar formation and enhancing intrinsic axonal growth.

M

1 Axonal Growth and Regeneration Group, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany. 2International Center for Spinal Cord Injury, Hugo W. Moser Research Institute at Kennedy Krieger, Department of Neurology, Johns Hopkins University, 707 North Broadway, Suite 523, Baltimore, MD 21205, USA. 3Department of Neuroscience, Erasmus Medical Center, Post Office Box 2040, 3000 CA Rotterdam, Netherlands. 4Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. 5The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, 1095 Northwest 14th Terrace, Miami, FL 33136, USA.

*To whom correspondence should be addressed: fbradke@ neuro.mpg.de

928

We first examined whether Taxol treatment reduced scarring after SCI. Adult rats underwent a dorsal hemisection at the eighth thoracic spinal

contribution or technical advice. This work was supported by the CNRS, Fondation Simone et Cino del Duca (B.B. and O.L.G.), Fédération pour la Recherche sur le Cerveau (B.B.), and the Agence Nationale de la Recherche (ANR grant 09-MNPS-005-01 to B.B.).

Supporting Online Material www.sciencemag.org/cgi/content/full/331/6019/924/DC1 Materials and Methods SOM Text Figs. S1 to S13 References 6 August 2010; accepted 16 December 2010 10.1126/science.1196164

cord level; Taxol was continuously delivered at the injury site using an intrathecal catheter connected to an osmotic minipump. Seven days after injury, the lesion of vehicle-treated animals was filled with laminin, fibronectin, and collagen IV, hallmarks of a fibrotic scar (Fig. 1, A and B, and fig. S1), which is a major impediment for axon regeneration (5–7). In contrast, in the lesion of animals treated with Taxol at 256 ng/day, a much lower dose than used for chemotherapy (8), laminin, fibronectin, and collagen IV were strongly reduced (Fig. 1, C and D, and fig. S1). At this Taxol concentration, astrocytes surrounded the injury as in vehicle controls (Fig. 1, B, C, and E); the injury size was equivalent in both groups (Fig. 1F), which suggests that astrocytes isolated the lesion from undamaged CNS tissue (9). Phosphohistone-H3 immunostaining and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) showed that the numbers of proliferating and apoptotic cells were similar between Taxol- and vehicle-treated animals (figs. S2 and S3). Thus, at low doses, Taxol reduced fibrotic scarring by

Fig. 1. Taxol decreases scarring induced by spinal cord injury. (A) Representation of lesioned spinal cord (box). (B and C) Midsagittal sections of lesion site from rats treated with (B) vehicle or (C) Taxol (256 ng/day) 7 days after injury. Scale bars, 300 mm. (D) Taxol significantly decreases fibrotic scarring (expressed as percentage of vehicle control; n = 12 rats per group; **P = 0.002; two-tailed t test) without affecting glial compaction (28 days after injury) (E) or injury size (F) (n = 7 to 10 rats per group; P = 0.951; two-tailed t test). Data expressed as mean T SEM. GFAP, glial fibrillary acidic protein.

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15. U. Frey, R. G. Morris, Trends Neurosci. 21, 181 (1998). 16. A. Barco, M. Lopez de Armentia, J. M. Alarcon, Neurosci. Biobehav. Rev. 32, 831 (2008). 17. G. M. Wittenberg, J. Z. Tsien, Trends Neurosci. 25, 501 (2002). 18. C. A. Smith, B. S. East, P. J. Colombo, Behav. Brain Res. 208, 243 (2010). 19. D. Tse et al., Science 316, 76 (2007). 20. T. L. Roth, J. D. Sweatt, Curr. Opin. Neurobiol. 19, 336 (2009). 21. C. G. Vecsey et al., J. Neurosci. 27, 6128 (2007). 22. M. W. Swank, J. D. Sweatt, J. Neurosci. 21, 3383 (2001). 23. W. B. Chwang, J. S. Arthur, A. Schumacher, J. D. Sweatt, J. Neurosci. 27, 12732 (2007).

REPORTS deed, in Taxol-treated animals, kinesin-1 was enriched in the microtubule fraction of the injury site extracts (Fig. 2B). In brain and spinal cord extracts, Smad2 directly bound to kinesin-1 (Fig. 2, C and D). Taxol strongly altered both kinesin-1and dynein-driven cargo transport in an intracellular trafficking assay (16) (Fig. 2, E and F), suggesting that Taxol would hinder Smad2 trafficking. Indeed, in TGF-b1–stimulated astrocytes, Taxol caused Smad2/3 to localize persistently to microtubules (fig. S4) and inhibited 70% of its translocation to the nucleus (Fig. 2, G and H). In time-lapse microscopy, overexpressed Smad2 fused to photoactivatable green fluorescent protein (PAGFP) moved into the nucleus within minutes after TGF-b1 stimulation, whereas Taxol treatment

abolished this movement (movies S1 and S2). In vivo, 7 days after SCI, phosphorylated Smad2/3 translocated into the nucleus in 95% of vehicletreated animals, compared with only 30% of the Taxol-treated animals (n = 13 animals per group) (Fig. 2I). This suggested that Taxol impairs TGF-b signaling-dependent processes. Indeed, in cultured meningeal cells, Taxol reduced the TGF-b1– stimulated production of fibronectin (Fig. 3A) and impaired TGF-b1–stimulated migration (Fig. 3, B and C). Thus, low doses of Taxol prevent fibrotic scarring after SCI by interfering with Smaddependent TGF-b signaling and reducing extracellular matrix secretion and cell migration. TGF-b signaling also regulates the production of the axon growth inhibitory chondroitin sulfate

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mechanisms independent of cell proliferation or apoptosis. A key event in fibrotic scarring after CNS injury is the activation of transforming growth factor–b (TGF-b) signaling. Following SCI, TGF-b expression dramatically increases, which favors fibrosis (10–12). Integrity of the microtubule network is crucial for the transduction of this signal (13). Smad2, the downstream effector of the TGF-b pathway, binds to microtubules through conventional kinesin-1 (14). We asked whether stabilizing the microtubule network hinders TGF-b signaling and attenuates fibrogenesis. In extracts from the injury site, Taxol treatment increased the level of detyrosinated microtubules (Fig. 2A), enabling kinesin-1 to bind tightly to microtubules (15). In-

Fig. 2. Taxol dampens TGF-b signaling. (A) Taxol treatment increases total tubulin and decreases tyrosinated tubulin in the lesion site. (B) Kinesin-1 enrichment in microtubule fraction of Taxol-treated lesion site. (C) His-Smad2 binds to kinesin-1 and endogenous Smad2 coimmunoprecipitates with kinesin-1 (D) of brain and spinal cord extracts. (E and F) Taxol alters microtubule-based cargo transport. (E) Overlay of colorcoded time series of red fluorescent protein (RFP)–labeled peroxisomes bound to the kinesin-1 (KIF5) motor domain upon Rapalog addition. Blue marks the initial distribution; the red gradient shows the distribution over time (30 min). Scale bars, 10 mm. (F) Time traces of radius of circle enclosing 90% of total fluorescence intensity for KIF5- or dynein adaptor (BICDN)–linked peroxisomes. Mean T SEM of 5 to 8 COS-7 cells per condition (P = 0.004; two-tailed t test for both KIF5 and BICDN). (G and H) In cultured astrocytes, Taxol counteracts the TGF-b1–induced nuclear translocation of Smad2/3 (arrowheads) causing cytoplasmic Smad2/3 accumulation (arrow). Results in (H) are mean T SD [three independent experiments; *P = 0.041; one-way analysis of variance (ANOVA)]. (I) At 7 days after injury, Taxol treatment interferes with the nuclear translocation of phospho-Smad2/3 induced by SCI (n = 13 rats per group). Scale bar, 20 mm. BSA, bovine serum albumin; DAPI, 4´,6´-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.

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Fig. 3. Taxol decreases meningeal cell migration and glycosaminoglycan release in vitro and in vivo. (A) Taxol decreases fibronectin deposits induced by TGF-b1 in meningeal cells. Scale bar, 300 mm. (B and C) Three days in vitro, Taxol (1 and 10 nM) decreases meningeal cell migration induced by TGF-b1. Arrows indicate the initial gap size. Scale bar, 300 mm; results in (C) are means T SD from three independent experiments; *P = 0.003; one-way ANOVA. (D) Taxol treatment decreases the amount of GAGs induced by SCI (n = 9 rats per group). Results are means T SEM; *P = 0.026; two-tailed t test. Taxol decreases GAGs released from control and TGF-b1– stimulated meningeal cells (E) and astrocytes (F). Results are means T SD from three independent experiments; **P = 0.008, *P = 0.035; two-tailed t test. (G) Carbohydrate epitopes of CSPGs (CS-56) are abundant within the ECM in the control group. After Taxol treatment, CSPGs remain cytoplasmic. Scale bar, 75 mm.

proteoglycans (CSPGs) (10). We asked whether Taxol decreases CSPGs after SCI. At 7 days after injury, Taxol decreased the amount of NG2, one of the most abundant CSPGs (17) (fig. S1). The inhibitory effects of CSPGs on axon growth reside in their Glycosaminoglycan (GAG) side chains (18–20). Lesion site extracts from Taxol-treated animals showed a significant reduction of GAGs compared with controls (Fig. 3D). The conditioned medium of cultured meningeal cells and astrocytes treated with 10 nM Taxol showed a 35% and 32% decrease of GAG levels, respectively (Fig. 3, E and F). Moreover, the CSPGs expressed in the Taxoltreated animals localized to the intracellular space instead of scaffolding the cells as observed in vehicle-treated animals (Fig. 3G). Thus, administration of low doses of Taxol decreases CSPGs at the lesion site after SCI. We next asked whether the Taxol-treated lesion site becomes permissive for regenerating axons in vivo by evaluating the regenerative response of dorsal root ganglion (DRG) neurons. These neurons are set into a growth-competent state by injuring their peripheral axon (conditioning) that allows them to regenerate their CNS axon, but only

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in a scar-free environment (21). We assessed whether the reduction of the scar induced by Taxol is permissive for conditioned axons to grow. Taxol was delivered at the lesion for 4 weeks; 2 weeks after central injury, we conditioned the lumbar L4-6 DRG neurons by transecting the sciatic nerve. Of the vehicle-treated animals, 76% had no regenerative response but rather showed fiber retraction (21) (Fig. 4A). In contrast, Taxol-treated animals had regenerative fibers growing along the edge of the lesion cavity into the injury site and beyond (Fig. 4A). The longest axons per animal grew 1199 T 250 mm in the Taxol-treated group versus 176 T 225 mm in the vehicle-treated animals (n = 13 animals per group; P = 0.002; twotailed t test) (Fig. 4B). The Taxol-treated lesion site thus becomes favorable for regeneration of growth-competent axons. Because Taxol also enhances intrinsic axon growth (4) and the elongation of cultured neurons plated on CSPGs or CNS myelin components (3) (fig. S5), we assessed whether Taxol treatment alone could promote growth of injured CNS axons. To this end, we examined the effect of 4-week continuous Taxol treatment on axonal growth of

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the Raphe-spinal tract after dorsal hemisection (22, 23). Immunostaining revealed an increase by a factor of 5 in the number of serotonin [5hydroxytryptamine (5-HT)]–positive fibers in the spinal cord caudal to the lesion in the Taxoltreated animals compared with vehicle (Fig. 4, C and D). The caudal edge of the lesion was enriched in 5-HT–positive axons (Fig. 4, C and D). Axonal tips showed growth cones rather than retraction bulbs (fig. S6) and, frequently, out of the retraction bulbs a new process emanated (fig. S6). Additionally, we observed an ectopic distribution of 5-HT fibers coursing along the dorsal part of the cord in Taxol-treated animals (Fig. 4C). Thus, Taxol induces growth of 5-HT axons after dorsal hemisection. We next examined whether Taxol treatment leads to functional recovery after moderate spinal cord contusion (24). Similarly to the dorsal hemisection injury, Taxol treatment increased the number of 5-HT–positive fibers caudal to the lesion 8 weeks after injury (Fig. 4E). We tested locomotor performance by analyzing paw placement using a grid walk (25). After 2 and 4 weeks, Taxol- and vehicle-treated animals performed

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REPORTS

equally on the grid walk (Fig. 4F). However, while vehicle-treated animals did not show additional recovery, Taxol-treated animals improved further to a 5% misstep frequency after 6 and 8 weeks, resulting in improvement by a factor of 3.4 (Fig. 4F and movies S3 and S4). Thus, Taxol-induced functional recovery correlates with its axon growth–inducing effect. Current attempts to elicit axonal regeneration in the injured spinal cord include interference with extrinsic growth inhibitory factors present in CNS myelin and scar tissue (18, 19, 26) and their various signaling pathways (27–30). Whereas these approaches aim to interfere with single inhibitory factors, we targeted the cytoskeleton, onto which growth inhibitory signaling pathways converge (28). Moreover, microtubules disassemble in injured CNS axons and, thereby, fail to provide protrusive force necessary for axon outgrowth (3, 4). Here, moderate stabilization of microtubules counteracted various cellular processes that prevent axon regeneration. Thus, Taxol has the potential to offer a multitargeted therapy for SCI. References and Notes 1. X. Liu et al., PLoS Med. 2, e354 (2005). 2. S. Westermann, K. Weber, Nat. Rev. Mol. Cell Biol. 4, 938 (2003).

3. A. Ertürk, F. Hellal, J. Enes, F. Bradke, J. Neurosci. 27, 9169 (2007). 4. H. Witte, D. Neukirchen, F. Bradke, J. Cell Biol. 180, 619 (2008). 5. N. Klapka, H. W. Müller, J. Neurotrauma 23, 422 (2006). 6. M. C. Shearer et al., Mol. Cell. Neurosci. 24, 913 (2003). 7. J. Silver, J. H. Miller, Nat. Rev. Neurosci. 5, 146 (2004). 8. E. K. Rowinsky, L. A. Cazenave, R. C. Donehower, J. Natl. Cancer Inst. 82, 1247 (1990). 9. S. Okada et al., Nat. Med. 12, 829 (2006). 10. C. Schachtrup et al., J. Neurosci. 30, 5843 (2010). 11. H. L. Moses, R. J. Coffey Jr., E. B. Leof, R. M. Lyons, J. Keski-Oja, J. Cell. Physiol. Suppl. 133 (S5), 1 (1987). 12. D. Lindholm, E. Castrén, R. Kiefer, F. Zafra, H. Thoenen, J. Cell Biol. 117, 395 (1992). 13. J. Batut, M. Howell, C. S. Hill, Dev. Cell 12, 261 (2007). 14. C. Dong, Z. Li, R. Alvarez Jr., X. H. Feng, P. J. Goldschmidt-Clermont, Mol. Cell 5, 27 (2000). 15. S. Dunn et al., J. Cell Sci. 121, 1085 (2008). 16. L. C. Kapitein et al., Biophys. J. 99, 2143 (2010). 17. L. L. Jones, Y. Yamaguchi, W. B. Stallcup, M. H. Tuszynski, J. Neurosci. 22, 2792 (2002). 18. E. J. Bradbury et al., Nature 416, 636 (2002). 19. A. Hurtado, H. Podinin, M. Oudega, B. Grimpe, Brain 131, 2596 (2008). 20. T. L. Laabs et al., J. Neurosci. 27, 14494 (2007). 21. B. Ylera et al., Curr. Biol. 19, 930 (2009). 22. J. Dill, H. Wang, F. Zhou, S. Li, J. Neurosci. 28, 8914 (2008). 23. D. D. Pearse et al., Nat. Med. 10, 610 (2004). 24. S. W. Scheff, A. G. Rabchevsky, I. Fugaccia, J. A. Main, J. E. Lumpp Jr., J. Neurotrauma 20, 179 (2003). 25. D. L. Behrmann, J. C. Bresnahan, M. S. Beattie, B. R. Shah, J. Neurotrauma 9, 197 (1992).

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Fig. 4. Taxol promotes axonal regeneration and functional recovery. (A) Spinal cord horizontal sections of L4-6 DRG axons labeled with cholera toxin B, 6 weeks after injury. Taxol treatment promotes regeneration of growthcompetent neurons (arrowheads). Scale bars, 200 mm. (B) Longest regenerating axon per animal T SEM (**P = 0.002; two-tailed t test). (C) Spinal cord sagittal sections stained with antibody to 5-HT 4 weeks after injury. After Taxol treatment, the caudal part of the cord is enriched in serotonergic fibers (C, arrowheads). Scale bars, 75 mm. (D and E) Quantification of 5-HT–positive fibers caudal to the lesion T SEM after dorsal hemisection [n = 16 rats per group; ***P = 0.0001; twotailed t test (D)] and contusion injury [n = 10 rats per group; **P = 0.002; two-tailed t test (E)]. (F) Taxol treatment improves locomotor performance over time (**P = 0.004, ***P = 0.0001; two-way ANOVA). n.s., not significant. P. Freund et al., Nat. Med. 12, 790 (2006). P. Dergham et al., J. Neurosci. 22, 6570 (2002). G. Yiu, Z. He, Nat. Rev. Neurosci. 7, 617 (2006). S. Neumann, F. Bradke, M. Tessier-Lavigne, A. I. Basbaum, Neuron 34, 885 (2002). 30. J. Qiu et al., Neuron 34, 895 (2002). 31. We thank C. Hill for the SMAD2-PAGFP plasmid; A. Ullrich for the HaCaT cells; Ariad Pharmaceuticals for FKBP/FRB plasmids and Rapalog; G. Morfini for technical advice; K. Dornmair, M. Hübener, R. Klein, D. Neukirchen, M. Stiess, S. Tahirovic, and H. Wekerle for critically reading the manuscript; and V. Ha, T. Irl, D.F. Wendell, and R. Zenke for technical assistance. This work was supported by the Max-Planck-Society, Deutsche Forschungsgemeinschaft, International Foundation for Paraplegia, and Human Frontier Science Program (F.B.); NIH ( J.B.); Netherlands Organization for Scientific Research (NWO)–Veni (L.C.K.); Netherlands Organization for Health Research and Development (ZonMW)–TOP, European Science Foundation Young Investigator Award, and NWO-VICI (C.C.H.); and a Marie Curie Fellowship (K.C.F.). The Max-Planck-Society has filed a patent on the use of Taxol after spinal cord injury.

26. 27. 28. 29.

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1201148/DC1 Materials and Methods Figs. S1 to S7 Movies S1 to S4 References 2 December 2010; accepted 12 January 2011 Published online 27 January 2011; 10.1126/science.1201148

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NEW PRODUCTS: PRODUCTS CELL/TISSUE CULTURE MicroBioreactor System

The new version of the ambr workstation automates the operation of 48 disposable microbioreactors and closely replicates the characteristics of benchtop bioreactors at microscale (10 ml to 15 ml working volume). The 48 microbioreactors are arranged in four culture stations containing sets of 12, each with a miniature internal impeller, as well as individual closed loop control of dissolved O2 and pH and a supply of gases. Each culture station has independent stirring speed and temperature control, allowing scientists to test up to four different temperature and stir speeds simultaneously, making sophisticated experimental designs much simpler to carry out with just one real-time parallel run. This new ambr system controls culture setup and inoculation, the addition of alkalis and feeds, and culture sampling for cell count and analysis such as antibody titre. The system is compact enough to fit in a standard laboratory biosafety cabinet.

Cell Growth Solutions ATCC Normal Human Primary Adipose-Derived Mesenchymal Stem Cells are cryopreserved in the second passage to ensure the highest viability and plating efficiency and are thoroughly tested for sample purity. They are performance tested together with media, growth supplements, and reagents to guarantee reliability as part of ATCC Primary Cell Solutions, a system of matched components designed to maximize growth, maximize functionality, and maintain normal morphology for specific cell types. The complete cell-specific ATCC Primary Cell Solutions growth system (consisting of the Mesenchymal Stem Cell Basal Medium and Growth Kit) creates the ideal cell system culture environment for propagation in low serum (2%) conditions and subsequent differentiation. Using the complete ATCC Primary Cell Solutions system, researchers will be able to produce stem cell cultures with functional expression of relevant biomarkers; superior growth and proliferation; normal morphology; and multipotent, lineage-specific differentiation. ATCC For info: 800-638-6597

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The EnPresso Tablet Cultivation Set delivers a slow, controlled release of glucose into culture medium. The EnBase Technology provides an enzymatic-controlled glucose supply in a fed-batch type manner, which allows for an extended growth period and promotes higher final cell density. The efficient EnBase technology has become even easier to use with the pre-sterilized EnPresso tablet. The longer shelf-life tablet can be added directly to sterile water and this instant microbial media is ready for inoculation in a shaking incubator. EnBase technology allows the efficient cell cultivation of recombinant bacteria in small-scale experiments. With the increased yield and ease-of-use obtained in the EnBase media, many typical experiments can be scaled down to increase laboratory testing capacity and effectiveness. EnBase can be used in microwell plates, deep well plates, shake flasks, and even in small-scale and disposable bioreactors. BioSilta Oy For info: +358-(0)-8-553-2361

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Driven to meet the changing needs of the market, Sheffield BioScience has introduced two high-quality, cost-effective animal component-free (ACF) recombinant supplements for cell culture media. Sheffield rAlbumin ACF is a replacement for HSA and BSA, and can be used to reduce/replace serum when used as a standalone supplement or when combined with other components, such as plant-derived protein hydrolysates. Sheffield rAprotinin ACF is a recombinant form of traditional bovine-derived aprotinin that is produced using exclusive technology. Aprotinin, also known as pancreatic trypsin inhibitor, acts as a serine protease inhibitor that forms stable complexes with and blocks the active sites of enzymes at near neutral pH conditions. Sheffield Bio-Science

New high throughput nanofiber culture dishes provide an environment that closely mimics the physical structure found in vivo. These 3D nanofiber substrates allow more accurate modeling of cell migration while still allowing for the critical need of highresolution live-cell imaging. Nanofiber Solutions develops synthetic nanofiber multiwell plates for cell culture applications ranging from drug discovery to stem cell expansion. The scaffold is composed of synthetic polymer nanofibers randomly arranged or aligned into parallel nanofibers which are placed on the bottom of standard cell culture dishes. This gives a 3D surface for cells to grow and attach to and allows for researchers to model metastasis and cell mobility in vitro. Investigators can coat the fibers with different biological coatings of interest (i.e., collagen and fibronectin), just like existing culture dishes, or upregulate/downregulate genes to investigate different cellular mechanisms. Nanofiber Solutions

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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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The Automation Partnership For info: 302-478-9060

POSITIONS OPEN

FACULTY POSITION The Center for Biomedical Informatics at Harvard Medical School The Center for Biomedical Informatics at Harvard Medical School announces the opening of one or two faculty positions in computational biology at the level of ASSISTANT or ASSOCIATE PROFESSOR. The goal of the center is to carry out first-rate research as well as to promote and facilitate collaborative activities in biomedical informatics among researchers at Harvard Medical School and its affiliated institutions. Current research interests of the faculty include bioinformatics, functional genomics, translational medicine, public health surveillance, and knowledge management. We seek scientists who combine exceptional quantitative skills with a deep knowledge of biology or medicine to carry out outstanding independent research program as well as to collaborate with colleagues in the biological and medical sciences in the Harvard Medical area. The search committee is interested in candidates with a strong record in bioinformatics and computational biology (such as functional genomics, systems biology, epigenomics, cancer, and stem cells), but others with superb record in areas of interest to CBMI mentioned will also be considered. The applicant should have a Ph.D. or equivalent degree, with relevant postdoctoral research training and a record of excellence in research as demonstrated through publications and funding. The successful candidate will be located at the Center for Biomedical Informatics at the Countway Library of Medicine on the Harvard Medical School campus and will have an academic appointment through the Informatics Program at Children_s Hospital Boston, an affiliate teaching hospital of Harvard Medical School. Applicants should submit curriculum vitae, a summary of current and future research programs, and arrange to have at least three letters of recommendation submitted. All materials should be electronically sent to Ms. Tracey Harik (e-mail: [email protected]). Review of applications will begin immediately and will continue until the position is filled. Harvard Medical School is an Equal Opportunity/ Affirmative Action Employer. Women and minorities are especially encouraged to apply.

QUANTITATIVE FISHERIES BIOLOGIST The Marine Science Program at Florida International University (FIU) is seeking applicants for a Quantitative Fisheries Biologist (rank open, senior applicants are welcomed) for a tenure-track position in Department of Biological Sciences. The Marine Science Program at FIU (website: http://casgroup.fiu.edu/ marine/) is a new and growing interdisciplinary initiative emphasizing research and teaching in coastal marine science. The Program is housed in a recently completed building that overlooks Biscayne Bay and is home to 12 research laboratories, two teaching laboratories, wet labs, a mesocosm facility, and running sea water systems. We are seeking a fisheries biologist with expertise in fisheries stock assessment or ecosystem management modeling. The successful candidate will teach courses in the areas of ecological statistics, population dynamics, fisheries modeling, or stock assessment. They will be expected to develop an externally funded research program and supervise graduate students in our Ph.D. program. To ensure full consideration, applications should be received by March 15, 2011. Screening of applications will begin on that date, and continue until a suitable candidate is selected. Only electronic applications will be accepted. Applications should include a cover letter, curriculum vitae, summary of their research interests and teaching goals, and at least three letters of reference. Letters should also be submitted electronically. Submit materials to Joel Trexler, Search Committee Chair; e-mail: trexlerj@fiu. edu; telephone: 305-348-1966. FIU is an Affirmative Action/Equal Opportunity Institution.

FACULTY POSITION in Infectious Disease The New England Primate Research Center (NEPRC) of Harvard Medical School is seeking outstanding candidates in the area of Infectious Disease for appointment at the faculty rank of INSTRUCTOR. Individuals with expertise in microbial pathogens and/or the immunologic basis of health and disease are encouraged to apply. Successful candidates will be appointed in an appropriate department of Harvard Medical School and will receive a three-year financial award to cover salary and defray research costs. Appointees are expected to develop an independent research program and to seek extramural research funding with guidance and mentorship from senior investigators on site at NEPRC. Applicants should include their curriculum vitae, summary of past accomplishments and future plans, and arrange for at least three references to be sent directly to the search committee. Applications should be submitted electronically as PDF files to e-mail: neprcidinstructor@hms. harvard.edu. Harvard Medical School and the New England Primate Research Center are Equal Opportunity Employers, committed to diversity in the workplace.

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NIH-funded POSTDOCTORAL OPPORTUNITY to investigate the cellular and molecular mechanisms of kidney injury, regeneration, and fibrosis in the laboratory of Dr. Katalin Susztak in the Department of Medicine and Genetics at the Albert Einstein College of Medicine. Current laboratory projects include high throughput genomic and epigenomic analysis of human kidney disease samples and investigating the role of DNA methylation in regulating gene expression and susceptibility to common diseases. In addition, we are also interested in determining the role of the Notch and Wnt/beta-catenin pathways in kidney disease development, renal epithelial cell homeostasis, and progenitor cell function differentiation. Visit website: http:// www.susztaklab.com-a.googlepages.com for qualifications, compensation, and application details.

POSTDOCTORAL POSITIONS at Brigham & Women_s Hospital Harvard Medical School One position involves positional cloning of mutants affecting organ and brain development in ENU-mutagenized mice. A second position involves genetic analysis of a complex trait (airway hyper-responsiveness) in a mouse model of asthma. Molecular biology experience necessary, genetics background preferred. English oral and written ability necessary. Please forward curriculum vitae and the names of three references to: David R. Beier, M.D.Ph.D., Genetics Division, NRB 458, 77 Ave Louis Pasteur, Boston, MA 02115. Fax: 617 525-4705; e-mail: [email protected]. edu. Brigham & Women_s Hospital is an Equal Opportunity Employer.

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POSITIONS OPEN

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The Philipps-Universität Marburg and the Max Planck Institute for Terrestrial Microbiology have recently established a center for Synthetic Microbiology, SYNMIKRO. Supported by the state of Hessen within its excellence program LOEWE, SYNMIKRO focuses on basic research in synthetic biology on all levels of microbial function, ranging from the development of regulatory circuits, genetic codes and metabolic pathways to the synthesis of new minimal cells and microbial communities. In its next phase of expansion, SYNMIKRO invites applications for up to five

Professorships (W2) in Synthetic Microbiology at the Philipps-Universität Marburg. We seek individuals with an outstanding track record in synthetic microbiology and with innovative research projects. We provide attractive start-up packages and research funding, and offer teaching associations within the faculties of biology, chemistry, or mathematics and computer science, depending on the successful candidate’s background. Preferred research areas include, but are not limited to,

(1) (2) (3) (4) (5)

Development of synthetic circuits Reprogramming of the genetic code Molecular evolution, metagenomics and prokaryotic biodiversity Quantitative analysis of metabolic networks Mathematical modelling of microbial systems

All appointments are for a period of 4 years, with a 1-year extension after a positive evaluation in the third year. Tenure track is in principle possible in all faculties according to the conditions described in § 61 (6) of the Hessen State Law on Higher Education (HHG) and is intended in the Faculty of Mathematics and Computer Science. The conditions of employment are set down in §§ 61, 62 HHG. Philipps-Universität is an equal opportunity employer, and applications from women are encouraged. Applications from candidates with children are welcome - the Philipps-Universität is dedicated to being a family-friendly university. Qualified disabled people are also encouraged to apply. Applications should be submitted by, March 18, 2011 to [email protected]. The application should contain the following information in a single pdf document: (1) cover letter, (2) curriculum vitae, including a list of publications, (3) summary of research achievements, (4) research plan, (5) statement of teaching interests and experience and (6) previous and current funding. Informal inquiries can be directed to Prof. Dr. Bruno Eckhardt, Director SYNMIKRO, Philipps-Universität Marburg, [email protected].

Faculty Positions in Virology Department of Microbiology and Immunology LSU Health Sciences Center, Shreveport, LA 71130 Tenure track positions at the rank of Assistant or Associate Professor are open for scientists with a Ph.D. and/or M.D. and postdoctoral research experience. Responsibilities include teaching graduate and medical students and directing a nationally competitive research program in molecular virology, tumor virology, or viral immunology. The Department faculty of 16 members directs 40 doctoral and postdoctoral trainees and an NIH-funded Center for Molecular and Tumor Virology staffed by 12 faculty principal investigators. The Department is well equipped for molecular research and is augmented by the LSUHSC Core Facility which offers biotechnologies such as ﬂow cytometry, confocal microscopy, laser capture microdissection, DNA array analyses, proteomics, etc. A Research Incentive Plan allows nationally funded investigators to receive unrestricted funds for research as well as salary augmentation. Applicants should send a curriculum vitae and statement of research goals and funding and arrange for three letters of reference to: Dr. Dennis J. O’Callaghan, Boyd Professor and Head, Department of Microbiology and Immunology, LSU Health Sciences Center, Shreveport, LA 71130. AA/EEO Employer.

Biomedical Scientist The University of Wisconsin School of Medicine and Public Health seeks a Biomedical Scientist with an established record of achievement, and active research program specializing in the biology of gastrointestinal inflammation, cancer, or both. The ideal candidate would be an MD, PhD, or MD/PhD with GI Board Certification desired but not required for candidates who hold an MD. Applicants must have a demonstrated record of extramural grant support, academic achievement, and a strong publication record. This faculty position is a tenure track with an appointment at assistant, associate, or professor level depending on experience. Substantial institutional support is available for qualified individuals. This includes a competitive compensation package, generous start up package, modern laboratory facilities, and protected research time. Send letter of interest with description of current research and future plans, and curriculum vitae to: Dr. Michael Lucey, MD, Chief, Division of Gastroenterology & Hepatology, School of Medicine and Public Health, University of Wisconsin, 1685 Highland Avenue (4245 MFCB), Madison, WI 53705. Phone: (608) 263-7322 The UW Madison is an EEO/AA employer. Minorities and women are encouraged to apply. Wisconsin Caregiver and Open Records laws apply. A background check will be conducted prior to employment.

Research Fellow Positions at Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences (CAS) Suzhou, Jiangsu, China

SIBET is situated in Suzhou, an ancient city near Shanghai in China. SIBET are focusing on the breakthrough of core technologies in detection based on spectroscopy, nuclear medical detector, micro-nano sensors and medical semiconductor laser by integrating high technology in fields of biology, photoelectricity, nuclear medicine and computer. SIBET is dedicated to develop mature products in the medical instrumentation, medical materials and biomedical reagents to meet the demands of public health and domestic medical industry development. Currently, the R&D fields include medical laser, biomedical measurement, medical imaging, biomedical electronics, biomaterials and biomedical reagents. We are seeking candidates for research fellow positions at Associate Professor/Professor levels to work in the fields of (1) medical laser, (2) medical imaging, (3) clinical diagnosis and analysis, (4) biomedical signal processing and (5) biomedical reagents. Generous startup packages to develop successful labs and competitive salaries and benefits will be provided. Candidates should have a Ph.D., an outstanding track record of scientific productivity, the ability to manage labs and research projects independently, and excellent communication skills in Chinese and English. Send curriculum vitae, a statement of research interests and future plans, and a list of three references to: Prof. Xuan Ming, President, Suzhou Institute of Biomedical Engineering and Technology (SIBET) Human Resource Department of SIBET 88 Keling Road, SND, Suzhou 215163, China Tel: 86-512-69588025; Fax: 86-512-69588088 Email: [email protected]

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Postdoc Opportunities UNITED STATES

Amgen is a science based biotechnology pioneer. Our cutting

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Discovery Driven. Patient Focused.

Physician-Scientist Investigator Recruitment Genetic Disease Research Branch, National Human Genome Research Institute

The Genetic Disease Research Branch (GDRB) of the National Human Genome Research Institute (NHGRI) provides unparalleled opportunities for investigators to develop world-class research programs in genetics and genomics. The Branch is pleased to announce that it is seeking to recruit a new clinical tenure-track or tenure-eligible investigator to pursue an innovative, independent program in molecular translational research as part of this group of highly interactive and supportive investigators. The successful candidate should have a strong interest in developing an active clinical research program that capitalizes on genetic or genomic approaches to understand the mechanisms of human disease. Current GDRB faculty members use a variety of approaches to study the regulation and function of genes involved in development and homeostasis in humans and model organisms, with the goal of providing insight into human diseases. We welcome applications from clinicians with a wide range of backgrounds and interests, including those that complement the interests of our current faculty. The successful candidate will take advantage of interactions with a highly collegial group of scientists within NHGRI and on the NIH campus as a whole. In addition, they will have access to NHGRI’s outstanding core laboratories and the unique resources of the NIH Clinical Center, the world’s largest hospital dedicated entirely to clinical research. This position includes a generous start-up allowance, an ongoing commitment of research space, laboratory resources, and positions for personnel and trainees. Candidates should have an M.D., an M.D.-Ph.D., or equivalent degree, as well as advanced clinical and laboratory training and a record of accomplishments. The preferred candidate will be expected to meet the requirements for authorization of patient care privileges by the Credentialing Services of the NIH Clinical Center. Medical graduates with substantial experience and achievements in clinical research are also encouraged to apply. Interested applicants should submit their curriculum vitae, a three-page description of proposed research, and three letters of recommendation through our online application system, at http://research.nhgri.nih.gov/apply.

Applications will be reviewed starting April 15, 2011 and will be accepted until the position is filled. For more information on GDRB and NHGRI’s Intramural Program, please see http://genome.gov/DIR. Specific questions regarding the recruitment may be directed to Dr. Steven Holland, the Search Chair, at [email protected]. Questions may also be directed to Dr. Leslie Biesecker, the GDRB Branch Chief, at [email protected]. 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

Department of Health and Human Services National Institutes of Health National Institute of Neurological Disorders and Stroke Director of the Extramural Ofﬁce of Translational Research Recent advances in neuroscience have created unprecedented opportunities to develop effective new treatments for the many disorders that affect the nervous system. The National Institute of Neurological Disorders and Stroke (NINDS) is at the forefront of efforts to translate discoveries in basic research into therapeutic interventions for neurological disease. NINDS seeks a Director of its Ofﬁce of Translational Research, to lead the development of new therapies and oversee ongoing translational programs. The Director will report directly to and advise the NINDS Director on translational programs, and will represent the Ofﬁce of Translational Research at an executive level within NINDS and on trans-NIH and trans-agency programs. Programs within the Ofﬁce of Translational Research will include a collection of high-risk, milestone-driven projects focused on obtaining IND or IDE approval for new diagnostic and treatment strategies. The Director will work with the NINDS Ofﬁce of Clinical Research to transition promising therapies into initial studies in patients, work closely with the extramural research community and build partnerships with nonproﬁt research organizations and companies, and set priorities for the Institute’s grants to small businesses (SBIR and STTR). The successful candidate for this position will have an international reputation for accomplishments in translational neuroscience research, and have demonstrated skills, knowledge, and experience in therapeutic development, project management, technology transfer, and public-private partnerships. Consistent with the required qualiﬁcations, the individual will be appointed as a Science Executive in Title 42. Application for this position should be sent to: Dr. Story Landis, Director, NINDS, c/o MaryAnn Sofranko, Building 31 Room 8A52 NIH, Bethesda, MD 20892. Evaluation of applications will begin April 2011. The NINDS is one of the Institutes of the National Institutes of Health, a component of the Department of Health and Human Services. HHS and NIH are Equal Opportunity Employers

Help Us Help Millions Assistant Director for HIV Therapeutics Research, NIAID, NIH, HHS Director, Therapeutics Research Program, DAIDS, NIAID The National Institute of Allergy and Infectious Diseases (NIAID) is seeking an exceptional and visionary leader to take on the dual role of NIAID assistant director for HIV therapeutics research and director of the Therapeutics Research Program (TRP) in the Division of AIDS (DAIDS).

NIAID

The TRP director is responsible for planning, implementing, and directing a global extramural research program, in excess of $120 million, for the preclinical development and clinical testing of therapies for HIV/AIDS and its associated co-infections and co-morbidities. This includes oversight of the NIAID-funded HIV/AIDS therapeutic clinical trials networks which have an annual budget of more than $70 million and conduct all phases of clinical trials at research sites around the globe. The director serves as a key scientific advisor to the directors of DAIDS and NIAID and is responsible for ensuring that the therapeutics research supported by DAIDS is integrated and complementary with the other programs within NIH, government agencies, and external organizations conducting HIV/AIDS therapeutics research. The director formulates an overall scientific agenda for the development and clinical testing of therapeutic interventions, recommends resource allocation across competing initiatives, and continually assesses and reorients program priorities and activities, both anticipating and responding to changing research needs or re-defined policies. This position also serves as the NIAID assistant director for HIV/AIDS therapeutics research, reporting to the director of NIAID. Responsibilities of this position include 1) advising the director on all aspects of the NIAID’s HIV/AIDS therapeutics research and development efforts to ensure a well-coordinated, seamless therapeutics research and development program; 2) representing the director in activities and discussions related to planning, implementing, conducting, and evaluating NIAID’s overall HIV/AIDS therapeutics research and development program; 3) serving as liaison among extramural components of NIAID; 4) representing the director in interactions with relevant constituencies and the media; and 5) maintaining ongoing relationships with relevant individuals and groups to further the HIV/AIDS therapeutics mission of NIAID. The successful candidate will be appointed under the Title 42(f) authority at a salary commensurate with experience. Maximum annual base salary is $200,000, with a maximum total annual compensation limit of $230,000. A full package of benefits is also available, including retirement; health, life, and long-term care insurance; annual and sick leave; and a thrift savings plan (401K equivalent). This position may be subject to public financial disclosure requirements.

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health

National Institute of Allergy and Infectious Diseases Qualifications: Applicants must possess an M.D. with extensive experience in 1) working both independently and collaboratively in planning, organizing, conducting, and/or overseeing clinical research in infectious diseases; 2) serving effectively in research program administration; and 3) effective communications and collaborations. Application Process: Provide curriculum vitae, bibliography, and a three-page statement explaining 1) your vision for HIV/AIDS therapeutics research; 2) your reasons for being interested in the position; and 3) the specific leadership skills and experience you would bring to the HIV/AIDS research programs at NIAID. Submit your application package to Mr. Robert Gulakowski, Office of the Director, DAIDS, NIAID, 6700-B Rockledge Drive, Room 4143, Bethesda, MD 20892-7620, and reference announcement number DAIDS-11-01. The deadline for receipt of applications is March 31, 2011. Direct any inquiries to Mr. Gulakowski at [email protected] or 301-496-0545. Additional information can be found at www.niaid.nih.gov/careers/dat. All information provided by applicants will remain confidential and will only be reviewed by authorized NIAID officials.

National Institute of Allergy and Infectious Diseases Proud to be Equal Opportunity Employers

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GRANTS Professor and Chair of Nutritional Sciences Rutgers University is seeking a scientist of international stature with visionary leadership skills to chair the Department of Nutritional Sciences. The university has targeted nutrition and health as a signature area for programmatic growth; the successful candidate will lead the expansion of the department and have an active role in this initiative. The Department of Nutritional Sciences has 18 faculty members who provide education and mentoring to over 300 undergraduate majors and a graduate program faculty of 45 members. The department also offers a Didactic Program in Dietetics, operates a child nutrition laboratory, and oversees outreach programs and the New Jersey Obesity Group. Research strengths span from basic metabolism to clinical and community nutrition (http://nutrition.rutgers.edu/). Nutritional Sciences is part of a vibrant life and social sciences research community at Rutgers, the land-grant institution of New Jersey, and plays an integral role in the newly established New Jersey Institute for Food, Nutrition, and Health. The New Brunswick campus is in central New Jersey, conveniently located between New York City and Philadelphia. Qualiﬁcations: Candidate should have a Ph.D., or the equivalent, a record of scientiﬁc leadership, a sustained record of peer-reviewed publications and funding, and a commitment to teaching. The candidate is expected to lead a successful and active research program and promote faculty development. The successful candidate will be provided with a competitive salary and start-up package. Applications: A letter of application, curriculum vitae, statement of research interests and leadership vision, and names of four professional references should be sent to Nutrisearch@aesop .rutgers.edu or Nutritional Sciences Chair Search, 65 Dudley Road, Rutgers University, New Brunswick, NJ 08901. Rutgers, The State University of New Jersey, is an Afﬁrmative Action/Equal Opportunity Employer and seeks to employ the best qualiﬁed individual without regard to race, religion, color, national origin, ancestry, age, sex, sexual orientation, physical or mental handicap or disability, or marital, military, or veteran’s status. Individuals covered by Section 503 of the Vocational Rehabilitation Act of 1973 or Section 402 of the Veteran’s Readjustment Assistance Act of 1974 may self identify. If you wish to self identify, please do so in the cover letter transmitting your curriculum vitae/resume. Employment eligibility veriﬁcation required.

Associate Professor Division of Reproductive Sciences The Division of Reproductive Sciences at Cincinnati Children’s Research Foundation (CCRF) invites applicants for a tenure-track position at the Associate Professor level. Candidates in all areas of reproductive sciences will be considered, while candidates with strong interests in the following areas will receive special consideration: Endocrine disruptors and uterine biology; epigenetics; embryo implantation and parturition; and the roles of noncoding RNAs and transcription factors in pre-implantation and implantation biology. Qualified candidates must have a PhD, MD or MD/PhD degree in biomedical sciences. Candidates should have an outstanding record of independent and collaborative research with federally funded grants. All candidates should have strong communication skills and leadership experience in developing innovative programs, collaborations and new initiatives, so they may help to lead the Division to excellence. Cincinnati Children’s is an outstanding place for growth and development and provides a very competitive start-up package. Cincinnati Children’s Research Foundation is one of the largest pediatric research programs in the nation and ranks 2nd among pediatric hospitals in funding from the National Institutes of Health. The Division is also a member of the Perinatal Institute, which brings together experts in basic and translational research and clinical care to provide a comprehensive approach to newborn health. CCRF has a strong collaborative relationship with the University of Cincinnati, and this position would provide the opportunity to work closely with the university’s many outstanding life sciences research programs, including the Environmental Health research program. Interested candidates should electronically send a letter of interest, current CV and contact information for three professional references to Dr. S. K. Dey, in the care of Julie Burns ([email protected]). Faculty appointments are with the University of Cincinnati College of Medicine. Cincinnati Children’s Hospital Medical Center is an Affirmative Action/Equal Opportunity Institution. Women and minorities are encouraged to apply.

RIKEN, one of Japan’s largest research organizations, carries out advanced basic and applied research in a wide range of fields, including physics, chemistry, medical science, biology, and engineering. RIKEN invites applications for the position of Associate Chief Scientist. 1. Research fields Research field is not specified. We require the applicant to pioneer his or her own curiosity-driven, internationally-oriented interdisciplinary research field. 2. Qualifications Self-disciplined scientists with a long-term vision of the next generation of scientific endeavor, and with the ability and research record to preside over their own autonomous laboratories will be considered as candidates for this position. 3. Remuneration Annual salary (with a bonus system) 4. Type of employment The position is tenured, subject to RIKEN’s mandatory retirement age of 60. 5. Starting date of employment April 1, 2012 (in principle)

Closing date: 5pm, May 27, 2011 (Japan Standard Time) For more information, please visit:

http://www.riken.jp/engn/r-world/research/ research/associate/2012/index.html

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The successful candidate will: • Bring substantial industrial experience, bioeconomy-related research expertise, and personal leadership to a young, entrepreneurially focused organization, with the aim of establishing nationally recognized R&D excellence relevant to private sector needs. • Recruit and maintain an internationally competitive, personally led R&D group. • Actively oversee the development of a diverse set of other independent R&D groups led by colleagues. • Liaise with corporate, economic development, government, and higher education collaborators, with the aim of ensuring that Institute programs support the emerging regional, state, and national bioeconomy. • Develop a sustainable funding base for Institute research through energetic and successful solicitation of peer-reviewed grants, cooperative research agreements, and industrial afﬁliate group memberships. • Build appropriate, mutually beneﬁcial collaborative relationships with faculty and units on the MSU campus and at other MSU sites throughout Michigan. Compensation will be competitive and commensurate with experience. Faculty appointment at an appropriate rank in any of several MSU colleges and schools/departments will be considered for interested candidates with requisite credentials. INSTITUTE BACKGROUND: In December 2007, Pﬁzer donated a modern, 138,000-square-foot research and development building with extensive chemical pilot plant production capabilities to MSU. MSU received possession of the Institute on March 14, 2009. The research component of the facility offers more than 31,000 square feet of modern labs with traditional and walk-in chemical fume hoods, a 105-seat auditorium, library space, plus professional and staff ofﬁces. The pilot plant contains stainless steel, glass-lined, and Hastelloy® reactor vessels with an aggregate 25,000-liter capacity, plus seven dryers, a Foxboro DCS control system, a glycol heat transfer medium system for temperature control, centrifuges, Niagra ﬁlters, and vacuum sources. MSU has contracted with Lakeshore Advantage Corporation, the regional economic development organization, to manage a start-up company incubator in one wing of the building. Current tenants pursue opportunities in biocomposites, biofuels, and specialty chemicals. In addition to the $5M+ CFHZA endowment for the support of the R&D Director, $4.8M has been received to date for the Institute in proposal-based grants from the Michigan Strategic Fund. For information about the Institute and this search, please visit http://research.msu.edu/stories/msu-holland-facility-offers-rd-pilot-plant or call MSU at (517) 432-4499. Nominations and applications, including curriculum vitae and references, may be sent to the search committee, c/o Brenda Hoten, [email protected]. MSU is an Afﬁrmative Action/Equal Opportunity Employer. MSU is committed to achieving excellence through cultural diversity. The university actively encourages applications and/or nominations of women, persons of color, veterans and persons with disabilities.

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MSU Bioeconomy Institute R&D Director With the very generous endowment support of regional donors to the Community Foundation of the Holland-Zeeland Area (CFHZA), Michigan State University (MSU) seeks a Research and Development (R&D) Director for the MSU Bioeconomy Institute in Holland, Mich.

online @sciencecareers.org

University of Pittsburgh FACULTY POSITIONS

FACULTY POSITIONS DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY SAINT LOUIS UNIVERSITY SCHOOL OF MEDICINE Saint Louis University, a Catholic Jesuit institution dedicated to student learning, research, health care, and service, is seeking outstanding applicants for tenure-track faculty positions at the ASSISTANT PROFESSOR level in the Edward A. Doisy Department of Biochemistry and Molecular Biology (http://biochem.slu.edu/). The Department has an outstanding faculty, a long tradition of excellence in research and education and is housed in the state-of-the-art Doisy Research Center completed in 2007. We are interested in candidates with demonstrated ability to develop strong and independent research programs that would complement existing strengths in the Department. Research programs addressing systems relevant to human health in terms of their structural and mechanistic components will be given high priority. A Ph.D. or M.D. degree, outstanding record of publications, evidence of current external funding, enthusiasm for teaching and mentoring research trainees are important criteria for selection. Interested candidates should submit a cover letter, current curriculum vitae, relevant publications, future research plans, a detailed 3-year budget and addresses of three references to http://jobs.slu.edu. Saint Louis University is an Afﬁrmative Action, Equal Opportunity Employer, and encourages nominations and applications of women and minorities.

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The Desert Research Institute (DRI), the non-profit environmental research institution of the Nevada System of Higher Education (NSHE), strives to be the world leader in environmental sciences through the application of knowledge and technologies to improve peoples lives throughout Nevada and the world. DRI has research divisions in Atmospheric Sciences, Hydrologic Sciences, and Earth and Ecosystem Sciences. The Division of Atmospheric Sciences within DRI is currently searching for an Executive Director to support, facilitate, and grow divisional research activities through strong leadership that utilizes the Divisions scientific and development skills. Recognized internationally for its air quality research, DAS also has a well-developed research component in the fire sciences as it relates to fire, weather, fuels, climate change, air quality, and characterization of emissions. DAS has approximately 80 faculty and support staff and over 70 graduate research assistants. Visit http://www.dri.edu/das for more detailed information. The DAS Executive Director reports to the Executive Vice President for Research (EVPR) and supports the scientific and programmatic needs of the Division. She/he will provide leadership by utilizing specific faculty and project activities and by facilitating cooperation in teaching and research with related departments at University of Nevada, Reno and the University of Nevada, Las Vegas as well as developing division-based projects with existing and new clients; and representing DRI to sponsors and government agencies. Additional responsibilities include leadership and direction to integrate the Divisional strengths and synergistic interactions among Division scientists, interact with funding agencies to market DAS capabilities, attract research funds and anticipate areas of scientific growth and opportunity, and prioritize allocation of divisional funds for development of research programs.

The Barrow Neurological Institute seeks new faculty members for a newly-initiated Brain Tumor Research Center to develop high-impact and sustainable research programs that advance our understanding of pediatric and adult brain tumors. Appointments may be at the Assistant, Associate or Full Professor level. Membership to the appropriate basic science or clinical department, appointment to graduate programs, excellent space, ongoing partial salary support, and startup funding will be provided. An advanced degree and substantial research background are required. The Barrow Neurological Institute, located in Phoenix, Arizona, is one of the busiest clinical neuroscience centers in the world and is ranked by US News & World Report as one of the top 10 Neurology and Neurosurgery programs in the country. Applicants may apply in any area of brain tumor research, although strategic interests include molecular neuro-oncology, epigenetics, neuro-epidemiology, stem cell biology, neuroﬁbromatosis, immunotherapy, and developmental neurobiology. Please send CV, summary of current research (one page), outline of future research (two pages), and 3 letters of recommendations to Debbie Nagelhout ([email protected]), academic assistant to Nader Sanai, M.D., Director, Barrow Brain Tumor Research Center, Barrow Neurological Institute. Applications will be considered starting January 1, 2011 and accepted until April 1, 2011.

Postdoctoral Fellow in Brain Tumor Research The BARROW NEUROLOGICAL INSTITUTE is seeking a postdoctoral fellow to compliment the preclinical neuroscience research in the laboratory of Nader Sanai, M.D. Qualiﬁed applicants holding an M.D., Ph.D., or equivalent degree will investigate the neurobiological basis of neural and glial CNS progenitors (see Nature 2004 February 19), particularly in the context of brain tumors. Successful candidates will have skill sets in the molecular biology, histopathology, biochemistry, tissue culture assays and mouse models to characterize stem and progenitor cells. Preference will be given to those who also have experience in molecular imaging of neural precursors and applying high ﬁeld-strength magnetic resonance spectroscopy. The Barrow Neurological Institute is a translationally oriented basic science research facility located in Phoenix, Arizona and located adjacent to the state’s largest tertiary care hospital. It is in an urban center of academic, biotechnology, and pharmaceutical research, neighboring Arizona State University and the University of Arizona School of Medicine. Initial appointment is for two years with the possibility of renewal. Interested candidates should e-mail a brief statement of research interests, CV and the names and contact information for three references to Debbie Nagelhout, Academic Assistant to the Director of the Barrow Brain Tumor Research Center, Barrow Neurological Institute and St. Joseph’s Hospital and Medical Center, 350 West Thomas Road, Phoenix, AZ 85013; [email protected] or apply online at http://www.stjosephs-phx.org/index.htm.

GRADUATE PROGRAM

Required: Ph.D. or equivalent graduate degree in atmospheric sciences or a related field. Broad understanding of atmospheric and air quality sciences. Evidence of success in building and leading research programs in governmental, private sector, or university settings. Demonstrated strong interpersonal, collaborative, and written communication skills. Creativity in identifying research issues and pursuing research avenues that will enhance DASs academic prestige and financial standing. Demonstrated ability to supervise/manage budgets for similar organizations along with the ability to supervise/manage personnel and human resource functions and issues. Preferred: Direct experience in the soft-money research environment, such as in a national laboratory, university institute, or high-level consulting firm. Salary is commensurate with experience and education. DRI offers an benefits package that includes medical, health, dental, vision, life and long-term disability insurance, 11.25% matching retirement by DRI, paid annual and sick leave, 11 paid holidays, tuition reduction at NSHE institutions for employee and dependents. Additionally, there is no state income tax and no social security deduction. For complete job description and to apply go to:

www.dri.edu/employment Review begins immediately and continues until the position is filled. Priority will be given to applications received by March 15, 2011. The Desert Research Institute is an Affirmative Action / Equal Employment Opportunity employer.

Sustainable Career Perspectives – The CECAD Graduate School.

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Faculty Position in Brain Tumor Research

AAAS is here – helping scientists achieve career success. Every month, over 400,000 students and scientists visit ScienceCareers.org in search of the information, advice, and opportunities they need to take the next step in their careers. A complete career resource, free to the public, Science Careers offers a suite of tools and services developed speciﬁcally for scientists. With hundreds of career development articles, a grants and scholarships database, webinars and downloadable booklets ﬁlled with practical advice, a community forum providing real-time answers to career questions, and thousands of job listings in academia, government, and industry, Science Careers has helped countless individuals prepare themselves for successful careers. As a AAAS member, your dues help AAAS make this service freely available to the scientiﬁc community. If you’re not a member, join us. Together we can make a difference.

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ScienceCareers.org/booklets • Business Sense: Starting an Academic Lab • Lab Management: The Human Elements • Mind Matters: In Defense of Downtime • Funding Your Future: Publish or Perish • If at First You Don’t Succeed, Cool Off, Revise, and Submit Again • Your Research in the Headlines: Dealing with the Media

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2 01 1 C o l d S p r i n g H a r b o r L aboratory Meetings & Courses Meetings

Honey Bee Genomics & Biology

Eukaryotic mRNA Processing

Systems Biology: Networks

May 8 - 10 abstracts February 18

August 23 - 27 abstracts June 3

The Biology of Genomes

Mechanisms of Eukaryotic Transcription

March 22 - 26

Computational Cell Biology March 29 - April 1

Cell Engineering & Cell-Based Therapies April 7 - 10

Synapses: From Molecules to Circuits & Behavior April 12 - 16

The Biology of Cancer: Microenvironment, Metastasis & Therapeutics

May 10 - 14 abstracts February 18

The Ubiquitin Family May 17 - 21 abstracts February 25

Retroviruses May 23 - 28 abstracts March 4

76th Symposium: Metabolism & Disease June 1 - 6 abstracts March 11

Single Cell Analysis July 22 - 24 abstracts April 29

Yeast Cell Biology August 16 - 20 abstracts May 27

Telomeres & Telomerase

October 11 - 15 abstracts July 22

Genome Informatics

August 30 - Sept 3 abstracts June 10

November 2 - 5 abstracts August 12

Eukaryotic DNA Replication & Genome Maintenance

Harnessing Immunity to Prevent & Treat Disease

September 6 - 10 abstracts June 17

November 16 - 19 abstracts Aug 26

Microbial Pathogenesis & Host Response

Plant Genomes & Biotechnology: From Genes to Networks

September 13 - 17 abstracts June 24

Nov 30 - Dec 3 abstracts Sept 9

Stem Cell Biology September 20 - 24 abstracts July 1

RNA & Oligonucleotide Therapeutics

Personal Genomes

December 4 - 7 abstracts Sept 16

September 30 - Oct 2 abstracts July 8

Neurobiology of Drosophila

April 26 - 30

Cell Death

Rat Genomics & Models December 7 - 10 abstracts Sept 16

October 3 - 7 abstracts July 15

May 3 - 7

Courses

Computational Cell Biology

Cell & Developmental Biology of Xenopus

Frontiers & Techniques in Plant Science July 1 - 21

July 1 - 21

April 8 - 19

Workshop on Autism Spectrum Disorders June 8 - 14

Advanced Bacterial Genetics June 8 - 28

Ion Channel Physiology June 8 - 28

Molecular Embryology of the Mouse June 8 - 28

Proteomics June 13 - 28

Neurobiology of Drosophila July 1 - 21

Imaging Structure & Function in the Nervous System July 26 - August 15

Yeast Genetics & Genomics

Advanced Sequencing Statistical Analysis of Genomic Data Technologies & Applications

August 2 - 15

Circuits & Connectivity in the Vertebrate Brain

October 19 - November 1

July 26 - August 15

Structure Function & Development Cellular Biology of Addiction of the Visual System August 9 - 15 July 6 - 19 Programming for Biology Advanced Techniques in October 17 - November 1 Molecular Neuroscience X-Ray Methods in Structural July 6 - 21

Live Cell Imaging, Super-Resolution Imaging and Image Analysis

November 8 - 21

C elegans November 8 - 21

Computational & Comparative Genomics November 9 - 15

Biology

The Genome Access Course

October 17 - November 1

April 19 - 20, November 29 - 30

July 6 - 21

Workshop on Pancreatic Cancer June 16 - 22

Molecular Neurology & Neuropathology June 27 - July 3

Biology of Memory July 21 - August 3

Eukaryotic Gene Expression July 26 - August 15

Photo above: Cocktails during the 2010 Symposium at the unveiling of the Darwin statue.

Cold Spring Harbor Laboratory Meetings & Courses Program, 1 Bungtown Road, Cold Spring Harbor, NY 11724 Phone 516 367 8346 email [email protected]

www.cshl.edu/meetings

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POSITIONS OPEN

POSITIONS OPEN

POSITIONS OPEN

FACULTY POSITION in Stem Cell Biology, Instructor Level Harvard Medical School The New England Primate Research Center (NEPRC) of Harvard Medical School (HMS) is seeking outstanding candidates in the area of Stem Cell Biology for appointment as Instructor. Instructor is an entry-level faculty rank. A three-year financial award will cover salary and defray research costs. Successful candidates will be expected to develop an independent research program and to compete for extramural grant support with guidance and mentorship from senior investigators on site. NEPRC has 18 HMS Faculty full time on site and currently employs monkey models for Parkinson_s disease, type-1 diabetes, and AIDS. Curriculum vitae, summary of past accomplishments, and future plans should be sent electronically to e-mail: scbneprc@hms. harvard.edu or by mail to: SCB Search Committee, NEPRC, P.O. Box 9102, Southborough, MA 01772-9102. Harvard Medical School and the New England Primate Research Center are Equal Opportunity Employers, committed to diversity in the workplace.

The Dickinson College Biology Department seeks applicants for two one-year sabbatical replacement positions to begin July 1, 2011. The successful candidates must complete the Ph.D. degree by fall 2011 and have demonstrated a high potential for excellence in undergraduate teaching. Position 1: VERTEBRATE BIOLOGIST/ EVOLUTIONARY ECOLOGIST to teach upper-level courses in ecology or evolution and in comparative vertebrate anatomy. Position 2: GENETICIST OR DEVELOPMENTAL BIOLOGIST to teach genetics and/or either developmental biology or developmental neurobiology. Responsibilities for both positions also may include participation in a topical introductory course. To apply, send letter of application, curriculum vitae, statements of teaching philosophy and research interests, and three letters of reference by March 1 through website: https:// jobs.dickinson.edu. Dickinson is a highly selective national liberal arts college with an emphasis on innovative science teaching and student/faculty research. Dickinson College is committed to diversity and we encourage candidates who will contribute to meeting that goal to apply. Applications and nominations of women and minorities are strongly encouraged.

UNIVERSITY OF FLORIDA Department of Medicinal Chemistry The Department of Medicinal Chemistry, College of Pharmacy, University of Florida, invites applications for a tenure-track ASSISTANT PROFESSOR position. The successful candidate will establish a strong independent research program in drug discovery and development and participate in professional and graduate instructional efforts of the college. Area of specialization within medicinal chemistry is open, but should complement the interests of the department and agree with institutional strategies adopted by the University of Florida to foster interdisciplinary research in cancer, infectious diseases, biotechnology, and age-related diseases. The department is located within the UF Health Science Center complex (website: http://www. health.ufl.edu/). This unique research environment offers excellent opportunities for synergistic collaborations. Applicants should submit a cover letter, curriculum vitae, summary of research experience and proposed research, teaching experience (if any) and three letters of recommendation to Dr. Hendrik Luesch, chair, Medicinal Chemistry Faculty Search Committee. Applications should be submitted electronically as PDF files to program assistant David Jenkins at e-mail: [email protected]. To ensure full consideration, materials should be submitted by April 1, 2011, when the search committee will begin reviewing applications. Applications received after this date may be considered at the discretion of the committee and/or hiring authority. The University of Florida is an Equal Opportunity Institution and encourages applications from qualified minority and female applicants.

POSTDOCTRAL/RESEARCH ASSOCIATE positions are available to study the regulation and function of guanylyl cyclase/natriuretic peptide receptor gene. The project 1 involves investigation of functional genomics by characterizing the genes involved in renal and cardiac hypertrophy and fibrosis in Npr1 gene-targeted mice. Experience in the in vivo studies of mice will be preferred. The project 2 involves studying the transcriptional regulation and expression of guanylyl cyclase receptor involving promoter analysis, EMSA, and CHIP. The project 3 involves analysis of receptor dynamics, protein phosphorylation, and intracellular trafficking using confocal microscopy. Strong background in biochemistry and molecular biology is highly desirable. The successful applicant(s) will be expected to have motivation and vision to develop independent research program. Opportunities are available for academic career advancement. Send resume and three references to e-mail: kpandey@ tulane.edu. Or to K.N. Pandey, Tulane University Health Sciences Center, Department of Physiology, 1430 Tulane Avenue, New Orleans, LA 70112. An Equal Opportunity/Affirmative Employer.

FACULTY POSITION in Integrative Developmental Biology The University of North Texas (UNT) seeks candidates at the ASSOCIATE or FULL PROFESSOR level preferably focusing on developmental aspects of functional/comparative genomics, physiology, neurobiology, cell biology, endocrinology, or genetics, with expertise in the cardiovascular system, respiration, stress response, and/or metabolism. The successful candidate must have an established international reputation, an externally funded research program, and will be an integral part of the BDevelopmental Physiology and Genetics Group[ (website: http://www.biol.unt. edu/dpgr), targeted by the UNT administration for support. Candidates are expected to have a Ph.D. in a Physiology related field and support the instructional goals of the university at both the undergraduate and graduate levels. Generous startup funding and salary will be provided. This will be one of four new faculty positions recruited to the group. Laboratory space is available in the newly constructed 80,000 square foot state-of the-art Life Sciences Complex. For further information, see websites: http://www.unt. edu, http://www.biol.unt.edu, and http://research. unt.edu/clusters. All applicants must apply online at website: http://facultyjobs.unt.edu. For questions, contact Pudur Jagadeeswaran, Search Committee Chair, at e-mail: [email protected]. Applications will be reviewed beginning March 15, 2011, and will continue until the search is closed. UNT is an Affirmative Action/ADA/Equal Opportunity Employer.

972

The Department of Neurology at Columbia University and the New York-Presbyterian Hospital has an open faculty position to develop a clinical and research program in neurogenetics. We are looking for an individual with outstanding experience and excellent references who will lead a program specifically dedicated to studying genetic diseases affecting the nervous system that includes clinical care, genetic counseling, and research. Protected research time and appropriate resource support will be provided. M.D. and board certified in neurology and medical genetics. Faculty rank is expected to be at the ASSISTANT or ASSOCIATE PROFESSOR level, commensurate with experience and achievement. Assistant professor level candidates must provide evidence of research accomplishments and have some teaching experience. External research funding is preferred. Qualified candidates should send a letter of interest and curriculum vitae in Word format to: Richard Mayeux, M.D., M.Sc. Gertrude H. Sergievsky Center 622 West 168th Street, P&S Box 16 New York, NY 10032 E-mail: [email protected] Apply electronically through website: https:// academicjobs.columbia.edu/applicants/Central? quickFind054294. Columbia University is an Affirmative Action/Equal Opportunity Employer.

St. John_s University currently has an ASSISTANT/ ASSOCIATE PROFESSOR, tenure-track, faculty position available in the Department of Pharmaceutical Sciences, at our Queens campus, in the area of toxicology/pharmacology. The incumbent will establish an active research program that will attract external funding, mentor students, and teach graduate and undergraduate courses. To qualify, you must have an earned Ph.D. in an appropriate biomedical science and postdoctoral experience. We offer a competitive compensation program and comprehensive benefits to meet the diverse needs of our workforce. Interested professionals should send letter of application, curriculum vitae, research plan and statement of teaching philosophy, and three letters of reference to: Dr. Louis D. Trombetta, Professor and Chair-Department of Pharmaceutical Sciences, St. John_s University, 8000 Utopia Parkway, Queens, NY 11439. E-mail: [email protected]. St. John_s is an Equal Opportunity Employer and encourages applications from women and minorities.

18 FEBRUARY 2011

VOL 331

SCIENCE

The Charles E. Schmidt College of Medicine is seeking a tenure-track/tenured FACULTY MEMBER at any rank to conduct research and teach microbiology and infectious diseases or a related topic to medical and graduate students. The applicant should have a well-developed research program focusing on molecular basis of human diseases and/or therapies. The successful candidate will have a Ph.D. and/or M.D. Preferred candidates will have research interests that complement those of current faculty, a demonstrated ability to conduct innovative research, a strong record of external grant awards, and must be currently funded with a NIH RO1 grant or equivalent. The position includes startup funds and a 12-month salary. Applications will be accepted until April 18, 2011 or until the position is filled. Application materials must be submitted electronically including: Administrative, Managerial, and Professional (AMP) application, cover letter, curriculum vitae, a one-page statement of teaching experience and philosophy, a one-page summary of research interests, and the names of three references to website: https://jobs.fau.edu (position 981310). Credentials will be subject to Florida Public Records Law. For accommodation, call telephone: 561297-4341. A background check is required for the candidate selected for this position. Equal Opportunity/Affirmative Action Employer.

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OSAK A I N F O CU S

Heritage for the Future Osaka builds on its legacy in the biosciences Leaders of the city of Osaka’s academic, industry, and government sectors are implementing ambitious plans to make the Northern Osaka Biomedical Cluster one of the top ﬁve bioscience regions worldwide within the next 10 years. Reporting from Japan, Adarsh Sandhu takes a closer look.

Osaka has been at the center of Japan’s pharmaceuticals industry since the 17th century when the Doshomachi district was a hub for merchants trading herbal medicines. The area has been an incubator for global companies like Takeda Pharmaceutical Co., Ltd. and Shionogi & Co., Ltd, as well as the predecessor of Osaka University’s School of Medical Sciences. “Osaka has a rich history of research in the life sciences and is a base for major pharmaceutical companies,” says former president of Osaka University and immunologist Tadamitsu Kishimoto, currently a professor at the Graduate School of Frontier Biosciences, Osaka University. “Biomedicine has tremendous potential here. We want to realize the dream of former president Yuichi Yamamura, who envisaged Northern Osaka as an international hub for life sciences.” Indeed, Osaka-based scientists have made outstanding contributions to the life sciences including the discovery by Kishimoto of interleukin-6 (IL-6), a protein governing the immune response. This led to the development and commercialization of the IL-6-inhibiting drug, tocilizumab, for the treatment of Castleman’s disease and rheumatoid arthritis. Kishimoto donates the proﬁts from the sale of tocilizumab to the

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Kishimoto Foundation to support research. “I want to contribute to nurturing the next generation of researchers in the life sciences,” says Kishimoto. In the area of infectious diseases, the discovery of Vibrio parahaemolyticus—a bacterium that causes food poisoning—was made at the university’s Research Institute for Microbial Diseases (RIMD), and its brother institute, the Research Foundation for Microbial Diseases (BIKEN), Japan’s largest manufacturer of vaccines for diseases such as chickenpox. Notably, scientists at RIMD are also developing vaccines to ﬁght malaria, a so-called neglected disease, which affects about 40 percent of the world’s population, mostly in poorer countries. The tradition of pioneering work in immunology, initiated by the late Yuichi Yamamura, is being continued by Osaka University’s Kishimoto and by Shizuo Akira, director of the Immunology Frontier Research Center (IFReC) whose research on Toll-like receptors, innate immunity, and viral recognition has made him one of the most highly-cited immunologists. Breakthroughs have also been made in transplant surgery at Osaka University Medical School, where surgeons performed Japan’s ﬁrst kidney transplant in 1964 and

the ﬁrst simultaneous heart-lung transplant in 2009. So what are the plans for Osaka’s return to center stage?

Developing a Strategy The core goals, which are contained in the so-called Osaka Bio Strategy, involve accelerating the development of new drugs and medical devices; promoting constructive alliances between industry, academia, and government throughout the Kansai area (which includes the cities of Osaka, Kyoto, and Kobe); and strengthening support for venture capital companies (via the Osaka Bio Fund’s ¥1.1 billion ($13 million) in capital assets). The establishment of a Comprehensive Global Strategic Special Zone of the Japanese government is included, with two core priority areas: the biomedical industry, and environment and new energy. Participation is being sought from some of the world’s largest electronics, chemical, and precision instrument companies, such as Panasonic and Sharp, both of which are headquartered in Osaka. “The biomedical industry relies on medical devices,” says Yasushi Sugimoto, director general of the Department of Commerce, Industry and Labor, Osaka Prefecture. “The

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OSAK A I N F O CU S

Tad a

Kansai region is Japan’s largest manufacturer of lithium ion batteries, generating ¥22 billion ($260 million) in revenue in 2009, and batteries are a critical component of medical equipment.” To promote interdisciplinary and crosssector collaboration, the Osaka Bio Headquarters has been set up to bridge the gap between research and commercial entities. “The Bio Headquarters consists of members from industry, academia, and government,” says Kishimoto, president of the organization. “Members of this organization devise plans to achieve our goals as part of the Osaka Bio Strategy framework.” To provide support for venture companies, the Osaka Bio Strategy includes the launch of a biotalent matching service catering to the human resource needs of startup companies.

CREDIT: (LEFT) © ISTOCKPHOTO.COM/SIMFO; (RIGHT) © ISTOCKPHOTO.COM/SHUNYUFAN

Promoting Research, Fostering Business To reduce both the time and cost of performing clinical trials, Osaka plans to make available the expert services of institutions such as the ﬁve hospitals of the Osaka Prefectural Hospital Organization to the pharmaceutical industry. A clinical trials website has also been set up, offering upto-date information about trial results. On the policy side, a request has been made by the Osaka Bio Headquarters to the central government and the Pharmaceutical and Medical Devices Agency to make positive reforms to clinical trial regulations and practices. Knowledge of the properties of proteins is extremely important for fundamental research on immunology, cancer treatment, and drug discovery. Osaka is renowned for

mitsu Kishimot

o

its expertise in protein research, exempliﬁed by more than 50 years of research conducted at Osaka University’s Protein Research Institute (PRI). Now, in an innovative development, the biomedical cluster launched the Protein Mall in May 2009 as an open access platform for drug discovery. It aims to create a ‘drug development value chain’ to foster business opportunities based on protein-related research. Knowledge is essential, but collaboration is also critical to achieve Osaka’s goals. As such, the Drug Seed Alliance Network Japan—managed by the Osaka Chamber of Commerce and Industry—is actively promoting partnerships between industries both within Japan and overseas. Recent international agreements include memoranda of understanding signed with France, Australia, Belgium, and the BioBusiness Alliance of Minnesota. A central and important facility in the Northern Osaka Bio Cluster is the National Institute of Biomedical Innovation (NIBIO), which the government set up in 2005 to act as a stepping stone for commercializing ideas from the laboratory and bringing them into the clinic. Research at NIBIO ranges from developing adjuvants for more effective vaccines to maintaining an ancestry database of cynomolgus monkey colonies, which is openly available to researchers. The strength of Osaka’s expertise in life sciences is based on decades of basic research conducted by imaginative and highly motivated scientists, historically from within Japan but now also from overseas. Shizuo Akira, director of IFReC—launched in October 2007 as one of only ﬁve World Premier Institutes chosen by the Japanese government—says that approximately 30 percent

of its principle investigators are foreign, a rare situation in Japan, but made possible through extensive support provided by IFReC staff for foreign scientists to help them overcome language and cultural barriers.

Looking to the Future There are some disturbing trends among young Japanese researchers that worry their senior counterparts. “Youngsters are reluctant to go overseas these days,” notes Kishimoto. “It’s probably because Japanese universities now offer world class research environments. But research is not only about equipment and clean rooms; meeting people from different backgrounds and developing international networks is equally, if not more, important.” Only time will tell how these trends will affect Japan’s research prowess, but the new research programs have attracted many bright researchers from overseas, which may compensate for the decreased willingness of Japanese scientists to travel internationally. Osaka and the Kansai region have a lot to offer the biomedical industry with new projects in neuroscience, human-machine interface research, cancer treatment, and robotic assistance in hospitals, all of which build on the legacy of Osaka's pharmaceutical and electrical industries. On balance, the important fact is that Osaka is still an energetic and forward-looking location for pursuing research and business in the biosciences.

Adarsh Sandhu holds joint professorships at the Tokyo Institute of Technology and Toyohashi University of Technology, Japan.

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OSAK A I N F O CU S: Excellence in Research

Establishing a Comprehensive Global Strategic Special Zone in Osaka

T

he last half of the 20th century saw Japan’s

automobile industry lead the nation to become a powerful ﬁrst-world economy. But this ‘unipolar economic structure’ has seen its day and the realities of the new millennium dictate different approaches

To pull Japan out of the current economic malaise, prominent members from academia, industry, and government of Osaka Prefecture— the heart of the Kansai region of Western Japan incorporating the cities of Kyoto, Kobe, and Nara—are devising new economic models based on the historical strengths of the region’s electronics, chemical, biomedical, and precision instruments sectors. In particular, a strategy that makes full use of Osaka's current expertise in the biomedical, and environment and new energy industries will be the driving force, with a potential economic upside of ¥700 billion ($8.3 billion). The history of Osaka’s biomedical industry goes back to the 17th century when the Doshomachi district in Osaka City was the national hub for the distribution of herbal medicine and related materials. Centered on Doshomachi, the Northern Osaka Biomedical Cluster covers an area 40 km in diameter, and includes university startups and pharmaceutical

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Osaka: Traditional Enterprising Spirit Is Still Alive companies as well as the world’s leading

government designation as a biomedical industry

research institutes. In recent years, the Northern Osaka Biomedical Cluster has enjoyed a reputation as one of the top biomedical clusters in Japan.

area in this region, Osaka could act as a catalyst to initiate the spread of ideas for life innovation throughout the rest of Japan. Osaka will submit a formal application in 2011.

Osaka Prefecture’s Proposal for the Comprehensive Global Strategic Special Zone

The proposal includes: (1) Establishment of a PMDA (Pharmaceuticals and Medical Devices Agency) ofﬁce in the Osaka region to accelerate the development of pharmaceuticals and medical devices, and resolve issues related to ‘drug-lag and devicelag’ (delays in drug and device development and approval due to stringent government regulations); (2) Extending the function of the National Cerebral and Cardiovascular Center (NCVC) by constructing new buildings to expand the Research and Development Initiative Center and related medical clusters; (3) Setting up a Comprehensive Special Zone

One of the pillars of the Japanese government’s strategy for economic growth is founded on a vision of so-called Life Innovation for becoming a leader in research and providing exceptional health care. Now, Osaka Prefecture will help to realize this national goal by integrating the unique potential of Northern Osaka with the world-class biomedical industries and abundance of highlyskilled employees available in this area. In September 2010, Osaka proposed a draft of the Comprehensive Global Strategic Special Zone to the national government. With a formal

CREDIT: © OSAKA CONVENTION & TOURISM BUREAU

for further economic growth.

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CREDIT: (TOP PHOTOS) © OSAKA CONVENTION & TOURISM BUREAU

Midosuji: Main Business and Shopping Street in Downtown Osaka based on the achievements of Osaka University, NCVC, and the Osaka Medical Center for Cancer and Cardiovascular Diseases to promote clinical trials and treatment of cancer and cardiovascular diseases; (4) Setting up a multidisciplinary medical and welfare robot demonstration experiment zone by bringing together local small and medium enterprises (SMEs) as well as global corporations such as Panasonic for the development of robot-assisted patient beds, automated drug mixing for injections under aseptic conditions, and autonomous delivery robots for transporting medical supplies and medicine; (5) Creation of a uniﬁed industry-academicgovernment approach to support the development of antibody and nucleic acid medicines based on shared ‘good manufacturing practice’

New Energy and Biomedicine

(GMP) facilities maintained at the Saito Life Science Park in the Northern Osaka Biomedical Cluster. In addition to contract-based manufacture of cutting-edge biomedical products, the GMP facilities would strengthen the research and development activities of startup companies by offering consultation, development of human resources, and collaborative research; (6) Increasing efforts to attract new vaccinerelated industries—the growth engines of medical industries—to form a strong foundation for the research, development, and manufacture of biomedical products. This policy takes advantage of Osaka University’s research institutes— the Immunology Frontier Research Center, the Research Institute for Microbial Diseases, and the Institute for Protein Research—and the National Institute of Biomedical Innovation (NIBIO), located in the Saito Life Science Park, all world leaders in vaccine research.

worldwide. The Research and Development Initiative for Scientiﬁc Innovation of New Generation Batteries, or RISING, program aims to understand the nature of the chemical reactions governing the lifespan of lithium ion batteries, and develop batteries with ﬁve times greater energy storage capacity than current technology. Future applications include not only automobiles, but also portable devices for improved health care. Taking the lead in the development and adoption of new energy technologies, Osaka will be hosting the Osaka New Energy Forum 2011, scheduled for 24–26 March, at the Osaka International Convention Center. Chaired by Osaka Governor Toru Hashimoto, the theme of the conference will be to review the role of electric vehicles in society. The organizers expect about 10,000 participants at the event.

Osaka's Bay Area as the New Administrative Center

The Comprehensive Global Strategic Special Zone also includes an area that addresses environmental and new energy industries. Osaka Prefecture hopes that the inclusion of academic and industrial initiatives on new energy as well as the integration of biotechnology will provide the impetus to improve Osaka’s economic potential and create a positive ripple effect throughout the rest of Japan. Osaka has a rich history of innovative contributions to the development of new energy resources. For example, Osaka is recognized globally for manufacturing 34 percent of the world’s supply of lithium ion batteries. In the area of photovoltaics, industries in Osaka manufacture 11 percent of solar cells sold

Panasonic's In-Hospital Delivery Robot, "HOSPI"

MORE INFO The Osaka New Energy Forum www2.convention.co.jp/oneforum/en/

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OSAK A I N F O CU S: Excellence in Research

Unsurpassed Pedigree in Translational Medicine Osaka University Medical School and afﬁ liated facilities set the example for a comprehensive approach towards taking cuttingedge research from the laboratory to the clinic, and into the home.

T

he Medical School at Osaka University, a part of the Faculty of Medicine within

Toshio Hirano

Masahiro Fukuzawa

back to the Tekijuku school established in 1838 by Ogata Koan, pioneer of Western medicine

and Hirano as well as Charles Dinarello, who

of other ailments such as cardiovascular and

in Japan, and the ﬁrst person in Japan to encourage a systematic approach to smallpox

discovered IL-1. “The discovery of IL-6 and more recent clari-

autoimmune diseases in order to develop better drugs and treatments.”

vaccination. “Our medical school has played a pivotal role

ﬁcation of the signal pathways involving Toll-like receptors by Shizuo Akira of the Immunology

The Osaka University Medical School is also recognized for pioneering transplant surgery, per-

not only in the development of medicine in this country but also in the growth of Osaka Univer-

Frontier Research Center are two examples of the research that have made Osaka University a

formed at Osaka University Hospital, starting with Japan’s ﬁrst kidney transplant in 1964, the ﬁrst

sity as a whole,” says Toshio Hirano, dean of the Graduate School of Medicine.

world-class center for immunology,” says Hirano. The Medical School also has world-class

heart transplant in 1999 (following the 1997 passage of new legislation on organ transplants by

In the early days the Medical School was renowned for the treatment of infectious diseases

medical imaging resources including an ultrahigh resolution semiconductor positron emission

the Japanese government allowing the harvest of organs from brain dead patients), and the ﬁrst

such as smallpox and pulmonary tuberculosis. A high standard of both innovative treatments and

tomography (PET) system commissioned in July 2010, the world’s ﬁrst permanent magnet PET/

simultaneous heart-lung transplant in 2009. Osaka University Hospital is the largest hospital

cutting-edge research continues today. The Medical School later evolved into an

MRI (magnetic resonance imaging), a real-time positron imaging system for monitoring the kinet-

in the Kansai region. “We have about 2,000 staff, including 800 doctors,” says Masahiro

immunology hub as the result of breakthroughs including the discovery of interleukin-6 (IL-

ics of drugs inside the body, a compact cyclotron system for the generation of radioisotopes and

Fukuzawa, director of the hospital. “In 2009, we had about 620,000 outpatients, 340,000

6)—a key immune stimulatory molecule—by Tadamitsu Kishimoto and Toshio Hirano. This led to the development of Japan’s ﬁrst humanized monoclonal antibody therapy, tocilizumab, which blocks the action of IL-6 and is used to treat rheumatoid arthritis, joint inﬂammation, and Castleman’s disease. Recently, the impact of the discovery of IL-6 and tocilizumab was acknowledged by the joint award of the 2009 Crafoord Prize by the Royal Swedish Academy of Science to Kishimoto

labeled compounds, and a 11.7 Tesla MRI facility at the Graduate School of Frontier Biosciences. In new developments, a 7 Tesla functional-MRI system will be available at the National Institute of Information and Communications Technology on the campus. “Osaka will use these imaging facilities to focus on the treatment of degenerative diseases—such as Alzheimer’s disease—and malignant diseases,” says Hirano. “In addition, we are working on clarifying the mechanisms governing the onset

inpatients, and performed 8,700 operations. We are an information technology-oriented hospital, with a heliport on the roof, and a world-class trauma and acute critical care center.” In 2008 an oncology center was set up at the hospital where cancer surgery is performed and clinical research is carried out on different therapy modalities, including drugs, radiation, and relaxation. Furthermore, as part of the Cancer Professional Training Program ﬁrst launched in 2007, education courses for patients and the general public were initiated on these same therapies. At the heart center, established in 2007, clinicians treat severe heart failure and intravascular disorders as well as perform heart surgery. “We are also developing advanced medical treatments for cancer and for partial organ transplants,” says Fukuzawa.

Osaka University Medical School

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Robotic Arm Operated by Electrocorticogram

The hospital collaborates with internal research groups including the Medical Center for Translational Research (MTR), set up in

CREDIT: (BOTTOM RIGHT) THIS WORK WAS SUPPORTED BY THE STRATEGIC RESEARCH PROGRAM FOR BRAIN SCIENCES OF MEXT.

the Graduate School of Medicine, is the oldest in Japan, with roots going

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Toshiki Yoshimine

Akira Myoui

Haruo Sugiyama

2002 to conduct research on topics including regenerative medicine, robotic surgery, and diagnostic imaging. “Being located within the hospital enables us to have ﬁrst-hand interaction with the patients and clinicians,” says Toshiki Yoshimine, director of the MTR. Patient conﬁdentiality and protection is a top priority. “Out of an abundance of caution we have established an ethics committee with whom we consult on decisions about exploratory clinical trials,” says Yoshimine. Innovative work is being done in tissue regeneration using a patient’s own cells. Examples include the use of myoblast sheets produced from femoral muscle for the treatment of cardiomyopathy and engineered cell sheets composed of oral mucosal epithelium for corneal reconstruction. “With this technology there is no risk of immunological rejection,” says MTR Vice Director Akira Myoui. “One of our most challenging projects is on the brain-machine interface,” says Yoshimine. “We are developing technologies to gauge human intent based only on brain activity as an alternative means of communication for people with severe motor impairments, such as ALS [Amyotrophic Lateral Sclerosis].” In recent trials, clinical researchers implanted electrodes directly onto the surface of the brains of patients with epilepsy

division is unique in Japan,” says Ohashi. “Our students have backgrounds in nursing science and medical technology science. They are studying in one of our three unique interdisciplinary programs, which are based on informatics, mechanics, and life sciences.” Yuko Ohno and Kenji Yamada are in the Department of Robotics and Design for Innovative Healthcare. “This department reﬂects a collaboration between Osaka University and the robotics center at Panasonic Corporation,” says Ohno. “Our nurses are working with robotics experts to develop a database of how robots could operate in hospitals.” One exciting project being conducted at the Osaka University, in collaboration with other groups, is looking at the potential of robotic beds, which will enable patients to move from the bed to a wheelchair without human assistance. “This is a unique experiment fusing engineering and nursing,” says Yamada. “Also, working directly with industry will enable rapid translation to actual applications in the future.” Immunology is another strong area of research at the Division of Health Sciences led by Haruo Sugiyama, developer of the WT1 (Wilms’ Tumor Gene 1) peptide cancer vaccine. “We completed phase one clinical trials at Osaka University Hospital in 2002,” says Sugiyama. “We are

or intractable pain in an attempt to treat these diseases. Analysis of information-based brain responses to simple grasp, pinch, and release hand movements in these patients has enabled them to control robots in real time. “These are very promising results,” says Yoshimine. “Ultimately we want to develop a commercial, fully-wireless implantable system.” Kazutomo Ohashi is head of the Division of Health Sciences at the Medical School. “Our

now pursuing phase 2 trials.” The WT1 gene is highly expressed in both hematopoietic and solid tumors. Sugiyama demonstrated that WT1 plays an oncogenic role via its antiapoptotic function and promotion of cell motility, and is thus a promising target for cancer therapy. The third major new program at the Division of Health Sciences is the Center for Twin Research, led by Kazuo Hayakawa. “This is the ﬁrst center in Japan to focus on collecting data from twins

Kazuo Hayakawa

Kenji Yamada and Yuko Ohno

Osaka University Hospital aged 65 to 100 to clarify the effects of genetics and environment on health,” says Hayakawa. “For example, why would one twin die at 70 and the other at 100?” The twin research project, which is one of the Japanese government’s ‘Life Innovation’ programs, will assess 1,000 twins separated in their youth who went on to live in different environments. Researchers will use NMR, PET, and other similar methods to examine the subjects. Results will be stored in a biobank accessible to scientists worldwide. “We want to use the results for preventative medicine, and perhaps even for anti-aging strategies,” says Hayakawa.

MORE INFO Osaka University Graduate School of Medicine www.med.osaka-u.ac.jp/index-e.html

Osaka University Hospital www.hosp.med.osaka-u.ac.jp/

Osaka University Hospital Medical Center for Translational Research www.hp-mctr.med.osaka-u.ac.jp/

Kazutomo Ohashi

A Laboratory at Osaka University Medical School

Graduate School of Medicine, Division of Health Sciences sahswww.med.osaka-u.ac.jp/eng/welcome.html

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OSAK A I N F O CU S: Excellence in Research

World-Class Interdisciplinary Research on Imaging the Human Immune System Scientists at the Immunology Frontier Research Center (IFReC) of Osaka University are using a large-scale, interdisciplinary approach to spatiotemporal imaging of the body’s immune system.

T

approximately 170 specialists of whom one-third are non-Japanese researchers. “We have made

IFReC Research Building

Shizuo Akira

Cevayir Coban

a conscious effort to establish an international center,” says Akira. “In fact, ﬁve of the principle investigators are from overseas.” In ﬁscal year 2009, the WPI-IFReC acquired funding totaling ¥1.35 billion ($15.9 million) and its researchers published 229 articles—statistics that underscore the high level of research activity at the center. Takao Kodama is administrative director at WPI-IFReC, with responsibilities that include supporting overseas researchers living in Japan. “One of the unique administrative aspects of this WPI-IFReC research project is that we deal

assistance with preparing applications for grants and other such matters.” “I am quite satisﬁed with the support we receive from the administration staff,” says Nicholas Smith, a principle investigator from Australia, who was attracted to the worldrenowned immunology research program at Osaka. “English is the de facto language for both administration and research,” says Cevayir Coban, a principle investigator who moved to Osaka from the United States and the recipient of a $100,000 research grant from the Bill and Melinda Gates

directly with MEXT and our decision-making process is top-down,” says Kodama. “We do not have to hold

Foundation. “The Japanese language is not a hurdle for conducting research here.” In addition to partnerships within Japan, WPI-

unnecessary faculty meetings. This enables a highly efﬁcient administrative process. In addition, the WPIIFReC administration has made a conscious effort to enable a smooth transition for overseas researchers moving to Japan, and to offer them convenient housing near the campus as well as

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IFReC is collaborating with overseas research institutions, including the National Institutes of Health; the University of California, San Francisco; the California Institute of Technology; Harvard University; New York University; Stanford University; POSTECH in Korea; and the Indian Institute of Science Education and Research in Bhopal, India. The research facilities of WPI-IFReC include the 10-story, 10,000 m2 Integrated Life Science building completed in June 2009, the four-story 2,500 m2 Animal Resource Center, and a new nine-story 8,000 m2 research space—scheduled for completion in March 2011—connected to the main research building. Notably, the WPI-IFReC is strategically located adjacent to the Research Institute for Microbial Diseases (RIMD) and the

CREDIT: (BOTTOM) BY J. SAKANOUE

he Osaka University Immunology Frontier Research Center (IFReC) was selected in 2007 by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) as one of only ﬁve coveted WPI (World Premier International) research centers. “IFReC is a unique interdisciplinary research center built on the foundations of our pioneering work in immunology,” says Shizuo Akira, director of WPI-IFReC. “We are taking a spatiotemporal approach to obtain a complete picture of the immune response in the human body. Our projects are a fusion of expertise in immunology, imaging technology, and bioinformatics.” One of the ultimate goals of the research at WPI-IFReC is to craft methods for controlling the dynamics of immune cells for the development of tailor-made vaccines and immunotherapy treatments. Research at WPI-IFReC is being conducted by

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“Clarifying the mechanisms of the innate immune response is important for the development of strategies for the treatment and prevention of malaria.”

Institute for Protein Research (IPR), forming a massive interdisciplinary research complex. WPI-IFReC is keen to recruit highly motivated postdoctoral researchers from overseas, and offers WPI-IFReC Kishimoto Foundation Fellowships of about ¥4,400,000 ($52,600) per year. The Kishimoto Foundation was set up in 2008 in honor of Tadamitsu Kishimoto, ex-president of Osaka University. To enhance its global visibility, WPI-IFReC organizes a wide range of international workshops and symposia to encourage further research and collaboration. “We organize a Winter School with colleagues in Singapore,” says Akira. “Postdoctoral researchers from overseas can apply for the 40 or so places available. The next Winter School is on January 16–21, 2012 and will be held on Awaji Island, in the Seto Inland Sea.” As part of its outreach programs, WPI-IFReC holds regular science cafés in the Osaka city center. “These are very popular,” says Kodama. “We have many regulars who attend.” In vivo imaging of cell movement is one of the central themes of research at WPI-IFReC. Principle investigator Masaru Ishii is an expert on intravital two-photon imaging of bone tissue in vivo. Ishii recently discovered a method for

rheumatoid arthritis and osteoporosis,” says Ishii. “Bone biology will become even more important as the number of aged people increases worldwide.” Label-free imaging offers several advantages over widely-used ﬂuorescent techniques, including a simpler protocol and elimination of spurious data related to nonspeciﬁc interactions between labels and targets. At WPI-IFReC, Smith focuses on label-free imaging of living cells using optical Raman scattering. “We record light scattered from a cell scanned with 532 nm laser light, which resonates with molecular states in a cell,”

another high-priority research area at WPI-IFReC. “Clarifying the mechanisms of the innate immune response—that is the body’s natural resistance to the Plasmodium parasite—is important for the development of strategies for the treatment and prevention of malaria,” says Coban. Coban is using mouse models of malaria to develop new vaccines to ﬁght the disease, in particular DNA vaccines incorporating adjuvants for efﬁcient immunogenicity. Speciﬁcally, Coban is studying hemozoin—a pigment produced by the malaria parasites as a byproduct of blood digestion and an essential compound for their

regulating the mechanism governing osteoclastogenesis by controlling the dynamics of movement of osteoclast precursors between blood and endosteum. “The signiﬁcance of our research is that we perform experiments in living animals, thereby giving us a real-life view of the dynamics of osteoclast cells, which are involved in bone tissue destruction and resorption,” says Ishii, who is one of the many physician-scientists at WPI-IFReC. Two of the major goals of Ishii’s research are to ﬁnd alternatives to bisphosphonates and vitamins for the treatment of bone resorptive disorders, and to develop in vivo visualization techniques to clarify the migration and differentiation of hematopoietic stem cells, which give rise to all bone marrow-derived cell types in the blood. “Our discovery that sphingosine-1-phosphate regulates the migration of osteoclast precursors is an important advance for the treatment of

says Smith. “The challenge is detecting extremely low-intensity scattered light. Our setup enables the extraction of well-deﬁned 3D images of molecular changes in cells.” Smith is also pursuing the use of nanoparticles to enhance the local light ﬁeld around these particles, allowing for ultrahigh-resolution laser imaging and even real-time modiﬁcation of cells. For example, particles can be heated using laser light to potentially ablate subcellular compartments, or the laser beam itself can induce changes in the cell directly. “Recently, we controlled the beating of heart muscles using pulsed laser irradiation,” says Smith. “This effect is due to the release of calcium ions during laser irradiation, and demonstrates how lasers are now being used in new ways for in situ measurement or control of biological functions.” Vaccine development to prevent malaria is

survival—for use as an endogenous vaccine adjuvant against malaria. “We found that hemozoin binds directly to TLR9 [Toll-like receptor 9] and activates it,” says Coban. “Synthetic hemozoin acts as an adjuvant in a canine anti-allergen vaccine, and we concluded that hemozoin affects immune responses to malaria infection.” These results have important implications for the development of vaccine adjuvants for malaria treatment in humans. The WPI-IFReC is aiming to break new ground in immunological research, and its staff are deeply committed to creating a unique and highly-imaginative research base in Japan.

Nicholas Smith

Masaru Ishii

MORE INFO Immunology Frontier Research Center (IFReC), Osaka University www.ifrec.osaka-u.ac.jp/index-e.php

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OSAK A I N F O CU S: Excellence in Research

'Brothers in Arms' in the Fight Against Infectious Diseases RIMD and BIKEN are two of Osaka University’s premier institutes with a common mission to eradicate infectious diseases.

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he Research Institute for Microbial Diseases (RIMD) and The Research Foundation for Microbial Diseases of Osaka University (BIKEN) were established as ‘brother’ organizations in 1934 following a donation from Gendo Yamaguchi, an Osaka entrepreneur. “Our roots can be traced back to 1934 and microbiologist Tenji Taniguchi, who was convinced of the need for a counterpart to the Institute of Infectious Diseases at Tokyo Imperial University, now the University of Tokyo,” says Shigeharu Ueda, director of BIKEN. Taniguchi strongly urged the head of the Osaka Imperial University to set up an institute in Osaka “to pursue basic and clinical medicine.” Both RIMD and BIKEN were established using funds from Yamaguchi, who was extremely impressed with Taniguchi’s ambitious suggestions. Proﬁts from BIKEN are used to fund research at RIMD in addition to supporting other academic research on microbial diseases. RIMD conducts research on microbiology,

HIV/AIDS and avian ﬂu.” Building on its success with RCC-ERI, in 2010 RIMD was selected to be a member of the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), a ﬁve-year program launched by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) as a successor to the RCC-ERI with the goal of establishing long-term research activities via further development of the RCC-ERI initiative. As

of the development of vaccines. Shortly after the construction of the Ishibashi production plant in 1935, BIKEN was given approval for the manufacture of seven vaccines, including those for cholera and pertussis. Then, at the end of World War II in 1945, BIKEN played a central role in restoring the health of the Japanese people by supplying large quantities of vaccines to ﬁght cholera and smallpox. In 1952, BIKEN received approval for the ﬁrst made-in-Japan inﬂuenza

oncology, and molecular biology. “We have more than 200 researchers here,” says Hitoshi Kikutani, RIMD director. Researchers at RIMD have made signiﬁcant contributions to the life sciences, examples being the discovery of cell fusion, which led to the development of monoclonal antibodies—important tools for drug discovery and basic research—and the discovery of the bacteria known as Vibrio parahaemolyticus, which causes food poisoning. Importantly, RIMD participates in international research and maintains collaborative research centers with the support of the Japanese government, including the Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), which was set up in Bangkok in 2005. “The RCC-ERI

one of activities of this program, RIMD set up the Mahidol-Osaka Center for Infectious Diseases (MOCID) at Mohidol University in Thailand in 2010. Speciﬁc objectives include ﬁnding treatment solutions for bacterial infections, HIV/AIDS, and mosquito-borne infectious diseases through collaborations with infectious disease research organizations from other countries. BIKEN is known internationally as Japan’s largest manufacturer of vaccines, and in line with government reforms of nonproﬁt foundations, it was restructured into a general incorporated foundation in April 2010. “The vaccines we manufacture are developed and produced based on the fundamental research conducted at RIMD,” says Ueda. “Recently, we were at the front line in the ﬁght against the pandemic H1N1

virus vaccine, followed by another ﬁrst in 1961 for inactivated polio vaccine. Between 1965 and 1970, BIKEN began manufacturing several forms of the measles vaccine, as well as a vaccine for chickenpox. Just recently, in 2009, BIKEN obtained approval for the manufacture of cell culture-derived Japanese encephalitis vaccine. “Cell cultured production is faster than procedures using eggs,” says Ueda. “The rapid spread of diseases requires equally rapid responses. We continuously strive to improve the development and manufacture of vaccines.” BIKEN may be an unsung hero in the ﬁght against measles, having supplied the CAM70 vaccine to UNICEF. “Over ten years we supplied about 200 million doses of CAM70,” says Ueda. “This vaccine eliminated measles in Brazil. In

is a partnership with Thailand’s National Institute of Health,” says Kikutani. “The facilities include biosafety level 3 laboratories for research on

inﬂuenza virus and produced 5 million doses of the vaccine.” Historically, BIKEN has been at the forefront

cooperation with JICA, the Japan International Cooperation Agency, we helped Indonesia to set up a manufacturing plant in Bandon, which is

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Shigeharu Ueda

Hitoshi Kikutani

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“Preventative medicine not only prevents the spread of disease but also reduces the cost of national health care budgets.”

Malaria Testing Clinic

Keizo Tomonaga

being used to manufacture CAM70 for export.” Ueda admits that, despite the success of CAM70, Japan still has not eliminated measles due to a lack of complete acceptance of vaccines by young mothers. In the 1990s, BIKEN transferred the know-how for the production of chickenpox vaccines to the U.S. company Merck & Co., Inc. “Our vaccines have had a dramatic effect on reducing chickenpox in the USA,” says Ueda. “Preventative medicine not only prevents

new production plant.” In addition to the manufacture of vaccines, BIKEN also offers JICA training courses on infectious disease-related topics approved by the World Health Organization. Plans for the future include a major effort to develop a malaria vaccine. “About 40 percent of the world’s population lives in malaria-endemic regions,” says Ueda. “We have initiated clinical trials in Uganda for a malaria vaccine based on the serine repeat antigen [SERA], a protein pro-

the spread of disease but also reduces the cost of national health care budgets. For BIKEN the patent royalties from the vaccine for chickenpox raised about ¥10 billion ($120 million) over ten years, which was used to fund construction of a

duced by the Plasmodium falciparum parasite, which is responsible for malaria.” Phase 1 trials on vaccine safety were completed in December 2010. “Malaria is present only in the jungles of Asia, but is everywhere in Africa,” says Ueda. “About one million people die annually due to brain malaria alone.” RIMD researchers are administrating the SE36 SERA vaccine in Uganda. If approval is obtained, the vaccine will be manufactured at the Kanonji Institute of BIKEN, which complies with strict international good manufacturing practice (GMP) standards. In a new initiative, BIKEN is funding a chair at RIMD to conduct research on dengue fever starting in April 2011, and will be building a laboratory at MOCID. A recent highlight of research at RIMD is the discovery by Keizo Tomonaga and colleagues of DNA remnants of the nonretroviral bornavirus in mammalian genomes, including

Bornavirus

humans. These endogenous viruses are so called because they become integrated into the genome of their host. “About eight percent of the human genome is known to be composed of endogenous viruses,” says Tomonaga. “But this discovery was a case of serendipity.” Bornaviruses cause neurological disorders in horses. The name originates from the city of Borna in Germany, where outbreaks of the disease were recorded to have occurred in 1885. These ﬁndings imply that RNA viruses are extremely old, with analysis suggesting that bornaviruses have coexisted with primates for about 40 million years. “This report generated a lot of international news coverage,” says Tomonaga. “In addition to newspaper reporters, the American Museum of Natural History contacted us about this discovery, and there is now a panel on display about so-called fossil viruses at the museum.”

MORE INFO Research Institute for Microbial Diseases (RIMD) www.biken.osaka-u.ac.jp/e/ Research Foundation for Microbial Diseases of Osaka University (BIKEN) www.biken.or.jp/english/index.html

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OSAK A I N F O CU S: Excellence in Research

Critical Bridge For Drug Development Located in the Saito Life Science Park in Northern Osaka, The National Institute of Biomedical Innovation fulﬁls the critical role of bridging the gap between basic research on drug discovery and drug development for commercialization.

“

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IBIO was set up by the Japanese government in April 2005 to act as a mediator between basic research at universities and commercialization by industrial corporations,” says Koichi Yamanishi, director general of The National Institute of Biomedical Innovation (NIBIO). “Our institute plays a unique role in the development of drugs in this country.” The institute’s three main functions are conducting basic research, sharing biological resources, and funding research. Furthermore, NIBIO’s Tsukuba Primate Research Center houses a large scale breeding colony of about 2,000 monkeys—mostly cynomolgus macaques— whose family history has been recorded for over 30 years, making it a valuable resource available to researchers in pharmaceuticals research and development. NIBIO’s budget for ﬁscal 2010 is ¥11.20 billion ($133 million). “We actively support innovative ideas,” says Yamanishi. “It is not widely known that NIBIO funded Shinya Yamanaka during the

Koichi Yamanishi In April 2010, NIBIO started its second phase of research (scheduled for completion in 2014) focusing on three areas: (1) Next generation vaccines with an emphasis on the development and understanding of adjuvants to enhance efﬁcacy; (2) Drug toxicity testing systems including a new method to induce differentiation in a variety of stem cell types; (3) Treatment of intractable or rare diseases by analyzing the molecular mechanisms governing these disorders and the development of relevant technologies for diagnosis and treatment. Since 2008, NIBIO has been working on two industry-academia-government collaborative projects based on Super Special Consortia programs for the development of cutting-edge medical care. They are the Next Generation

center is the only comprehensive research facility in Japan conducting research on the cultivation and biochemical analysis of medicinal plants,” says Kawahara. The center cultivates and preserves more than 4,000 species and groups of medicinal plants, and supplies seeds and advice to other research institutes. Notably, only 12 percent of the medicinal plants used in Japan are produced in the country—the remainder being imported from China and other countries. “We want to expand the sources of these ‘second type of rare-earths’—the ﬁrst kind being metals such as lithium used in batteries—by working with other countries,” says Kawahara. Recently, a research group at RCMPR produced liquorice root by hydroponic culture— growing plants using mineral nutrient solutions

Infectious Disease Vaccine Innovation Program, and the Development of a Novel Drug Toxicity Testing System using human iPS cells. Notably, these projects were two of only 24 such programs approved by the government. “The development of a drug takes about 15 to 20 years,” says Yamanishi. “NIBIO was set up only ﬁve years ago, and during the second phase of research I want to push for actual commercialization of some of the drugs being studied in our projects.” NIBIO also works with international groups including Cambridge University (on bioinformatics), as well as Peru and the Solomon Islands (on medicinal plants).

in water and without soil. Liquorice root contains the sweet-tasting compound glycyrrhizin and is widely used as an herbal medicine, as well as a sweetener for food products. Furthermore, another medicinal plants group has developed medicinal treatment for Leishmaniasis, a disﬁguring disease transmitted by sand ﬂies, based on screening of medicinal plants in countries including Myanmar, Peru, and the Solomon Islands. “We used the local knowledge to produce a solution for this disease,” says Kawahara.

early stages of his research on induced pluripotent stem (iPS) cells.” NIBIO also funds venture companies and offers grants and consultation on so-called orphan products—pharmaceuticals and medical devices for rare diseases—which large corporations can be reluctant to invest in because of the small numbers of affected patients and consequently limitations in recouping development costs. In spite of the relatively small size of NIBIO, statistics underscore the signiﬁcant scientiﬁc contributions of its 50 full-time researchers and their groups. For example, in ﬁscal year 2008 each NIBIO researcher received on average ¥16 million ($190,000) from competitive grants—the highest grants per person ratio for researchers at Japan’s approximately 30 independent administration

Medicinal Plant Research

agencies. Furthermore, in 2009 each researcher published an average of three peer-reviewed papers—about twice the number in 2005.

Nobuo Kawahara is director of the Research Center for Medicinal Plant Resources (RCMPR). “Our

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Tetsuji Naka

SOCS for Intractable Diseases Tetsuji Naka is the project leader of the Immune Signaling Project. “We need to work on

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“We hope that our research will enable a reduction in both time and cost of drug development, as well as the development of safer drugs.”

Nobuo Kawahara people,” says Naka, “and not [only on] cells or lab animals. So we are working with colleagues at the Osaka University Graduate School of Medicine on the treatment of intractable diseases such as cancer using SOCS [suppressor of cytokine signaling] gene delivery.” SOCS is a family of genes involved in inhibiting the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway. “Abnormal expression of SOCS molecules can cause dysregulated cytokine signaling, which leads to the development of disease,” says Naka. Recently, Naka and colleagues found overexpression of SOCS3 in cancer cells to be an effective new treatment for malignant pleural mesothelioma—a form of lung cancer caused by exposure to asbestos. This is an important development considering that asbestos-related diseases are expected to reach a peak in around 2030.

Predicting the Toxicity of Drugs Tetsuro Urushidani is project leader of the Toxicogenomics/Informatics Project, which was recently awarded The President’s Prize from the Science Council of Japan for the development of new biomarkers for human safety. “This is a large project involving academia, government, and 13 companies, including Astellas Pharma, Daiichi Sankyo, and Takeda Pharmaceutical Company,” says Urushidani. The project has yielded a toxicology database, called the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (or TGGATEs), containing millions of data points collected by observing in vitro changes in gene expression following administration of more than

Tetsuro Urushidani 150 drugs. The database can be applied in the early stages of drug development to predict toxicity risk and characterize the molecular mechanism of this toxicity, without the need for expensive animals testing. “We hope that our research will enable a reduction in both time and cost of drug development, as well as the development of safer drugs,” says Urushidani.

Ken Ishii

Treatment of malignant pleural mesothelioma

Vaccines and Innovative Adjuvants Ken Ishii is the project leader of the Laboratory for Adjuvant Innovation. He is developing vaccines and their adjuvants—substances added to vaccines to increase the response of the immune system. Ishii joined NIBIO because he wanted to take his ideas from the lab to real world applications, and “not just write high-impact papers.” “We lack a fundamental understanding of the mechanisms by which viral preparations affect the immune response of the body,” says Ishii. “In October 2010, we established the Next Generation Adjuvant Research Group to address these issues.” Notably, the Adjuvant Group includes members from vaccine manufacturers and venture capital companies. Recently, Ishii reported on why some inﬂuenza vaccines work better than others, and determined that a rare immune system cell type, plasmacytoid dendritic cells, modulate the effect of inactivated ﬂu virus vaccines, but not live virus vaccines. “These results highlight the importance of our research,” says Ishii. “We need a scientiﬁc approach to understanding adjuvants, otherwise interest in adjuvant research may dwindle, similar to the demise of gene therapy with viral vectors, which saw a dramatic decrease in activity following the death of patients on this therapy.”

Hierarchical clustering of genes and chemicals

MORE INFO National Institute of Biomedical Innovation www.nibio.go.jp/english

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OSAK A I N F O CU S: Excellence in Research

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"One plus one equals three and a little more" at the National Cerebral and Cardiovascular Center The NCVC launches the Research and Development Initiative Center for an integrated approach to translational research in the 21st century.

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he National Cerebral and Cardiovascular Center (NCVC) was established in 1977 by the Japanese government for the treatment of cardiovascular disease and to investigate its causes. “Our founding fathers had great foresight in setting our mission as being the treatment of both cerebral and heart diseases,” says Nobuo Hashimoto, president of the center, “because a malfunction of, say, the heart can lead to a stroke due to clogged blood vessels in the brain.” Invoking a common Japanese expression, Hashimoto further illustrates the beneﬁt of the center’s broad mission: “Our integrated approach to treatment utilizes all our extensive resources, where one plus one equals three and a little more.” The NCVC is internationally renowned for its scientiﬁc contributions, including the discovery of bioactive peptides such as atrial natriuretic peptide and brain natriuretic peptide, the development of artiﬁcial hearts and lungs, and research into the rapid treatment of acute cerebral thrombosis using the thrombolytic drug tissue

proach for translating basic research and clinical trials into real-life applications.” Notable areas covered by the RDIC include preventative medicine and epidemiologic informatics, a biological and clinical data bank, and intellectual asset management. “We want to share our wealth of assets with the public and industry,” says Taenaka, “including our knowledge of how to prepare healthy diets for patients suffering from speciﬁc ailments. We also have research facilities for drug and medical device trials, which we are willing to share with industry.” These resources include the NCVC Hospital. “Our hospital has 640 beds of which about 30 percent are for treatment of diseases requiring intensive care,” says Hiroaki Naito, director general of the hospital. Staff at the RDIC are committed to Director Taenaka’s vision. “My mission is to support clinical trials,” says Haruko Yamamoto, director,

Japanese cities show higher occurrences of heart disease compared with other Japanese cohorts. “This reﬂects the possible effects of modern urban life in Japan,” says Miyamoto. When prevention fails, rapid treatment is essential. “We established the ﬁrst stroke care unit and earliest coronary care unit in Japan,” says Kazuo Minematsu, deputy director general of the NCVC Hospital. The NCVC multidisciplinary team, including Takeshi Nakatani, director, Department of Transplantation, has made tremendous contributions to heart transplants and clinical applications of the ventricular assist device (VAD) including the NCVC-type, for long-term circulatory support. In addition to reducing treatment time during emergencies, the NCVC is initiating trials of a ‘mobile telemedicine’ system that will transmit essential medical data from ambulance to

plasminogen activator (t-PA) in combination with, or without, catheter procedures. In April 2010, the Japanese government reorganized the NCVC into an independent administrative institution, thereby giving Hashimoto and his staff greater autonomy to hire specialists—especially from industry—and initiate new wide-ranging collaborative research activities. The NCVC has continued to evolve. “We set up the Research and Development Initiative Center (RDIC) in April 2010,” says Yoshiyuki Taenaka, director of the RDIC. “This center was set up by the NCVC and offers a one-stop ap-

Department of Advanced Medical Technology Development. “We are testing drugs for efﬁcacy against so-called orphan diseases, which large pharmaceutical companies often ignore because they are not commercially viable.” An important mission of the RDIC is the secure collection, storage, and analysis of data on cardiovascular disease for preventative medicine. “Clarifying the cause of stroke and cardiovascular disease is important as a ﬁrst step in prevention,” says Yoshihiro Miyamoto, director, Department of Preventative Medicine and Epidemiologic Informatics. Studies of residents in highly populated

hospital, aiding physicians in their diagnosis prior to the patient reaching the hospital. Recently, Hiroshi Nonogi, chairman of the Department of Cardiovascular Medicine was awarded the ﬁrst International Collaboration in Resuscitation Science Award by the American Heart Association, underscoring the high quality of hyperacute care at NCVC. But NCVC is not resting on its laurels. “We plan to integrate the hospital, research laboratories, and the new RDIC into a single building to create an environment even more conducive for translational research,” says Hashimoto.

Nobuo Hashimoto

Yoshiyuki Taenaka

Hiroaki Naito

Kazuo Minematsu

MORE INFO National Cerebral and Cardiovascular Center www.ncvc.go.jp/english/

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Haruko Yamamoto

Yoshihiro Miyamoto

Takeshi Nakatani

OSAK A I N F O CU S: Integrating Research and Business

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Realizing the Potential for Life Sciences in Osaka

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he Northern Osaka Biomedical Cluster

consists of an area covering a 20 km radius, centered on the Doshomachi district, Osaka City. The origin of the district goes back to 17th century when it became the national center of pricing and distribution of raw materials for herbal medicines.

The cluster was established based on the

Saito Life Science Park and Nearby Research Institutes

ambitious vision of Yuichi Yamamura, former president of Osaka University, to make Northern Osaka a destination for the life sciences. This vision is now coming to fruition. A signiﬁcant development was the opening in September 2008 of the Osaka Bio Headquarters at the Senri Life Science Center building to act as a bridge in coordinating bioscience projects involving academic, industrial, and governmental organizations. Additionally, a number of startup companies have been established in the area, over 300 Osaka Pharmaceutical Manufacturers Association (OPMA) member companies are conducting business around Doshomachi, and world-class research is being carried out at institutes such as the National Institute of

Institute of Advanced Industrial Science and Technology (AIST). In the cluster, the Saito Life Science Park (LSP) is becoming increasingly important as a place to conduct biotechnology research and business, with 40 companies and approximately 1,300 researchers and support staff working on site. Small and medium sized companies and university startups will be able to expand their business at the Saito Bio Incubation Facilities, which include wet labs and animal research facilities. Additionally, a new industrial park neighboring the Saito LSP is scheduled to begin operation in the spring of 2014. It is expected that innovative industries, including those in the life sciences, will move to this area and join those already

called the Osaka Bio Strategy has been laid out. These goals—including that the Osaka Bio Cluster should rank at least 5th worldwide in the biomedical ﬁeld within 10 years—were devised by the Osaka Bio Strategic Promotion Conference, headed by Toru Hashimoto, governor of Osaka, and chaired by Tadamitsu Kishimoto, former president of Osaka University. The Osaka Bio Strategic Promotion Conference consists of 10 organizations (listed on page xv). The policies of the Conference are implemented by the Osaka Bio Support Group, made up of 20 members from the front line of the biotechnology ﬁeld, led by Isao Teshirogi, president and representative director of Shionogi & Co., Ltd. In an effort to support startup companies,

Biomedical Innovation, the National Cerebral and Cardiovascular Center, Osaka University, Osaka Prefecture University, and the National

established in the Saito LSP. To accelerate biotechnology development in the Osaka region, an ambitious set of goals

the Osaka Bio Strategy made provisions for the Osaka Bio Fund—¥1.1 billion ($13 million) in assets to provide ﬁnance for new continued »

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OSAK A I N F O CU S: Integrating Research and Business

“World Medical Polis, Minnesota” Event in Osaka

Osaka Bio Strategic Promotion Conference Member Organizations Ministry of Economy, Trade and Industry (METI), Kansai Bureau National Cerebral and Cardiovascular Center National Institute of Biomedical Innovation Osaka Bioscience Institute Osaka Chamber of Commerce and Industry Osaka Pharmaceutical Manufacturers Association Osaka Prefectural Government Osaka Prefecture University Osaka University Senri Life Science Foundation

MORE INFO Osaka Bio Headquarters www.osaka-bio.jp/en/

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Osaka Booth at MD&M in Minneapolis

Institut Pasteur has signed an MOU with OCCI

ventures in pharmaceutical products and medical devices. Making the most of another of Osaka’s advantages—a highly skilled biotech workforce from world-class universities—Osaka Bio Headquarters provides human resources matching programs to introduce postdoctoral researchers and senior specialists to startup companies. On the clinical side, Osaka Bio Headquarters organizes the Osaka Clinical Trials website in cooperation with OPMA. The website releases the results of clinical trials run by Osaka’s 13 highly specialized hospitals. Established in May 2009, the Protein Mall Kansai (PMK, protein-mall.osaka-bio.jp/en/) is Japan’s largest assembly of protein-related research institutes and companies, organized by the Osaka Bio Headquarters and the Senri Life Science Foundation. The 75 members of PMK aim to create commercial opportunities through the practical application of protein-related research, the promotion of collaborative research, and the support of contract research, all of which

tionally, having organized successful meetings for European and Korean companies on Alzheimer’s disease. The Medical Device Forum holds regular monthly meetings where professional medical researchers and doctors from medical institutions propose joint projects for industry to develop, such as medical devices, biomedical protocols, and equipment. OCCI conducts the World Forum for BIO/MEDICAL Device in Kansai for stimulating alliances between Japanese and overseas companies in medical device ﬁelds. Utilizing the Kansai region's strengths in developing advanced medical platforms, these initiatives are seen as economic models that can be applied nationwide in the drug discovery and the medical device industries.

contribute to innovative technology and new business in the Kansai region, which includes Osaka and ﬁve other Prefectures. The Osaka Chamber of Commerce and Industry (OCCI) promotes open innovation by providing business-matching programs. Speciﬁc programs include the Drug Seeds Alliance Network Japan (DSANJ, www.dsanj.jp/e/) with OPMA as a co-organizer, formed to accelerate technology transfer in the following areas: drug candidates and targets; foods and cosmetics; biomarkers, diagnostics, and reagents; and platform technologies. OCCI maintains the DSANJ database and conducts partnering events categorized by disease area, country, or region. There are already 164 organizations networked as technology providers, and 55 pharmaceutical companies with a presence in Japan have registered as technology searchers. DSANJ is active interna-

with the goal of forming an alliance between Japanese and Minnesota-based companies to promote the development and commercialization of medical devices. Furthermore, an MOU between OCCI and Institut Pasteur in Paris, France in the ﬁeld of drug development was signed June 2010. Institut Pasteur maintains a presence on the DSANJ database, posting technology information that serves to promote alliances with Japanese pharmaceutical companies. The Kansai Bio Promotion Conference has also signed MOUs with partners in France, Belgium, and the State of Queensland in Australia. These alliances are possible because of the historical accomplishments of the life sciences community in Osaka. This community plans to continue building upon this strong foundation, creating greater potential for life science-related research and business in the future.

International Partnerships In February 2010, OCCI signed a memorandum of understanding (MOU) with the Bio Business Alliance of Minnesota, United States—one of the top medical device clusters in the world—

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Dynamic and Evolutionary Research on Protein Science The Institute for Protein Research was set up in 1958 as a joint-use research facility of Osaka University and has played a central role in protein science both in Japan and around the globe.

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n the early days this institute was addressing issues related to nutrition,” says Toshiharu Hase, director of the Institute for Protein Research (IPR). “Now we conduct fundamental research on protein science—P450 was discovered here. We also support the development of the Worldwide Protein Database and the Protein Data Bank of Japan [PDBj, www.pdbj.org].” The IPR has state-of-the-art research facilities, including equipment for X-ray analysis and a powerful 950 MHz nuclear magnetic resonance spectrometer. There are also plans to install a cryo-electron microscope. Notably, IPR has a dedicated beamline at the SPring-8 synchrotron facility in Hyogo, which is available for collaborative research. IPR has inter-faculty agreements with a number of academic institutions, including Peking University in China, Yonsei University and Seoul National University in South Korea, National Tsing Hua University in Taiwan, Indian Institute of Chemical Biology in India, and the University of

Manchester in the United Kingdom. The institute also often welcomes international scientists, for example it hosted eight researchers from seven overseas countries in 2009. IPR is also the source of new, enabling technologies. Junichi Takagi is a structural biologist at the Laboratory of Protein Synthesis and Expression. “Our group has developed a high purity recombinant protein expression/puriﬁcation system for a wide range of proteins,” says Takagi. “We have created an inexpensive and fast afﬁnity puriﬁcation method, which has played a vital role in many structural biology projects.” Recently, Takagi was the focus of international attention when he

Toshiharu Hase

Junichi Takagi

clariﬁed the 3D structure governing semaphorin signaling through the plexin receptor. “We used X-ray analysis to determine the 3D structure of Semaphorin 6A and Plexin A2, before and after signal transmission,” says Takagi. “Our discovery will contribute to the understanding of the mechanism underlying many serious diseases and the development of new drugs.” Indeed, this report resolves the structural mechanism governing receptor activation, namely the transmission of signals across membranes. Takagi is also conducting research on the structure of proteins that are implicated in neuronal synapse functions through ultrastructural analysis of proteins using electron microscopy and tomography. “I want to use our know-how to tell the whole biological story based on the 3D structures of

“Malfunction of homologous recombination leads to cancer and infertility in humans,” says Shinohara. “My research interests are to clarify the mechanism of homologous recombination in eukaryotes and the mechanism of meiotic recombination.” Shinohara is studying the role of protein/genes in the RAD52 family both in vivo and in vitro, and in particular, researching Rad51—a homolog of bacterial RecA. Notably, Rad51/RecA is known to form a right-handed helical ﬁlament on singlestranded (ss) DNA and carry out homology search and strand exchange during the recombination process. Since the Rad51-mediated exchange event is inefﬁcient in vitro, other factors must be required to promote this reaction, including Rad52-mediated formation of a ring-like structure on the ssDNA, which facilitates the

proteins,” says Takagi. Also interested in structural biology is Akira Shinohara at the Laboratory of Genome and Chromosome Functions, who is investigating the molecular mechanisms of homologous recombination using molecular, genetic, and biochemical methods.

binding of Rad51 to the DNA. “We are also studying recombination and chromosome dynamics during meiosis—a type of cell division that occurs during the production of gametes [eggs and sperm],” says Shinohara. Recent work has implicated the Csm4 protein in the speciﬁc movement of DNA ends during recombination. “Our results imply that chromosome movement promotes a variety of biochemical reactions on chromosomes including meiotic recombination,” says Shinohara. “More recent experiments indicate that physical forces may be regulating chromosome functions—that is, physics may regulate biochemistry.”

Akira Shinohara

MORE INFO Institute for Protein Research, Osaka University www.protein.osaka-u.ac.jp

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Excellence in Interdisciplinary Research and Education Osaka University’s Graduate School of Frontier Biosciences offers a stimulating environment for cutting-edge interdisciplinary research on biological system dynamics.

chemistry. “We welcome students from overseas with appropriate TOEFL [test of English as a foreign language] and GRE [Graduate Record Examination] scores,” says Namba. In addition, the FBS holds annual symposia, biannual summer schools, and annual retreats— which are open to overseas participants—and supports collaborative visits of students and postdocs to universities and research institutes in the United States, Europe, and Asia. Namba is a biophysicist specializing in biological nanomachines. “We analyze the structure and dynamics of macromolecular assemblies using techniques including electron

MORE INFO Graduate School of Frontier Biosciences, Osaka University www.fbs.osaka-u.ac.jp

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Keiichi Namba

Structure of F-actin and Flagellar Motor

cryomicroscopy and X-ray diffraction,” says Namba. “Recently, we used cryomicroscopy to successfully visualize the structure of F-actin in a few days.” F-actin is the helical polymer of a protein, actin, which is an essential component of muscle ﬁbers and cell cytoskeletons. These results offer a deeper understanding of the cellular mechanisms governing life. Namba is also investigating the source of energy for the torque of the bacterial ‘ﬂagellar motor’—a rotary nanomachine composed of assemblies of proteins—and how the tiny helical propellers connected to the rotary motor propel bacteria through liquids. Toshio Yanagida, who is the leader of the Soft Biosystems Group at Osaka University and a pioneer of single molecule imaging, is now focused on the dynamics of living cells.

and RIKEN,” says Yanagida. “We will focus on the basic elements regulating biological systems while developing new experimental and theoretical techniques. The infrastructure for the project includes a new 7000 square meter building, 15 principle investigators, 60 postdoctoral researchers, 20 technicians, and more than 20 graduate students. We aim to double this manpower by 2013,” says Yanagida. The other initiative is the Brain Information Transmission Interdisciplinary Research Center, to be launched in 2012 in collaboration with the National Institute for Communication Technology (NICT, in Hyogo) and the Advanced Telecommunications Research Laboratory (ATR, in Kyoto). “This project focuses on how the brain processes internal and external information, and

“Molecular machines use ﬂuctuations such as

how to transmit this data to a computer. This could revolutionize human-machine communication and network system technologies,” emphasizes Yanagida. The project is based on three main pillars: heart-to-heart science (HHS), brain–machine interface (BMI), and brain-function installed information network (BFI). The aim of these collaborations is to analyze the ﬂow of information, energy, and materials within living organisms and interface them with an external control system. ”We will start with single molecules and work up towards the whole brain,” explains Yanagida. “Ultimately, such studies are expected to

Brownian motion,” says Yanagida. “A deeper understanding of how living cells use such ﬂuctuations—or yuragi—to process enormous amounts of information will enable the design of more energy-efﬁcient and robust machines.” In 2011 and 2012, Yanagida is launching two major interdisciplinary projects. One is the Quantitative and Computational Biology Center (QBiC), which begins in April 2011 and aims to develop a comprehensive understanding of biological system dynamics. “This is a multi-institute project with Osaka University

Toshio Yanagida

lead to the construction of highly energy-efﬁcient machines and advanced medical diagnostics.”

CREDIT: (TOP RIGHT) PROFESSOR KEIICHI NAMBA, OSAKA UNIVERSITY

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he Graduate School of Frontier Biosciences (FBS) at Osaka University was established in 2002. “Our mission is to educate graduate students in an interdisciplinary environment,” says Keiichi Namba, dean of FBS. “Our 70 or so faculty members are from diverse backgrounds including mathematics, physics, chemistry, medicine, and information science. And our approximately 250 graduate students interact with these specialists. It’s a unique graduate school, and very popular with students from both Japan and overseas.” Doctoral students at FBS receive support from the prestigious Global Center of Excellence (GCOE) program running from 2007 to 2011. As part of this program, students spend one month doing their research in the laboratory of another research group. “This enables students to gain ﬁrsthand experience in interdisciplinary research,” says Namba. The graduate school entrance examination at FBS is ﬂexible, offering applicants the choice of sitting exams in physics/mathematics, biology, or

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Pharmaceutical Approach to Alleviating Human Disorders Scientists at the Graduate School and School of Pharmaceutical Sciences of Osaka University are dedicated to investigating a range of pharmaceutical and health sciences, including cancer and psychiatric disorders. Here, we talk to two prominent members devoted to translational research.

K

azutake Tsujikawa is known for the discovery of prostate cancer antigen-1 (PCA-1), a gene highly expressed in prostate cancer and pancreatic cancer. “Prostate cancer is one of the biggest killers in the world,” says Tsujikawa. “We are using mouse models to study whether this gene can act as a molecular marker, a biomarker, for this type of cancer, which may allow early diagnosis.” Currently the biological roles of this novel gene remain unclear. Tsujikawa and colleagues found that in vitro silencing of PCA-1 induced apoptosis in cancer cell lines and signiﬁcantly reduced tumor formation in vivo. They concluded that PCA-1 may serve as a promising molecule target for prostate cancer and pancreatic cancer therapy. Tsujikawa has recently developed an enzymebased assay for PCA-1 activity to aid in screening possible therapeutic agents. “PCA-1 is a member of the hABH [human AlkB homologs] family,” says Tsujikawa. “We have also discovered that the expression of hABH family proteins may correlate with speciﬁc cancers. The hABH family of molecules could be targets for cancer therapy. We are working on a joint industry-academia-government project to clarify the cancer mechanisms for translational research.” This research is being conducted in collaboration with the medical school of Osaka University, Nara Medical University, Kagoshima University, and Hyogo University of Health Sciences. Tsujikawa is also pursuing research as part of the Ministry of Education, Culture, Sports, Science and Technology's Knowledge Cluster Initiative (second stage) and the Program for Promotion of Fundamental Sciences in Health Sciences of the National Institute of Biological Innovation. Tsujikawa is also working on calcitonin generelated peptide (CGRP) that is released from sensory nerve endings. CGRP is known to play a central role in migraine and diseases such as heart failure. “We have generated mice deﬁcient

Kazutake Tsujikawa in RAMP-1 [receptor activity modifying protein 1], a speciﬁc subunit of CGRP receptors. Interestingly, RAMP-1-deﬁcient mice showed recovery from experimental allergic encephalomyelitis. The result indicates that CGRP plays a crucial role in controlling the body’s immune system,” says Tsujikawa. The research is supported by the Japan Chemical Industry Association as part of the International Council of Chemical Associations Long-Range Research Initiative. Hitoshi Hashimoto is at the Laboratories of Molecular Neuropharmacology where he investigates the molecular mechanisms for psychiatric disorders and drugs for their treatment. “We are using molecular biology and animal models to identify new molecular targets for the development of drugs,” says Hashimoto. Hashimoto is investigating the molecular pharmacology of pituitary adenylate cyclase-activating polypeptide (PACAP); identifying and evaluating hyperplasia-associated gene of beta cell (HPGB), regenerating islet-derived protein 3 (RegIII), and prostaglandin receptor CRTH2 as molecular targets for drug discovery; and developing methods to differentiate embryonic stem cells into neurons. “In 1993, we successfully cloned the cDNA of MRI images of diminished tumor growth in PCA1-knockdown prostate cancer cells in a xenograft system (left, control; right, PCA-1 knockdown). The arrow indicates a tumor.

Hitoshi Hashimoto the PACAP-selective receptor, PAC1, in rat brain,” says Hashimoto. “Since then we have investigated the role of PACAP in the nervous system.” Hashimoto’s research includes in vivo studies on PACAP signaling, which led to the development of mice lacking the PAC1 receptor gene and/or PACAP gene and a transgenic mouse line overexpressing the PACAP gene. Notably, mice lacking PACAP (PACAP-/-) exhibited behavioral abnormalities, including explosive jumping, suggesting that PACAP has a role in the regulation of psychomotor functions. Furthermore, clinical genetic experiments showed variants of the genes encoding PACAP and PAC1 receptor to be associated with schizophrenia, implicating PACAP-mediated signaling pathways in psychiatric disorders. Recently, Hashimoto used PACAP-deﬁcient mice as a model to study how light intensity affects resetting of the circadian clock when light cycles are altered. Intriguingly, PACAP-deﬁcient mice also showed depression-like behavior, making them potentially useful for studies on the mechanistic association between an altered biological clock and depression. Hashimoto’s research has an international perspective. “I collaborate with David Vaudry at INSERM, France; Herbert Meltzer at the Vanderbilt University Psychiatric Hospital; Dóra Reglödi at University of Pécs, Hungary; and many colleagues elsewhere.”

MORE INFO Graduate School and School of Pharmaceutical Sciences, Osaka University www.phs.osaka-u.ac.jp/en/

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Breaking the Mold: Changing Traditional Views of Dentistry The Graduate School of Dentistry at Osaka University plans to introduce ambitious new programs for a molecular biology-based approach to training dentists in the 21st century.

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hat is a dentist?” asks Toshiyuki Yoneda, dean of the Graduate School of Dentistry at Osaka University. “I am afraid that dentistry is still viewed as a surgical-based vocation involving ﬁlling cavities and pulling teeth. We want to destroy this antiquated view because we believe that the future skill sets of dentists must cross many disciplines, including pharmacology, law, and even the arts.” The heart of the problem in Japan is the rapidly aging population. “The majority of patients in Japan are middle-aged or above, and traditional ailments such as tooth decay are no longer the main issues for dentists,” says Yoneda. “For example, periodontal disease in an elderly patient can be linked to other diseases such as diabetes and cardiovascular disease, so dentists must be able to understand these other systemic disorders. We must revise the education curricula for dental schools to include a deeper understanding of molecular biology and pathophysiology.” Yoneda also says that he wants Osaka University—a comprehensive university with schools of medicine, pure sciences, engineering, and arts—to introduce new curricula for training dentists. As a ﬁrst step, Yoneda has submitted a proposal to the Ministry of Education, Culture, Sports, Science and Technology to establish a program on ‘Life Innovation’. The six-year program would focus on intractable diseases of the mouth and their relationship with other diseases of the body. Distinctive features of the program include: the awarding of a Master of Oral Diseases to graduates of a post-residence course,

MORE INFO Graduate School of Dentistry, Osaka University www.dent.osaka-u.ac.jp/english

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Graduate School of Dentistry introduction of special lectures on ‘biodentistry,’ creation of a personal genome database using ‘oral bioinformatics,’ and the establishment of an ‘intractable diseases international station.’ In addition to these plans, Yoneda and his colleagues are also involved in cutting-edge research. Recent highlights include studies on bone biology in which the researchers discovered a link between stress signals in the endoplasmic reticulum (ER)—an important cellular compartment in which protein folding occurs—and activation of vesicular trafﬁcking out of the ER. In an equally high-impact contribution, Yoneda’s group conducted experiments in an attempt to quantify pain. “Pain is a subjective

Approach for Periodontal Tissue Regeneration results showing that teeth weakened by periodontal problems can be made stronger by drug therapy using ﬁbroblast growth factor-2 [FGF-2].” At the Cell Processing Center, the group led by Shinya Murakami, head of the Department of Periodontology, is extracting fat cells to generate periodontal tissue. “This is the ﬁrst such center in a dental school in Japan and, although we are still in the early stages, we have had encouraging results that indicate the possibility of producing periodontal tissue from fat cells,” says Yoneda. The Osaka University Graduate School of Dentistry holds many joint symposia with universities and institutes in the United States, United Kingdom, Korea, and Thailand. Osaka

experience, but our experiments show promise students can go to the United States for two– as a means of producing an objective view,” says month ‘home stays’ to improve their English. Yoneda. Experiments on inﬂammatory pain in The dental school holds regular community rats showed the importance of proton sensing outreach forums to emphasize the importance receptors, such as TRPV1, in the transmission of of dentistry, and its evolution, for the young pain. When activated, and old. Surgery at the Translational TRPV1 appears to Osaka University was Research Center upregulate calcitonin the ﬁrst governmentgene-related peptide run comprehensive (CGRP) expression, a university to establish a scenario associated school of dentistry. “We with inﬂammatory pioneered dentistry in pain. Japan,” says Yoneda. “We have also been “I am conﬁdent that our working on using new initiatives based drugs to treat oral on a molecular biology diseases, instead of approach will inspire periodontal surgery,” education and research says Yoneda. “We at dental schools in have convincing Japan and globally.”

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Evolution from Molecular Biology to Atomic Biology Seiki Kuramitsu believes that thermophilic bacteria, which live in water temperatures close to boiling, offer an excellent model for creating a new system biological ﬁ eld of life sciences that predicts biological function through atomic-level analysis.

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n spite of the tremendous advances in the life sciences, it is still not possible to accurately predict the results of medical treatment,” says Seiki Kuramitsu, professor in the Department of Biological Sciences, Graduate School of Science, Osaka University. This department was established in 1949 for education and research in the biological sciences, based on advances in science at the boundary of chemistry and physics. “I believe that it is necessary to construct a new ﬁeld of life science whereby we can predict the response of an organism based on chemistry.” Kuramitsu's laboratory and many research groups are investigating whether biological phenomena underlying different environmental stimuli can be deﬁned in single cells by studying the 3D structure and function of all the intracellular molecules at an atomic level.

Seiki Kuramitsu “Thermus thermophilus HB8, an extremely thermophilic bacterium that thrives at 80ºC, is an excellent model for structural and functional studies. Its constituent proteins are stable, and most of its approximately 2,200 genes are common to many other organisms, including humans,” says Kuramitsu.

The four aspects required for understanding the whole cell system are: (1) structural genomics, (2) functional genomics, (3) functional analysis of each system, and (4) simulation and prediction of all biological phenomena in the cell. “The goal of our so-called Whole Cell Project is prediction, as opposed to explanation,” says Kuramitsu. “I want to move from molecular biology to atomic resolution biology.” “This is a human project, not just an Osaka project,” adds Kuramitsu. “Also, this is not a subject just for medical school researchers; it is an interdisciplinary subject. We welcome the world’s scientists to join us.”

MORE INFO The Whole Cell Project www.thermus.org/e_index.htm

Cognitive Neuroscience Robotics at Osaka Osaka University launches an innovative program in cognitive neuroscience robotics, supporting education and development of technology at the human-machine interface.

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he goal of the Global Center of Excellence Program (GCOE) based on cognitive neuroscience robotics is to develop new information and robot technology systems to provide information and services based on cognitive neuroscience. “This highly competitive ﬁve-year GCOE program was launched in 2009,” says Hiroshi Ishiguro, leader of the program. “I believe that we were awarded the project because of our interdisciplinary approach towards cognitive science and robotics.” The project involves experts from robotics, neuroscience, and human sciences. “Cognitive neuroscience is related to meta-level brain functions such as memory and reasoning,” says Ishiguro. “Conventional information and robot technology, IRT, has produced a more conve-

safe and adaptable IRT systems.” One of the goals of the program is to establish a new graduate school department for students to study cognitive neuroscience robotics. “There are many hurdles to overcome to achieve this goal, with the highest being the need to scrap old and low-performing research areas,” says Ishiguro. “The big question is whether Osaka University can do this.” The research and educational activities of the GCOE program are coordinated with the Advanced Telecommunications Research Institute International (in Kyoto) and the National Institute of Information and Communications Technology (in Hyogo) in Japan, and institutes overseas including the Italian Institute of Technology and Bielefeld University in Germany. “We welcome doctoral candidates from

nient world but without regard for effects on our cognitive functions. We want to establish new paradigms based on cognitive neuroscience for

overseas to join us to create human-friendly robots based on cognitive neuroscience,” says Ishiguro.

Professor Hiroshi Ishiguro and his Android “We welcome doctoral candidates from overseas to join us to create human-friendly robots based on cognitive neuroscience.”

MORE INFO Global COE Program “Center of Human-friendly Robotics Based on Cognitive Neuroscience” www.gcoe-cnr.osaka-u.ac.jp/english/

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OSAK A I N F O CU S: Integrating Research and Business

Innovative Approaches to Neuroscience and Microﬂuidics AIST Kansai continues to excel in producing ideas for a sustainable society of the future.

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he National Institute of Advanced Industrial Science and Technology (AIST) is one of Japan’s largest public research organizations with about

3,000 researchers working at nine locations throughout Japan. Takahisa Taguchi is the director of AIST Kansai. “Our activities here are focused on three

Hidenori Nagai

Microﬂuidic Device for High-Speed PCR

areas: energy, life sciences, and information communications technology,” says Taguchi. “One of

forces to move droplets of the sample through

that BDNF induces de novo cholesterol bio-

our main goals is to commercialize our research for the beneﬁt of society.”

the various zones in the microcapillary network. “We successfully used this device to detect

synthesis in cultured cortical and hippocampal neurons, thereby indicating a role for BDNF in

Researcher Hidenori Nagai is focusing on creating compact analytical devices using

anthrax down to a concentration of 10 cells per microliter after a ﬁve-minute ampliﬁcation,” says

cholesterol metabolism. “Depression is a serious illness affecting mil-

microfabrication technology. “I am working on technology for the rapid and highly sensitive

Nagai. Masami Kojima is studying the effect of

lions of people worldwide, and BDNF may have a role to play,” says Kojima. “So, recently I have

detection of target genes,” explains Nagai. “I have developed a new method for detecting

brain-derived neurotrophic factor (BDNF) on brain function. “In 2003, I was with the group at

been working with clinicians on a national project [JST/CREST] on mental illnesses—including

DNA based on rapid ampliﬁcation.” Compared with ampliﬁcation by polymerase chain reaction

the National Institutes of Health [in the United States] that identiﬁed BDNF as playing a role

depression—focused on molecular dysfunction of BDNF.”

(PCR), which can take several hours, Nagai’s approach enables DNA ampliﬁcation in just

in memory formation,” says Kojima. “We found that a common polymorphism [Val66Met] in

Kojima works with many groups overseas, including those in the United States and China.

two to ﬁve minutes. “This is the fastest method reported to date,” says Nagai. “Our compact and fast approach will enable point of care testing [POCT] on a tailor-made basis.” Nagai is collaborating with academia to develop a commercial POCT system for genome assays. The new high-speed PCR system consists of a pressure sensitive polyoleﬁn ﬁlm and cyclooleﬁn polymer substrate containing microﬂuidic channels. During operation, the device is placed onto heated aluminum blocks maintained at 95°C (denaturation), 72°C (extension), and 55°C (annealing). When the sample is injected into the device, differences in vapor pressure due to the thermal gradient generate sufﬁcient driving

the BDNF gene affects human memory and the secretion mechanism of the growth factor.” More recently, Kojima and colleagues reported that BDNF regulates cholesterol synthesis for synapse development. Speciﬁcally, they showed

“One of the major goals of AIST is to ﬁnd applications for our research,” says Kojima. “I often discuss my work with colleagues at AIST and at other institutes to achieve this goal, which would be difﬁcult to accomplish by myself.”

MORE INFO National Institute of Advanced Industrial Science and Technology (AIST), Kansai www.aist.go.jp/index_en.html

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Masami Kojima

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Cancer Research at Osaka Prefecture University Five contrasting and innovative approaches to diagnosing and curing cancer.

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saka Prefecture University (OPU) is one of the largest public universities in Japan. Established about 130 years ago to train veterinarians, the university employs 707 faculty who teach the approximately 8,000 students—164 from overseas—attending classes at three campuses: Nakamozu, Habikino, and Rinku. Here, we introduce the research activities of ﬁve members of the OPU faculty who are actively involved in the Osaka Prefecture University Bio Strategy Committee on the development of bioindustries in the Osaka Bay area. Kikuya Sugiura, a veterinarian at the School of Life and Environmental Sciences explains his research: “I am developing immunology-based therapy for tumors using dendritic cells [DC]. The function of DC is to present antigens to T lymphocytes, activating the immune response. We are obtaining these cells in culture by inducing the differentiation of monocytes isolated from blood, which can then be used to ﬁght cancers.”

development of a similar treatment in humans. Ikuo Fujii, based at the Graduate School of Science, is an active member of the Osaka Prefecture Bio Strategy Committee and is committed to making Osaka a center for life sciences. Fujii has recreated in the laboratory the process by which the immune system makes antibodies and is developing low molecular weight peptides known as microantibodies. This will allow him to “contribute to molecular–targeted medicine and advanced medical diagnostics,” says Fujii. In particular, Fujii is addressing problems associated with antibody medicine. These problems include antigenicity, manufacturing costs, and cell membrane permeability issues. “I am reproducing the functions of antibodies using small peptides,” says Fujii. “I have developed a unique

Using dendritic cells (DC) developed from peripheral

This method alone, however, is not effective in completely destroying tumors. In a novel approach, Sugiura injects interferon γ (IFNγ)—a

phage-display peptide library of peptide surfaces, which are stable against proteases in the blood, show high binding activity and speciﬁcity,

tumor cells using a gene delivery system (GDS).

potent inducer of the T lymphocyte-mediated immune response—along with DC into dogs with naturally occurring tumors, brought to the OPU veterinary school for cancer therapy. “This treatment induced complete healing in the dogs,” says Sugiura. “They are an almost perfect model for these experiments because breast cancer, lymphoma, and osteosarcoma occur spontaneously. So if the treatment is effective in dogs, there is a very high probability that it will work in humans.” Sugiura is also testing drug delivery directly into the tumor microenvironment by introducing cytokine genes into tumor cells using a gene

and are highly membrane permeable.” These development of human cancer therapies. exciting results are expected to contribute to the development of the MRI-detectable dendron thermosensitive polymer biocompatible polymer next generation of antibody-based therapeutics and diagnostics for the detection and treatment of cancer. The precise and reproducible delivery of bioactive molecules to speciﬁc targets is essential for the development of medical Detection of liposome treatment protocols. Chemist Kenji accumulation with MRI Kono is developing biofunctional nanomaterials for the delivery of drugs to treat cancer. “The development of accurate drug

delivery system. Applying IFNγ and DC together appears to create a strong synergistic immune response against tumors, providing hope for the

Kikuya Sugiura

delivery technology is critical for personalized chemotherapy,” continued »

blood monocytes (Mo) by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4, DC-based immunotherapy has provoked immune responses against cancer in human patients. However, improvements are necessary in order for the immune response to be sufﬁcient for a satisfactory clinical outcome. Interferongamma (IFNγ), a potent inducer for DC maturation and T helper type 1 response, is an important tool for achieving such improvements. Inoculation of canine IFNγ with DC into the microenvironment of canine tumors signiﬁcantly improved the clinical outcome, including complete remission of mammary carcinomas. For obtaining local-limiting and longlasting effects of IFNγ, methods are being developed for in vivo transfection of the canine IFNγ gene into

Results from the study of dogs with common human tumor types may provide useful information for the

Detection of liposome accumulation of mouse tumor with MRI

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OSAK A I N F O CU S: Integrating Research and Business

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“We are aiming to lead research in the ﬁeld of high-precision, liposome- and dendrimer-based drug delivery.”

says Kono. “We are aiming to lead research in the ﬁeld of high-precision, liposome- and dendrimer-based drug delivery.” In one set of experiments, Kono and colleagues loaded anticancer drugs into temperature-sensitive 100 nm-diameter liposome capsules that are designed to break down at elevated temperatures. They injected the capsules into a tumor, applied an RF microwave to locally heat the capsule to about 45°C, and monitored the delivery of the anticancer drugs using magnetic resonance imaging. “Having a way to visibly monitor the delivery, release, and spread of drugs is important for the development of safe procedures,” says Kono. Other drug delivery systems developed by Kono and his team include nanoparticles made up of dendrimers, artiﬁcially-created branched molecules, that can respond to stimuli by virtue of surface modiﬁcations or loading of gold nanoparticles. These can be used for stimulus-induced drug release or heat generation upon laser light irradiation. Kono also created drug-carrying liposomes composed of a new type of functional, dendron-bearing lipid. Boron neutron capture therapy (BNCT) is a high-energy physics approach to cancer therapy that combines two treatments that individually

be used in positron emission tomography imaging of BNCT-treated tumors. “We have set up a consortium to make the Osaka Bay Area an international center for BNCT research and treatment,” says Kirihata. “The new, compact, and relatively inexpensive accelerator for BNCT at Kyoto University is a major step towards the proliferation of BNCT treatment.” The drug development process is both time consuming and expensive, with many compounds being shelved during early testing due to difﬁculties in handling, and in many cases because the compound is simply not sufﬁciently soluble in water. These unused compounds could be potentially useful therapies if modiﬁed to improve solubility and therefore more easily

are virtually harmless, but together can be lethal. A beam of low-energy (or thermal) neutrons directed at a tumor interacts with boron-10 isotopes injected into the patient and targeted to tumor cells using specialized antibodies. “This interaction produces boron-11 particles, which rapidly decay to produce alpha particles and lithium-7 ions,” says OPU’s Mitsunori Kirihata. “This reaction is local and destroys cancer cells without harming adjacent healthy ones.” Kirihata has developed a method for the mass production of highly concentrated boron-10 compounds (in collaboration with locally-based Stella Chemifa Corporation), as well as the synthesis of boron-10-antibody compounds, which can be injected into patients and directed to speciﬁc tumor sites. Kirihata is also investigating further applications of BNCT by developing a new method for synthesizing ﬂuoride-labeled boronophenylalanine that can

School of Life and Environmental Sciences has developed a new method of drug delivery that makes use of previously abandoned therapeutic compounds. “I spent many years in the pharmaceuticals industry and am aware of the vast number of unused compounds,” says Inui. “I have successfully loaded low-solubility drugs inside L-PGDS [lipocalin-type prostaglandin D synthase] carrier cavities. I call this a drug delivery system based on intelligent-type artiﬁcial proteins.” The drug molecules exhibit selective molecular biorecognition and can be used for targeting cancer tissues. Inui used mass spectroscopy, SAXS (small-angle X-ray scattering), and NMR to identify that the drug was indeed inside and bound to the carrier capsule. “This is a world ﬁrst, and I expect these results will have a tremendous effect on drug discovery,” says Inui.

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Ikuo Fujii

Takashi Inui

Kenji Kono

reach target cells. Takashi Inui at the Graduate

Mitsunori Kirihata

MORE INFO Osaka Prefecture University www.osakafu-u.ac.jp/english/index.html

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LIFE SCIENCE TECHNOLOGIES

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NEW PRODUCTS PRODUCTS: CELL/TISSUE CULTURE MICROBIOREACTOR SYSTEM The new version of the ambr workstation automates the operation of 48 disposable microbioreactors and closely replicates the characteristics of benchtop bioreactors at microscale (10 ml to 15 ml working volume). The 48 microbioreactors are arranged in four culture stations containing sets of 12, each with a miniature internal impeller, as well as individual closed loop control of dissolved O2 and pH and a supply of gases. Each culture station has independent stirring speed and temperature control, allowing scientists to test up to four different temperature and stir speeds simultaneously, making sophisticated experimental designs much simpler to carry out with just one real-time parallel run. This new ambr system controls culture setup and inoculation, the addition of alkalis and feeds, and culture sampling for cell count and analysis such as antibody titre. The system is compact enough to ﬁt in a standard laboratory biosafety cabinet. The Automation Partnership For info: 302-478-9060

www.automationpartnership.com

CELL GROWTH SOLUTIONS

GLUCOSE SUPPLY TABLET

ATCC Normal Human Primary Adipose-Derived Mesenchymal Stem Cells are cryopreserved in the second passage to ensure the highest viability and plating efﬁciency and are thoroughly tested for sample purity. They are performance tested together with media, growth supplements, and reagents to guarantee reliability as part of ATCC Primary Cell Solutions, a system of matched components designed to maximize growth, maximize functionality, and maintain normal morphology for speciﬁc cell types. The complete cell-speciﬁc ATCC Primary Cell Solutions growth system (consisting of the Mesenchymal Stem Cell Basal Medium and Growth Kit) creates the ideal cell system culture environment for propagation in low serum (2%) conditions and subsequent differentiation. Using the complete ATCC Primary Cell Solutions system, researchers will be able to produce stem cell cultures with functional expression of relevant biomarkers; superior growth and proliferation; normal morphology; and multipotent, lineage-speciﬁc differentiation. ATCC

The EnPresso Tablet Cultivation Set delivers a slow, controlled release of glucose into culture medium. The EnBase Technology provides an enzymatic-controlled glucose supply in a fed-batch type manner, which allows for an extended growth period and promotes higher ﬁnal cell density. The efﬁcient EnBase technology has become even easier to use with the pre-sterilized EnPresso tablet. The longer shelf-life tablet can be added directly to sterile water and this instant microbial media is ready for inoculation in a shaking incubator. EnBase technology allows the efﬁcient cell cultivation of recombinant bacteria in small-scale experiments. With the increased yield and ease-of-use obtained in the EnBase media, many typical experiments can be scaled down to increase laboratory testing capacity and effectiveness. EnBase can be used in microwell plates, deep well plates, shake ﬂasks, and even in small-scale and disposable bioreactors. BioSilta Oy

For info: 800-638-6597

NANOFIBER CULTURE DISHES

www.atcc.org

For info: +358-(0)-8-553-2361

www.biosilta.com

Driven to meet the changing needs of the market, Shefﬁeld BioScience has introduced two high-quality, cost-effective animal component-free (ACF) recombinant supplements for cell culture media. Shefﬁeld rAlbumin ACF is a replacement for HSA and BSA, and can be used to reduce/replace serum when used as a standalone supplement or when combined with other components, such as plant-derived protein hydrolysates. Shefﬁeld rAprotinin ACF is a recombinant form of traditional bovine-derived aprotinin that is produced using exclusive technology. Aprotinin, also known as pancreatic trypsin inhibitor, acts as a serine protease inhibitor that forms stable complexes with and blocks the active sites of enzymes at near neutral pH conditions. Shefﬁeld Bio-Science

New high throughput nanoﬁber culture dishes provide an environment that closely mimics the physical structure found in vivo. These 3D nanoﬁber substrates allow more accurate modeling of cell migration while still allowing for the critical need of highresolution live-cell imaging. Nanoﬁber Solutions develops synthetic nanoﬁber multiwell plates for cell culture applications ranging from drug discovery to stem cell expansion. The scaffold is composed of synthetic polymer nanoﬁbers randomly arranged or aligned into parallel nanoﬁbers which are placed on the bottom of standard cell culture dishes. This gives a 3D surface for cells to grow and attach to and allows for researchers to model metastasis and cell mobility in vitro. Investigators can coat the ﬁbers with different biological coatings of interest (i.e., collagen and ﬁbronectin), just like existing culture dishes, or upregulate/downregulate genes to investigate different cellular mechanisms. Nanoﬁber Solutions

For info: 800-833-8308

For info: 614-975-6646

ANIMAL COMPONENT-FREE RECOMBINANT SUPPLEMENTS

www.shefﬁeldbioscience.com

www.nanoﬁbersolutions.com

Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.dtl for more information. Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are featured in this space. Emphasis is given to purpose, chief characteristics, and availability of products and materials. Endorsement by Science or AAAS of any products or materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.

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VOL 331

SCIENCE

www.sciencemag.org/products

AAAS is here – promoting universal science literacy. In 1985, AAAS founded Project 2061 with the goal of helping all Americans become literate in science, mathematics, and technology. With its landmark publications Science for All Americans and Benchmarks for Science Literacy, Project 2061 set out recommendations for what all students should know and be able to do in science, mathematics, and technology by the time they graduate from high school. Today, many of the state standards in the United States have drawn their content from Project 2061. Every day Project 2061 staff use their expertise as teachers, researchers, and scientists to evaluate textbooks and assessments, create conceptual strand maps for educators, produce groundbreaking research and innovative books, CD-ROMs, and professional development workshops for educators, all in the service of achieving our goal of universal science literacy. As a AAAS member, your dues help support Project 2061 as it works to improve science education. If you are not yet a AAAS member, join us. Together we can make a difference.

To learn more, visit aaas.org/plusyou/project2061

Making improvements in life possible

QIAGEN is making improvements in life possible in: ■ Companion diagnostics ■ Molecular diagnostics ■ Pharma ■ Animal and veterinary testing ■ Food testing CORPORATE0211S1WW

■ Genetic identity and forensics ■ Life science research Visit www.qiagen.com for more information.

Sample & Assay Technologies