Scendgfrytzyau

CONTENTS Volume 330 Issue 6009
SPECIAL SECTION
Metabolism
INTRODUCTION 1344 Autophagy and Metabolism
1337 Metabolism Is Not Boring J. D. Rabinowitz and E. White
1349 Circadian Integration of Metabolism
PERSPECTIVE and Energetics
1338 On Getting There from Here J. Bass and J. S. Takahashi
S. L. McKnight 1355 Manufacturing Molecules Through
Metabolic Engineering
REVIEWS J. D. Keasling
1340 The Control of the Metabolic Switch in
>> Science Translational Medicine p. 1281 and Science page 1306
Cancers by Oncogenes and Tumor
Signaling at www.sciencemag.org/special/metabolism/
Suppressor Genes
A. J. Levine and A. M. Puzio-Kuter
EDITORIAL LETTERS PERSPECTIVES

1287 Policy-Making Needs Science 1316 Fishing for Data in the Ross Sea 1326 Cryptic Links in the Ocean
Bruce Alberts L. K. Blight et al. A. Teske
Assisted Colonization: >> Report p. 1375
NEWS OF THE WEEK Move Ahead with Models 1327 The DNA Damage Road Map
1298 A Powerful and Perplexing M. R. Stanley Price N. Friedman and M. Schuldiner
New HIV Prevention Tool Assisted Colonization: >> Report p. 1385
1301 New HIV Infections Drop, Facilitate Migration First 1328 Dynamic Metabolons
But Treatment Demands Rise J. B. Ruhl B. L. Møller
1302 What Poison? Bacterium Uses Arsenic Assisted Colonization: 1330 Opening the Cellular Poison Cabinet
to Build DNA and Other Molecules Protect Managed Forests S. J. Martin
>> Science Express Research Article J. F. Fernández-Manjarrés and L. Tschanz >> Report p. 1390
by F. Wolfe-Simon et al.
1318 CORRECTIONS AND CLARIFICATIONS 1331 Dedicated to Memory?
1303 From Science’s Online Daily News Site H. Eichenbaum
1304 Panel Explores New Funding Pact BOOKS ET AL. >> Report p. 1408
With Washington 1320 Brain Storm 1332 High-Temperature Rubber Made
1305 With Money Tight, White House Panel R. M. Jordan-Young; from Carbon Nanotubes
Offers New Path to Energy Research Delusions of Gender Y. Gogotsi
C. Fine, reviewed by D. F. Halpern >> Report p. 1364
1305 From the Science Policy Blog
1322 Photograph 51
GE PRIZE ESSAY
NEWS FOCUS A. Ziegler, reviewed by B. Juncosa
1334 A New Approach to Fluorescence
1306 SCIENCE IN BRAZIL
POLICY FORUM Microscopy
Brazilian Science: Riding a Gusher
M. Bates
Tapping a Deep, ‘Pre-Salt’ Bounty 1324 Developing Health Workforce Capacity
Talented But Underfunded: in Africa
Brazil’s Future Scientists F. S. Collins et al.
CONTENTS continued >>
>> Science Podcast
COVER DEPARTMENTS
Autophagy, the process by which cells digest their own components, 1283 This Week in Science
takes place in vesicles called autophagosomes (white membrane 1289 Editors’ Choice
structures), the size of which are determined by the amount of 1292 Science Staff
a protein called Atg8. Basal amounts of Atg8 (small membrane- 1297 Random Samples
associated spheres) generate a small vesicle (middle left), whereas 1419 New Products
larger amounts cause formation of a full-fledged autophagosome
1420 Science Careers
(upper right). Autophagy provides fuel for cellular metabolism,
the topic of the special section beginning on page 1337.
Painting: David S. Goodsell; Scientific Design: Zhou Du
and Daniel J. Klionsky
www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1277

Published by AAAS
CONTENTS
RESEARCH ARTICLE 1390 BID, BIM, and PUMA Are Essential for
1359 How Learning to Read Changes Activation of the BAX- and BAK-Dependent
the Cortical Networks for Vision Cell Death Program
and Language D. Ren et al.
S. Dehaene et al. Proapoptotic proteins act directly on
Reading changes the mind. mitochondrial “gatekeeper” proteins
to initiate apoptotic events during
mouse development.
REPORTS >> Perspective p. 1330
1364 Carbon Nanotubes with 1393 Arabidopsis Type I Metacaspases Control
Temperature-Invariant Viscoelasticity Cell Death
from –196° to 1000°C N. S. Coll et al.
M. Xu et al. An ancient link between cell death control
A dense carbon-nanotube network shows and innate immune receptor function
nearly constant viscoelastic properties over has been discovered in plants.
an exceptionally wide temperature range. pages 1332 & 1364
>> Perspective p. 1332 1397 An Antagonistic Pair of FT Homologs
Mediates the Control of Flowering Time
1368 Video-Rate Molecular Imaging in Vivo in Sugar Beet
with Stimulated Raman Scattering P. A. Pin et al.
B. G. Saar et al. A homolog of a flowering time gene has
Raman spectra can be acquired rapidly evolved a flowering repression function,
from samples that otherwise would scatter affecting the seasonal cold response in beets.
the usable signal.
>> Science Podcast 1400 Alleviating Neuropathic Pain
Hypersensitivity by Inhibiting PKMζ
1371 The Role of Particle Morphology in in the Anterior Cingulate Cortex
Interfacial Energy Transfer in CdSe/CdS X.-Y. Li et al.
Heterostructure Nanocrystals Nerve injury increases the activity of an
N. J. Borys et al. enzyme in the brain and contributes to
Single-particle spectroscopy suggests that chronic pain–related cortical sensitization.
non-uniform geometries favor efficient
charge separation for light harvesting. 1404 Micro-Optical Sectioning Tomography
to Obtain a High-Resolution Atlas
1375 A Cryptic Sulfur Cycle in of the Mouse Brain
Oxygen-Minimum–Zone Waters A. Li et al.
off the Chilean Coast Acquisition of light microscopic data at pages 1326 & 1375
D. E. Canfield et al. 1-micrometer resolution for an entire
Bacterial sulfur reduction and oxidation mouse brain has been developed.
accompanies nitrogen cycling where
oxygen levels at depth are low. 1408 Paradoxical False Memory for Objects
PHOTO CREDIT (BOTTOM): JEAN-MARIE BOUQUET AND JIRI SLAMA/SARS INTERNATIONAL CENTRE, BERGEN, NORWAY
>> Perspective p. 1326 After Brain Damage

S. M. McTighe et al.
1378 Dynamical Response of the Tropical Impaired recognition may be due to treating
Pacific Ocean to Solar Forcing During novel objects as familiar, rather than treating
the Early Holocene familiar objects as novel.
T. M. Marchitto et al. >> Perspective p. 1331
Enhanced solar activity caused the tropical
Pacific to cool into a La Niña–like state
1410 Frequent Mutation of BAP1 in
during the mid-Holocene. Metastasizing Uveal Melanomas
J. W. Harbour et al.
1381 Plasticity of Animal Genome Architecture A gene implicated in the control of protein
Unmasked by Rapid Evolution of degradation is mutated at high frequency
a Pelagic Tunicate in a metastatic eye cancer.
page 1381
F. Denoeud et al.
1413 Direct Exchange of Electrons Within
A metazoan genome departs from
the organization that appears rigidly
Aggregates of an Evolved Syntrophic
established in other animal phyla. Coculture of Anaerobic Bacteria
Z. M. Summers et al.
1385 Rewiring of Genetic Networks in Response Direct cell-to-cell electron transfer occurs
to DNA Damage between two related species of bacteria.
S. Bandyopadhyay et al.
A network comparison of genetic interactions CONTENTS continued >>
mapped at two conditions reveals genetic
responses to DNA damage in yeast.
>> Perspective p. 1327

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CONTENTS
SCIENCEONLINE
SCIENCEXPRESS SCIENCESIGNALING
www.sciencexpress.org www.sciencesignaling.org
A Bacterium That Can Grow by Using Arsenic The Signal Transduction Knowledge Environment
Instead of Phosphorus RESEARCH ARTICLE: The Extracellular Domain
F. Wolfe-Simon et al. of Fibroblast Growth Factor Receptor 3 Inhibits
A bacterium has been found that can swap a toxic Ligand-Independent Dimerization
metalloid for a key molecular building block L. Chen et al.
in its nucleic acids and enzymes. The extracellular domain of a receptor tyrosine kinase
10.1126/science.1197258 has opposing effects on dimerization, depending on
>> News story p. 1302; Science Podcast whether ligand is present or not.
Cytoplasmic Partitioning of P Granule PERSPECTIVE: Compartment-Specific Control of
Components Is Not Required to Specify Signaling from a DNA-Sensing Immune Receptor
the Germline in C. elegans A. Engel and G. M. Barton SCIENCESIGNALING
C. M. Gallo et al. The endosomal compartment in which TLR9 is Trafficking inflammation.
Germ granules do not need to be segregated activated determines which cytokines are produced.
asymmetrically during cell division to specify GLOSSARY
germ cell fate. Find out what BAPTA, CaSR, and DOCK mean in the RESEARCH ARTICLE: IL-22+ CD4+ T Cells Are
10.1126/science.1193697 world of cell signaling. Associated with Therapeutic Trichuris trichiura
Vernalization-Mediated Epigenetic Silencing NETWATCH: Peptide Atlas Infection in an Ulcerative Colitis Patient
by a Long Intronic Noncoding RNA Access a database of peptides identified in mass M. J. Broadhurst et al.
J. B. Heo and S. Sung Infection with a nematode worm decreases
spectrometry analyses; in Protein Databases.
Spring flowering enabled by a winter chill is inflammation in an ulcerative colitis patient.
regulated by interplay between protein-coding NETWATCH: BioInteractive
Take advantage of online videos and interactive
RESEARCH ARTICLE: Replacing the Enzyme
and noncoding RNA transcripts.
lessons for teaching and learning about biology; α-L-Iduronidase at Birth Ameliorates Symptoms
10.1126/science.1197349
in Educator Sites. in the Brain and Periphery of Dogs with
Large Variations in Southern Hemisphere Mucopolysaccharidosis Type I
Biomass Burning During the Last 650 Years SCIENCECAREERS A. D. Dierenfeld et al.
Z. Wang et al. Replacing α-L-iduronidase at birth ameliorates
www.sciencecareers.org/career_magazine
Large variations in the degree of biomass burning symptoms in dogs with a lysosomal storage disorder.
Free Career Resources for Scientists
in the Southern Hemisphere occurred during
the past 650 years. Battling Cyber Threats SCIENCEPODCAST
10.1126/science.1197257 S. Carpenter www.sciencemag.org/multimedia/podcast
A critical shortage of highly skilled cybersecurity
SPORE SERIES WINNER Free Weekly Show
technicians and engineers means wide-ranging
Science 101: Building the Foundations opportunities for scientists. Download the 3 December Science Podcast to hear
for Real Understanding about a bacterium that uses arsenic instead of
A. Thanukos et al. Becoming ‘MacGyvers’ phosphorus, imaging the skin in vivo, science
S. Carpenter in Brazil, and more.
10.1126/science.1186994
For University of Tulsa Cyber Corps students,
homework means picking through Dumpsters
SCIENCENOW SCIENCEINSIDER
and hacking computer systems.
www.sciencenow.org news.sciencemag.org/scienceinsider
Taken for Granted: Choosing Between Science Policy News and Analysis
Highlights From Our Daily News Coverage
Science and Caring?
Black Hole May Offer Clues to Extra Dimensions B. L. Benderly
Controversial idea relies on string theory and Research suggests that many able women
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Published by AAAS
EDITED BY STELLA HURTLEY
written words, rather than being able to rely An Upside of Asymmetry

upon evolutionarily ancient modifications of
the visual system pathways. Dehaene et al. Advances in synthetic techniques have enabled
(p. 1359, published online 11 November) the preparation of nanometer-scale semiconduc-
examined the neural response to a range of tors in a wide range of precise shapes and sizes,
visual stimuli in three groups: illiterate adults, including core-shell morphologies that layer
adults who learned to read as children, and several different materials in the same particle.
adults who learned to read as adults. Reading Such two-in-one motifs are promising for light-
induced a greater facility in processing horizon- harvesting applications because they
tally oriented stimuli at early stages in the allow optically induced charge separation
visual pathway and was also associated with the across the internal interface. Borys et al.
appearance of an area specialized for words. (p. 1371) studied a series of rod-shaped cad-
This gain of function appeared to occur at a mium sulfide–cadmium selenide hybrid particles
Downloaded from www.sciencemag.org on December 2, 2010

cost—the area in the temporal cortex devoted using single-particle–resolved optical spectros-
to face processing shrank. copy and found that smooth versus bulbous
The Yin and Yang geometries produced distinct emission spectra.
of Plant Caspases Shake It to Wake It Further analysis of more complex, tetrapodal

particles (with four arms aligned tetrahedrally)
Viscoelastic materials combine the recoverable suggested that nonuniform geometries facilitate
The function of plant metacaspases, iden- stretchiness found in elastic materials with the interfacial charge transfer by reducing the likeli-
tified by limited sequence homology
slow-flowing behavior of a thick fluid, like honey. hood of electronic band misalignment.
to the animal caspases that control cell
When subjected to an oscillatory motion, the
death, has remained elusive. Coll et al.
(p. 1393) have now elucidated the actions
response will depend on the frequency. At low
frequencies, the viscous behavior will dominate
Cryptic Sulfur Cycling
of two metacaspases in the small plant Ara-
bidopsis. One metacaspase, AtMC1, pro- and lead to a dissipation of the applied energy Aerobic bacteria and ocean circulation pat-
moted cell death, and the other, AtMC2, as heat, while at fast frequencies the elastic terns control the formation and distribution of
acted antagonistically to stall cell death. behavior dominates. Xu et al. (p. 1364; see the oxygen-minimum zones at moderate depth in
The results help to elucidate the mecha- Perspective by Gogotsi) developed a viscoelastic the oceans. These habitats host microorganisms
nisms by which plants control cell survival material with an exceptionally broad operating that thrive on other metabolic substrates in the
during development and defend against temperature range, based on a network of carbon absence of oxygen—most commonly, metabo-
pathogen attack. nanotubes. The responsiveness of the material lizing thermodynamically favorable nitrogen
was probably caused by the “zipping” and “un- compounds like nitrate. Off the coast of Chile,
zipping” of the nanotubes at points of contact. however, Canfield et al. (p. 1375, published
CREDITS (TOP TO BOTTOM): DR. PETRA EPPLE/THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL; SAAR ET AL.
Sunny and Cool online 11 November; see the Perspective by

Teske) suggest that bacteria may often reduce
Changes in solar output cause changes in the Skin-Deep Raman sulfate as well. Metagenomic sequencing
amount of radiation that Earth receives from the
Sun, which in turn can cause climate variations.
Spectroscopy revealed the presence of both sulfate-reducing
and sulfide-oxidizing bacteria. With the coinci-
The effects of solar variations are not uniform over Raman spectroscopy allows for dence of sulfate and nitrate reduction, the sul-
the globe—owing to the complexity of the climate molecular identification via vi- fur and nitrogen cycles may be intimately
system, larger solar fluxes may produce warming brational spectra at optical linked; for example, sulfate reduction
in one area but cooling in another. Marchitto wavelengths. However, could provide nitrogen-rich ammo-
et al. (p. 1378) present a record of Holocene sea if the optical signal is nium for bacteria that ultimately
surface temperature in the eastern equatorial scattered, as occurs transform it into nitrogen gas.
Pacific Ocean that shows cooling as solar output when trying to image
increased and warming as the Sun dimmed. These
temperature changes resulted from dynamical
tissue, the signal be-
comes very weak, and
Deadly Trio
control of El Niño and La Niña episodes by solar it becomes difficult to The proteins BAX and BAK act as a
radiative forcing of Earth’s climate. image a sample with high key decision point, regulating apopto-
time resolution. Saar et al. sis by controlling the permeability of the
(p. 1368) now show that by mitochondrial outer membrane. Evidence
Reading, Writing, and improving the optics and electronics has been presented for two mechanisms of
Face Recognition of the acquisition of the backscattered signal,
stimulated Raman scattering spectroscopy
activation of BAX and BAK: an indirect mecha-
nism where proapoptotic proteins neutralize
Reading, not to mention writing and texting, can be performed at video rates on human the antiapoptotic effects of the protein BCL-2
is a relatively recent invention, and hence it is skin, which should enable label-free studies of and its relatives; or direct activation of BAX
believed that a preliterate brain must adapt on tissues and, for example, the tracking of the and BAK by BIM, BID, or PUMA. Analysis of the
the fly, so to speak, in learning how to process delivery of a drug. Continued on page 1285

Published by AAAS
This Week in Science Call for
Continued from page 1283
Papers
situation in vivo is complicated by the overlapping function of BIM, BID, and PUMA. Ren et al. (p.
1390; see the Perspective by Martin) thus analyzed triple-knockout mice lacking BIM, BID, and
PUMA. Apoptosis during mouse development required a direct effect of one of these proteins to
activate BAX or BAK, thereby promoting cell death.
Better Brain Maps

A high-resolution atlas of the complete neuronal connectivity in a whole brain should fundamentally Science
Si
Signaling
advance our understanding of the organization and function of animal nervous systems. Now, A. Li. et
al. (p. 1404, published online 4 November) describe an automated system, micro-optical sectioning to-
mography, that allowed three-dimensional mapping of the morphology and spatial location of neurons
and traces of neurites in a whole, intact mouse brain. Science Signaling, from the
publisher of Science, AAAS,
Wired for Life features top-notch, peer-

reviewed, original research
Syntrophic bacteria live on the metabolic by-products weekly. Submit your manu-
of a partner species. The exchange of the by-products scripts in the following areas
accompanies a flow of electrons in the opposite direction of cellular regulation:
that helps some species grow in conditions that would
otherwise be unfavorable. In mixed anaerobic cultures of • Biochemistry
two related Geobacter species, Summers et al. (p. 1413) • Bioinformatics
observed that one species evolved to promote the trans- • Cell Biology
fer of electrons directly to the other, in large aggregated • Development
cell clusters, without coupling to common anaerobic • Immunology
by-products such as hydrogen or formate. Selection pres- • Microbiology
sures in nine parallel populations all resulted in a point • Molecular Biology
mutation that truncated a protein involved in the produc- • Neuroscience
tion of small hairlike projections involved in intercellular
• Pharmacology
communication—pili—and indirectly increased the
• Physiology and
expression of a c-type multiheme cytochrome responsible
Medicine
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• Systems Biology
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sible alternative route to anaerobic syntrophy rather than interspecies hydrogen transfer; indeed, Subscribing to Science Signaling
deleting a gene that encodes a hydrogenase involved in hydrogen transfer conferred a growth ensures that you and your lab
advantage in the cocultures. have the latest cell signaling
resources. For more information
Novel or Familiar? visit www.ScienceSignaling.org
Amnesia is characterized by a number of memory deficits, including the apparent inability to distin-
guish between novel and familiar stimuli. McTighe et al. (p. 1408; see the Perspective by Eichen-
baum) observed that the recognition memory of brain-damaged rats in a standard model of amnesia
Chief Scientific Editor
was impaired not because previously experienced objects seemed to be novel, but because objects Michael B. Yaffe, M.D., Ph.D.
not previously experienced seemed to be familiar. Furthermore, simply placing the animal in a visu- Associate Professor, Department of Biology
ally deprived environment during the delay, reducing visual interference, completely rescued the Massachusetts Institute of Technology
impairment. This counterintuitive finding contradicts the predominant “multiple memory systems”
Editor
model in which amnesia is usually considered and forces a reconsideration of fundamental assump-
Nancy R. Gough, Ph.D.
tions underlying our understanding of amnesia. AAAS
An Eye on Metastasis
CREDIT: ZARETH SUMMERS AND KAITLYN RUBIN
Despite the considerable progress being made in elucidating the cell biology of metastasis, little is Submit your research at:
known about the genetic alterations that promote metastasis of human tumors, the cause of most www.sciencesignaling.org/
cancer deaths. A potentially important clue now emerges from the work of Harbour et al. (p. 1410, about/help/research.dtl
published online 4 November), who used an exome-sequencing approach to search for genetic mu-
tations in uveal melanomas, an eye cancer associated with a high rate of fatal metastasis. Remark-
ably, over 80% of tumor samples with a high metastatic risk had inactivating somatic mutations
in the gene encoding BAP1 (BRCA1-associated protein 1), a nuclear protein involved in controlling
protein degradation. Thus, in this tumor type, mutational inactivation of BAP1 may be a key event in
the acquisition of metastatic competence.
www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010

Published by AAAS
EDITORIAL
Bruce Alberts is Editor-

Policy-Making Needs Science
in-Chief of Science. OVER THE LONG RUN, ANY NATION THAT MAKES CRUCIAL DECISIONS WHILE IGNORING SCIENCE
is doomed. Consider, for example, the decision about how much arsenic should be allowed in
drinking water supplies. There is no one “right answer” to this or many other policy questions,
but it is critical that national legislation be based on what science knows about potential harm. It
is therefore disturbing that so many lawmakers elected to the new U.S. Congress reject the over-
whelming scientific consensus with respect to human-induced climate change. It will be diffi-
cult to make wise choices with such attitudes. The question now facing the United States is not
only how to effectively reinject the facts of climate science back into the core of this particular

debate, but also how to ensure that good science underlies all legislative decisions.
For 12 years, I served as the president of the U.S. National Academy of Sciences.
As part of a larger nongovernmental organization known as the
National Academies, it produces more than 200 reports a year, aimed
at making the current scientific consensus on important issues avail-
able to policy-makers and the public. In major reports released this
spring, the National Academies strongly reiterated its position that
climate change, caused largely by human activities, poses signifi-
cant risks to the world’s future.* This conclusion is nevertheless chal-
lenged by numerous politicians, as well as by a substantial fraction of
the public. There is only one effective solution for this type of prob-
lem: Scientists must make both science education and community
outreach a much more central part of the scientific culture.
Most Americans have never met a scientist, and despite having
been “taught science” at school, most have no real idea of how a
scientific consensus is reached through continuous open debate
and experiment. Every adult should have a base of scientific under-
standing about how the world works. But understanding the process through which scien-
tific knowledge develops is equally critical. By the end of any introductory college science
class—which can be an adult’s final exposure to science—a student should have a realistic
understanding of the nature of science. Scientists are taught to challenge authority, and their
responsible challenges to a consensus help science advance. Thus, adults should expect to
find some scientists who disagree with the scientific consensus on an issue. And they should
appreciate why a strong scientific consensus, such as that about climate change, must never-
theless form the basis for making wise personal and community decisions, representing by
far the best bet for predicting the future consequences of present actions.
In addition to education, an energetic community outreach to schools, the public, and
decision-makers is key. Both established scientists and those in training can be highly effec-
tive in putting a human face on science and conveying optimistic, honest attitudes toward
grappling with society’s problems. Week-long science festivals, to which local institutions
based on science and engineering contribute ideas and personnel, should become an annual
event at hundreds of sites around the nation. And programs that encourage and facilitate out-
reach into nonscientific communities need to become a standard part of every university and
CREDITS: (TOP) TOM KOCHEL; (RIGHT) THINKSTOCK
science-based industrial establishment.

The environment in which decisions are made in a democracy will always be highly
politicized, but it is crucial that both sides of any argument pay close attention both to what
science knows and how that knowledge has been gained. Attaining this goal in every nation
will require that scientists vigorously reach out to their communities, informing them not
only about their new discoveries, but also about the path they took to get there.
– Bruce Alberts
10.1126/science.1200613
*http://americasclimatechoices.org. T. E. Bowman et al., Science 330, 1044 (2010).

Published by AAAS
EDITORS’CHOICE
EDITED BY KRISTEN MUELLER AND JAKE YESTON
EVOLUTION
Good for You, Good for Me, Too

In most examples of cooperative breeding,
reproductive individuals are assisted by closely
related helpers. Although helpers sacrifice
their own ability to reproduce, they obtain an
indirect fitness benefit from improving survival
in their kin (kin theory). Increasingly, how-
ever, cooperative breeding has been revealed
among unrelated individuals. In greater anis
(Crotophaga major), multiple pairs of birds

will cooperate to build nests and raise and
defend young. Riehl has now shown that these
pairs are relatively unrelated, thus cooperation
ECOLOGY
cannot be explained by kin theory. Instead,
Migration-Based Monitoring she finds that the individual fitness of all the
pairs is increased when pairs cooperate, despite
Counts of birds passing traditional hawk-watching sites provide a means for estimating
initial reproductive competition generated
population trends in migrant raptors. To gauge the effectiveness of such efforts, Farmer et
during egg-laying. Three pair groups were able
al. explore the degree to which the existing network of sites could sample the autumn tracks
to obtain and defend prime nesting sites better
of 57 adult ospreys (Pandion haliaetus). Birds from four breeding areas across the United
than two pair groups, and pairs attempting to
States were fitted with satellite transmitters. An average of 1.5 relocations per day were
breed alone failed in all observed instances.
observed per bird. To analyze flight trajectories, the authors identified migration paths that
Furthermore, there was no cost to individual
connected locations with 3-km- or 6-km-wide corridors. Across North America, 12% of the
fitness on the basis of the number of eggs laid,
narrow and 23% of the broader pathways intersected one or more active watch sites; these
most likely because females changed their lay-
values rose to 21 and 36% in the eastern region (through which the majority of the con-
ing order across years. In unrelated cooperative
tinent’s population probably passes). Models that assume a conditional random walk be-
breeders, what’s good for “the many” is most
tween consecutive positional fixes estimate that 95% of the birds’ utilization distributions
likely also good for “the one.” — SNV
cross at least one observation area. Disparities in mean regional detection probabilities for
Proc. R. Soc. London Ser. B 10.1098/
individuals (east, 34%; midwest, 5.8%; northwest, 4.7%) reflect the relative distribution
rspb.2010.1752 (2010).
of hawk watches. The satellite tracks also show southbound ospreys concentrating along
narrow fronts and avoiding large water crossings. These results suggest that migration
GEOCHEMISTRY
counts do offer a practical means of monitoring populations of the North American osprey
and perhaps many other hawks. — SJS Reflecting on Mica
Auk 127, 863 (2010).
Trace metals in soils and sediment predomi-
nantly exist as either tiny mineral grains or
mobile aqueous ions in pore water. In both
cases, adsorption and desorption reactions at
the surface of mineral
BIOCHEMISTRY structure of two protofilaments of grains control their
CREDITS (TOP TO BOTTOM): ISTOCKPHOTO.COM; GC MINERALS/ALAMY
the tubulin/FtsZ-like protein, TubZ, fate and transport

Acting Like Actin
which mediates plasmid segregation. through the subsur-
In a dividing cell, DNA must be replicated and A combination of crystallography face. Because the size
accurately partitioned between the two daugh- and electron microscopy revealed of ions and strength
ter cells. In eukaryotes, microtubules, which that instead of forming tubulin-like of the surround-
are linked to the centromeric DNA by kineto- straight protofilaments that organize ing hydration shell
chores, separate the replicated chromosomes into microtubules, TubZ forms twisted are highly variable,
with the help of motor proteins. Prokaryotic double filaments that can bundle, predicting the way
plasmid-partitioning systems are similar in that reminiscent of structures formed in which metals are
they involve an adaptor protein that links DNA by ParM, an actin-like prokaryotic distributed even at
to a filament-forming protein; however, no partitioning filament. That two disparate one specific mineral/water interface is challeng-
motor proteins are involved, and nucleotide- proteins have evolved to perform a similar ing. To systematically address this question,
hydrolysis dependent filament dynamics are function using similar superstructures suggests Lee et al. measured—using an X-ray reflectiv-
responsible for movement. To gain insight into strong evolutionary constraints on partitioning ity technique sensitive enough to determine
the segregation of the Bacillus thuringiensis systems. — VV the thickness of a single layer of water—the
pBToxis plasmid, Aylett et al. determined the Proc. Natl. Acad. Sci. U.S.A. 107, 19766 (2010). Continued on page 1291

Published by AAAS
EDITORS’CHOICE
Call for
Continued from page 1289 Papers
manner in which divalent metal ions adsorbed is absorbed by the sea and by the terrestrial
on a hydrated muscovite surface. As expected, biosphere. Studies have suggested that North
metal ions with weak hydration shells lost water America constituted a terrestrial carbon sink
and bound tightly to muscovite within just 2.5 during the past several decades, although
Å; stronger hydration shells remained intact,
pushing other metal ions further out (3.5 to 4.5
how much carbon it has accumulated remains
debatable. Crevoisier et al. have now estimated Science
Å from the surface). The reflectivity profiles also
revealed, surprisingly, an unexpected additional
land-atmosphere fluxes across North America
during the period from 2004 to 2006 by bal- Translational
Medicine
broad layer of metal ions 5 to 10 Å away from the ancing the inflow and outflow of carbon dioxide
mineral surface. Computational models of similar from the troposphere, using a simple budgeting
interfaces may need to be adjusted to account for approach and vertical profiles of atmospheric
this extended layer. — NW carbon dioxide to construct the distribution Integrating Medicine
Langmuir 26, 16647 (2010). over the continent in three dimensions. They and Science
find that the coterminous United States pro-
The new journal from the publisher

PHYSICS vided a moderate sink and that the distribution
of Science stands at the forefront
of carbon uptake agrees well with agricultural
Hiding in the Pseudogap of the unprecedented and vital
and climate patterns during that time. Their collaboration between basic
When cuprate superconductors, which can technique thus also provides a potential inde- scientists and clinical researchers.
conduct electricity without resistance up to pendent method to link regional carbon uptake
temperatures on the order of 150 K, were first to climate drivers. — HJS • Cardiovascular Disease
discovered almost 25 years ago, it was expected Proc. Natl. Acad. Sci. U.S.A. 107, 18348 (2010). • Neuroscience/Neurology/
Psychiatry
that the explanation for their extraordinary prop- • Infectious Diseases
erties was around the corner and would lead to SIGNAL TRANSDUCTION • Cancer
even higher transition temperatures Tc. Today this • Health Policy
mechanism remains a mystery; however, most
Cell as Corporation
• Bioengineering
researchers agree that the so-called pseudogap Systems biology provides insight into the • Chemical Genomics/
region, which lies above Tc but is characterized complex regulatory networks within a cell, with Drug Discovery
by a non-zero excitation gap that disappears at the hope that such analysis will yield insight into • Other Interdisciplinary
Approaches to Medicine
a higher temperature T *, is key to understand- how biological control systems work and how
ing the entire phase diagram. Along the doping they may be modified for therapeutic benefit.
axis, the pseudogap region borders an antifer- Some network models suggest that the extent Submit your research at
romagnetic phase, the remnants of which can be of connectivity of a transcriptional regulator www.submit2scitranslmed.org
detected by neutron scattering as a resonance indicates the essentiality of that protein in that
centered around the corner of the Brillouin system. Bhardwaj et al. propose an alternative
zone. Li et al. use polarized neutron scattering view. They organized transcription factors in
to detect another magnetic excitation yeast or bacteria in
that is present at all wave vectors and, a pyramidal hierar-
surprisingly, has an integrated spectral chy and then used
weight an order of magnitude higher information regard-
than that of the resonance. Because ing the effects of the
the studied compound, HgBa2CuO4+δ, various factors on cell
is thought to be representative of fitness to determine
the cuprates, and the new magnetic that the importance of
excitation appears at the same T * a transcription factor
determined from prior transport and is more related to its
neutron diffraction measurements, it placement in the hier-
is tempting to interpret this result as archy than to its total
a corroborating piece of evidence that direct connectivity to
a true phase transition, rather than a target genes. Thus, Chief Scientific Adviser
crossover, takes place at T *. — JS proteins at the top of Elias A. Zerhouni, M.D.
Nature 468, 283 (2010). the hierarchy exerted Former Director,
the greatest influence National Institutes of Health
C L I M AT E S C I E N C E over cell fitness. The relative ease of testing
CREDIT: ISTOCKPHOTO.COM
network perturbations in biological systems

The American Sink
suggests that this type of analysis may also be
Understanding how fast the concentration of useful in understanding similar corporate, gov-
carbon dioxide in the atmosphere will rise as ernment, or social networks, where one cannot
we continue to burn fossil fuels depends on so easily intervene. — LBR
how well we know what fraction of emissions Sci. Signal. 3, ra79 (2010).
ScienceTranslationalMedicine.org
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Robert W. Boyd, Univ. of Rochester Martin Heimann, Max Planck Inst., Jena Helga Nowotny, European Research Advisory Board Jonathan Zehr, Ocean Sciences
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David Clary, Oxford University David Holden, Imperial College Reginald M. Penner, Univ. of California, Irvine Angela Creager, Princeton Univ.
J. M. Claverie, CNRS, Marseille Lora Hooper, UT Southwestern Medical Ctr at Dallas John H. J. Petrini, Memorial Sloan-Kettering Cancer Center Richard Shweder, Univ. of Chicago
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1292 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

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RANDOMSAMPLES
EDITED BY LAUREN SCHENKMAN
Nepal, Goldfarb hopes that the record motion. Fujii’s own data combines multi-
Poop Scoop will highlight how science, like music, electrode recording and motion capture to
New baby? Feeling like you’re is a creative enterprise. “We are not study adaptive behavior in primates.
waist deep in dirty diapers? trying to compete with the Beatles Many animal researchers who put data online
Forget diaper-collection or Motown,” he says, “but to try and cloak it in technical jargon or hide it behind
services; just volunteer your interest young people in physics.” Imagine Paul academic firewalls to forestall attacks by activists.
infant for a poop study McCartney’s relief. Neurotycho, by contrast, highlights the infor-
and researchers will take mation with simple explanations and cartoon
them off your hands illustrations. “I thought the benefit of starting
for free. Dirty diapers, it Thanks for Sharing Neurotycho was much larger than the risks,” Fujii
seems, hold the key to mea- The phrase “copyright-free repository of says. “We need new ideas from different fields for
suring infant hormone levels. neuroscience data” doesn’t exactly roll off the understanding neural mechanisms.” The site’s
Sex hormones, such as tongue. So when he
estrogen, are important for babies’ healthy decided to share his A cartoon from neurotycho.org.

development. But some endocrinologists worry data, Naotaka Fujii
that children are exposed to too much additional of the RIKEN Brain
estrogen via soy formula, plant fertilizers, and Science Institute in
even plastics, which could cause faster-than- Wako City, Japan,
normal development and future problems with dubbed his collection
reproduction. However, few infants tolerate a “Neurotycho” (http://
frequent finger or heel prick, and so “very little neurotycho.org). The
is known about hormone levels in infants,” name pays homage to the 16th century Danish usefulness is catching on: In the first 10 days
explains Michelle Lampl, an anthropologist at astronomer Tycho Brahe, whose collected data after it was launched last month, 150 neuro-
Emory University in Atlanta. inspired Johannes Kepler’s laws of planetary science experts from around the world signed up.
O SCIENCE!
Diapers, however, can be collected frequently
GOOO O O
and over a long period of time, perfect for a
longitudinal study. Practicing on eight to 10 dia-
pers collected from each of 32 largely breast-fed
infants over 6 months, Lampl’s group perfected a It was just another Monday night for patrons gathered at a Philadelphia sports bar on 15 Novem-
technique for extracting hormone levels from the ber to watch the Eagles play the Washington Redskins. And then a squad of cheerleaders burst
poop, they reported online 11 November in through the door and, armed with megaphones and pompoms, explained football in terms of
Frontiers in Systems Biology. vector physics.
They also perfected their diaper-collection The Science Cheerleaders strike again. Like all superheroes, they lead double lives: All of them
technique. “It took years to find the right nappy are either professional cheerleaders working on a scientific degree or former cheerleaders who
and work out how you get the diaper fresh from switched to science-related careers. “I am very fortunate that I have the opportunity to fulfill both
the home to the lab,” says Lampl. The secret: a passions: science and dance,” says Alyson, a Tennessee Titans cheerleader who plans to start medi-
cotton diaper, a Ziploc bag, and an ice pack. cal school next year. (Like pro cheerleaders, squad members don’t use their full names as a precau-
CREDITS (TOP TO BOTTOM): THINKSTOCKPHOTOS.COM; NEUROTYCHO.ORG; COLELLAPHOTO.COM
tion against stalking.) In October, they performed at the U.S.A. Science & Engineering Festival on
the National Mall in Washington, D.C. “Some people feel we are trying to improve the reputation
The Sound of Science of cheerleaders, while others feel this is helping scientists,” says Darlene Cavalier, a former Phila-
Two years ago, particle physicists at the Euro- delphia 76ers cheerleader who manages the Science Cheerleaders (www.sciencecheerleader.com)
pean laboratory CERN near Geneva, Switzerland, and other science outreach programs. “I just hope it inspires as many people as possible!”
rapped about the start-up of their enormous
new atom smasher, the Large Hadron Collider
(LHC). Now, they’re taking it up a notch: On
6 December, scientists from the collaboration
that takes data with LHC’s ATLAS detector will be
releasing a double album featuring everything
from classical and medieval music to blues and
heavy metal.
Resonance (www.atlas-resonance.ch) will be
released on homegrown label Neutralino Records
(named after a hypothetical subatomic particle)
and includes a song about the detector’s work-
ings by physicist Steven Goldfarb’s Canettes
Blues Band. In another tune, guitar band the
TLAs (Three Letter Acronyms) laments that “when
black holes destroy the Earth, I’ll be in a meet-
ing.” Besides raising money for an orphanage in

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NEWS>> Bacteria that A gathering storm
thrive on arsenic for U.S. universities
1302 1304
HIV/AIDS CLINICAL TRIALS a treatment in AIDS drug cocktails, which
means doctors can immediately start pre-
A Powerful and Perplexing scribing it “off-label” as a preventive.
Yet PrEP’s role in public health remains
New HIV Prevention Tool anything but clear. “We don’t think, ‘Let’s
just sprinkle Truvada in the water supply
and it solves the problem,’ ” says Kenneth
On 2 October, two dozen AIDS research- bottom line. “It was very, very dramatic,” Mayer, who headed an iPrEx study at Fen-
ers gathered at the Eden Roc hotel on Mil- says Robert Grant, a virologist with the way Health in Boston, one of two U.S. sites.
lionaire’s Row in Miami Beach, Florida, to J. David Gladstone Institutes at the Univer- (Nine other trial sites were in Peru, Ecuador,
learn whether an HIV prevention study they sity of California, San Francisco (UCSF), South Africa, Brazil, and Thailand.) “It’s an

had just completed would become a mill- who headed the trial. “People were over- imperfect tool.”
stone or a milestone for the field. In the six- joyed to be among one of the few preven- First, iPrEx shows only that the drug
country study, 1251 men and transgender tion trials to have shown a protective effect.” works in the specific high-risk population
women who have sex with men and were not The New England Journal of Medicine studied. Mayer and others stress that, as was
infected with HIV at the study’s start took published a full report of the iPrEx results done in the study, the drug should be offered
an antiretroviral pill each day to see whether on 23 November. The global response was as part of a prevention package that includes
it could ward off infection with the virus. ecstatic, prompting no less than U.S. Presi- condom promotion and counseling. Experts
Another 1248 carefully matched participants dent Barack Obama to issue a statement. “I worry that PrEP might lead people to take
took a placebo. Failure would cast a pall over am encouraged by this announcement of more risks than they would otherwise, off-
the whole concept of oral pre-exposure pro- groundbreaking research on HIV preven- setting the benefit of the pill, although this
phylaxis (PrEP), which is now being tested in tion,” said Obama. “While more work is wasn’t seen in the study.
four other efficacy trials that involve 16,000 needed, these kinds of studies could mark PrEP could do harm, too, if it drove the
heterosexuals and injecting drug users (see the beginning of a new era in HIV preven- evolution of Truvada-resistant HIV strains.
table). Success would offer a powerful new tion.” But the good news was tempered by a When used as treatment, Truvada alone,
option to dodge the virus—and might even dizzying array of complicated issues about versus in combination, is not strong enough
to keep the virus
ONGOING PRE-EXPOSURE PROPHYLAXIS EFFICACY TRIALS >>> in check. Theo-
retically, if people
Trial Location Sponsor Population Intervention Expected Results did not know they
Bangkok Tenofovir Study Thailand U.S. CDC 2400 injecting drug users Daily oral TDF 2012 were already HIV-
Partners PrEP Kenya, Uganda Gates Foundation 4700 serodiscordant Daily oral TDF; daily oral 2013 infected and took
heterosexual couples TDF/FTC Truvada, the virus
VOICE South Africa, Microbicide Trials 5000 heterosexual women Daily oral TDF; oral TDF/FTC; 2013 could mutate around
Uganda, Zimbabwe Network, NIH topical 1% TDF gel the drug. An increase
FEM-PrEP Kenya, South Africa, FHI, USAID, 3900 heterosexual women Daily oral TDF/FTC 2013 in Truvada-resistant
Tanzania, Zimbabwe Gates Foundation strains, which are
TDF = tenofovir; FTC = emtricitabine
already circulat-
More news soon. Large PrEP studies in different populations will start reporting results by 2012. ing at low levels,
could under mine
slow HIV’s spread in entire communities. human behavior, ethics, resources, risk, and both treatment and PrEP. Again, such drug
Unlike the many HIV prevention tri- public health. resistance did not surface in iPrEx, but the
als that have failed or had positive but The iPrEx study, which cost $43.6 mil- researchers were rigorous in their efforts to
barely significant results, the study—called lion and was conducted between July 2007 exclude HIV-infected people and tested par-
the Pre-Exposure Prophylaxis Initiative and December 2009, has one clear-cut ticipants for new infections every 4 weeks.
CREDIT: AIDS VACCINE ADVOCACY COALITION
(iPrEx)—showed unequivocally that the message: A pill can dramatically lower the For policymakers, the PrEP results raise
treated group had 44% fewer infections chances of transmission of HIV through difficult ethical and practical questions.
after an average of 1.2 years. More encour- receptive anal intercourse in men who Funding for HIV/AIDS is already insuffi-
aging still, most of the failure seemed to have many partners—on average, 18 in the cient: Ten million infected people who need
occur among those who did not take the 12 weeks preceding the start of the trial— treatment currently have no access to drugs.
pill as directed: A small substudy found and frequently do not use condoms. The pill, “I think it’s going to be quite a while before
that risk of infection plummeted by 92% Truvada, made by Gilead Sciences in Fos- we’d start using oral antiretrovirals for pre-
in people who had measurable drug levels ter City, California, combines two anti-HIV vention,” says epidemiologist Salim Abdool
in their blood. The researchers applauded drugs, tenofovir and emtricitabine. Truvada Karim of the University of KwaZulu-Natal
and some even cried when they heard the is already on the market and widely used as in Durban, South Africa, which has more

Published by AAAS
Special report:
Science in Brazil
1306
infected people than any country.
In wealthy countries, no one knows
whether insurance companies will cover the
use of Truvada as a preventive, and there are
no guidelines for prescribing PrEP, although
in the next few weeks, the U.S. Centers for
Disease Control and Prevention says it will
publish interim guidance.
Anthony Fauci, director of the U.S.
National Institute of Allergy and Infec-
tious Diseases (NIAID), which paid for

two-thirds of iPrEx (the Bill and Melinda
Gates Foundation picked up the balance),
says Truvada’s success as PrEP could have
far-reaching impact on other HIV preven-
tion studies. Ethical principles demand that
researchers minimize risk for people who
participate in studies, which means offer-
ing known effective interventions in the
CREDITS (TOP TO BOTTOM): MALCOLM LINTON; ADAPTED FROM R. M. GRANT ET AL., N. ENGL. J. MED. (23 NOVEMBER 2010) © 2010 MASSACHUSETTS MEDICAL SOCIETY
control arms of trials of vaccines or other

PrEP compounds. Fauci says NIAID will
now review “virtually every study that we
have ongoing or planned” to assess whether
trials that have placebo controls should 0.10 Fielding questions. Robert Grant (right) spoke
instead offer Truvada. “It’s going to create Placebo about potential risks and benefits with communi-
Cumulative probability
0.08
welcome work for everyone in HIV pre- ties in Peru before launching iPrEx. Positive results
of HIV infection
vention science,” says Jeremy Sugarman, a 0.06 (left) mean clinicians everywhere must do the same.
bioethicist at the Johns Hopkins Bloomberg
0.04 FTC-TDF
School of Public Health in Baltimore, Mary- Ultimately, PrEP’s popularity may be
land. Sugarman, who chairs the ethics work- 0.02 P=0.005 tied to whether Gilead asks the U.S. Food
ing group of the NIAID-funded HIV Preven- and Drug Administration (FDA) for a label
0.00
tion Trials Network, stresses that he’s thrilled 0 12 24 36 48 60 72 84 96 108 120 132 change to include its use as a preventive.
about the positive results. “If they make our Weeks since randomization Although not required, insurers will often
ethical questions harder to answer, so be it.” reimburse only for indications approved by
UCSF’s Grant contends that the iPrEx tion in the price of the drug, which ranges regulatory bodies, and many countries fol-
results create new opportunities that might from $11 per month for a generic version low the lead of FDA. Gilead says it wants
mitigate some of the potential downsides. sold in poor countries versus up to nearly to have “frank” talks with FDA and other
First, he wonders whether real-world adher- $1000 per month for the retail Gilead prod- stakeholders before it decides to seek licen-
ence might be better than in a clinical trial uct. And PrEP costs would fall if people do sure for Truvada as a preventive. “We’ll
setting, as people know that they are taking not need to take it every day: Truvada has a have, I imagine, a very interesting discus-
an active drug and that it works. Adherence long half-life, and ongoing and future stud- sion about the potential risks and benefits
also could differ in different populations. Epi- ies will assess whether it will work with less associated with this kind of a modality, and
demiologist Connie Celum of the University frequent dosing. CAPRISA 004, a PrEP trial I think that will govern what we choose to
of Washington, Seattle, is currently heading with a vaginal microbicide that contained do,” says Howard Jaffe, president of the Gil-
a PrEP study in 4700 Kenyan and Ugandan tenofovir, revealed in July that inserting the ead Foundation, a nonprofit started by the
heterosexual couples in which only one part- gel before and after sex alone cut transmis- company to help poor communities combat
ner is known to be infected. The Partners sion by 39%. HIV and hepatitis B and C.
PrEP Study carefully monitors drug adher- In keeping with a push to link prevention Confusing as PrEP’s fate might seem,
ence, and preliminary evidence suggests it and treatment—treated people are likely less Fenway Health’s Mayer stresses that the
is much higher than in iPrEx, which Celum infectious—PrEP might encourage more iPrEx results are an important milestone
suspects is because the uninfected long-term people to undergo HIV tests. “No one is get- for the failure-weary HIV prevention field.
partners are highly motivated. ting tested hoping to be positive and start “This plus CAPRISA means we’ve crossed
As for resource limitations sidelining treatment,” says Grant. “People are hoping the Rubicon,” says Mayer. “Antiviral chemo-
PrEP, prophylactic use of Truvada could cre- to be negative. This becomes a potent moti- prevention works, no question.”
ate more competition and lead to a reduc- vator to get a test.” –JON COHEN

Published by AAAS
NEWS OF THE WEEK
N E W S M A K E R I NT E RV I E W: M I C H E L S I D I B É
New HIV Infections Drop, But Treatment Demands Rise

Two years ago on World AIDS Day, 1 Decem- Q: What do you think of regions that aren’t
ber, Michel Sidibé was appointed the new stabilized, like Eastern Europe and Central
executive director of the Joint United Nations Asia, which saw incidence increase by 25%
Programme on HIV/AIDS (UNAIDS). An between 2001 and 2009?
economist by training and a native of Mali, M.S.: In these regions, prevention services
Sidibé came into the job when funding for for drug users are below 35%, which is far
HIV/AIDS was still flush. But the global short of what’s needed. If we have an epi-
economic crisis that fall threatened to end demic mainly driven by drug use and we
the era of extraordinary largesse, which don’t have prevention services available, it
enabled many poor countries to rapidly will be impossible to see the reduction we’re
increase the number of people who were seeing in other parts of the world. In Ban-
receiving antiretroviral treatment. On World gladesh and Indonesia, we’re having diffi-
AIDS Day today, that rapid growth has come culty reaching the sex worker and the client

to a standstill, and Sidibé is wrestling to with HIV prevention services. It will also
renew the momentum and turn the funding be a question of how we deal with laws and
situation around. stigma and prejudice that are making people
In its new 359-page Global Report on not have access to the services.
the epidemic, UNAIDS spells out the chal-
lenges in detail, documenting the country- Q: Where have you seen success that you
by-country prevalence that adds up to an M.S.: I think it’s mainly due to prevention didn’t expect?
estimated 33.3 million HIV infections. The combined with treatment. To give you a good M.S.: I was visiting Iran and was very sur-
report, as usual, is a sweet-and-sour dish, example, in 59 countries, fewer than 25% of prised to see one of the most progressive
emphasizing that despite increasing con- the men reported having sex with more than programs in a prison setting where they were
cern about funding, 33 countries—two-thirds one partner in the last 12 months, which is having condom distribution and methadone
of them in sub-Saharan Africa—have seen spectacular. A few years ago we were seeing maintenance for prisoners.
rates of new HIV infections drop by more the opposite trend. Condom use and avail-
than 25% since 2001. Sidibé spoke with ability have also increased significantly. And Q: What do you think of the 1-year retention
Science from New York City, where he was young people are leading the prevention rate of people on antiretrovirals? It’s 95%
visiting the U.N. headquarters. –JON COHEN effort by delaying the first time they have sex. overall but falls to 47% in one country, and
I don’t think it’s just saturation. after 2 or 3 years it plummets in many places.
Q: What’s the main news from the report? M.S.: We need a serious monitoring of the
M.S.: For me the most exciting news is we Q: The new report has scorecards that sum- dropout rate. Dropouts have become one of
have broken the trajectory of the AIDS epi- marize the prevention, treatment, funding, the critical issues to deal with because we’re
demic. It’s the first time through data and and human rights situations in each coun- going to have increasing drug resistance and
analysis we can show that 56 countries have try. It doesn’t look like everyone is achieving a more complex problem to deal with.
stabilized or significantly slowed the rate of what you want.
new HIV infections. M.S.: I tried to introduce this scorecard con- Q: On human rights, are we making prog-
cept to better analyze the gaps. We’re closing ress or sliding backward? Are you con-
Q: How confident are you that these stabi- the gap between treatment and prevention. cerned about the homophobic laws being
lizations are due to prevention efforts as Just 2 years ago, for every two people we were pushed in Uganda?
opposed to HIV having infected the most putting on treatment we were having five new M.S.: Without any doubt. Uganda is one of
vulnerable people years ago and saturating infections. Now it’s one to two. That’s a very the model countries for us. It’s one of the first
populations? good signal. countries to break the conspiracy of silence,
putting people with AIDS at the front line. I
raised this issue with Uganda’s government,
CREDITS (TOP TO BOTTOM): J. COHEN/SCIENCE; UNAIDS
and I certainly hope they will take the deci-

sion that is most appropriate for protecting
those people.
Q: Do you think we’re at a point where we

cannot sustain people who have already
started ARVs?
M.S.: We’re at that point, and it’s very, very
Changes in Increasing >25% scary. Most of the programs will have dif-
Incidence Rate Stable ficulty increasing the number of people on
Decreasing >25%
of HIV Infection treatment. Most just are trying to maintain
Not included
(2001–2009) in analysis what already is being implemented.

Published by AAAS
B I O C H E M I ST RY forming each fraction. They also separated
out the DNA from the bacteria and analyzed
What Poison? Bacterium Uses Arsenic its composition using a technique called high-
resolution secondary ion mass spectrometry;
To Build DNA and Other Molecules the isolated DNA contained arsenic.
Tests utilizing the intense x-rays at a syn-
From elephants to the bacterium Escherichia arsenic. Others were skeptical. The arsenic- chrotron facility offered additional support,
coli, all forms of life on Earth depend on the containing compound arsenate is much more indicating that at least some of the arsenic in
same six elements: oxygen, carbon, hydrogen, unstable than phosphate in water, and no cell the bacteria was in the form of arsenate with
nitrogen, phosphorus, and sulfur. “The para- would be able to cope with that, critics argued. the appropriate molecular bonds to carbon
digm is that the chemistry of life is so specific To test her hypothesis, Wolfe-Simon col- and oxygen atoms to replace the phosphates
that any change in chemistry also changes lected mud from Mono Lake, California, a in DNA and other molecules.
molecular stability and reactivity, which desert body of water known for having high Such work has convinced many that
would not be tolerated,” says Clara Chan, a arsenic levels, and grew the microorganisms Wolfe-Simon’s team has isolated a bacterium
geomicrobiologist at the University of Dela- from it in increasing concentrations of arse- that uses arsenic to grow. “The organization
ware, Newark. nate. She didn’t add any phosphate or other of the experiments presents convincing and
In a paper published online by Science phosphorus-containing compounds to the exhaustive results,” says Milva Pepi, an envi-

(www.sciencemag.org/cgi/content/abstract/ growth medium, as is typically done to sus- ronmental microbiologist at the University
science.1197258) this week, however, an tain microbes. Instead, she periodically trans- of Siena in Italy. But not everyone agrees.
exception to that rule makes a surprising ferred the growing cultures to a new dish to Rosen finds the study “believable” but says
debut. Meet GFAJ-1, a bacterial strain that reduce the concentration of any original phos- he still has lingering concerns that the arse-
researchers say can replace the phospho- phorus to the point that any microbe making nic is simply concentrated in the bacterial
rus in its key biomolecules, including DNA, new DNA or other biomolecules would need cell’s extensive vacuoles and not incorpo-
with the legendary poison arsenic. “This is a to use the arsenic to survive. rated into its biochemistry. He would like to
very impressive and exciting discovery,” says Like others, says Wolfe-Simon, she didn’t see Wolfe-Simon’s team demonstrate a func-
Barry Rosen, a biochemist at Florida Inter- really expect to find any survivors. So she was tional arsenic-containing enzyme, for exam-
ple. Steven Benner, an astrobiologist at the
Foundation for Applied Molecular Evolution
in Gainesville, Florida, is more skeptical:
That GFAJ-1 uses arsenic as a replacement
for phosphorus, “is, in my opinion, not estab-
lished by this work,” he says.
Wolfe-Simon isn’t arguing that GFAJ-1
prefers, or even naturally uses, arsenic.
Mono Lake has a lot of phosphorus as
well as arsenic, and the strain grows better
when supplied with phosphorus. But to her
Search for unusual life. Felisa Wolfe-Simon and others, GFAJ-1 is proof that phospho-
collects samples from Mono Lake (left), where rus-free life forms can exist and may do so
high arsenic levels proved conducive to the somewhere on Earth. Next, Wolfe-Simon
evolution of arsenic-using microbes. wants to collect samples from places with
high arsenic but low phosphorus concen-
national University in Miami. “The impli- thrilled and surprised when one evening she trations in hopes of finding microbes that
cation of this work is that life can be quite checked the latest cultures under the micro- depend solely on the former.
different from what we know,” agrees Chan. scope and saw fast-moving bacteria. She Wolfe-Simon speculates that organisms
In 2009, Felisa Wolfe-Simon, a geomicro- rechecked the components of the culture like GFAJ-1 could have thrived in the arsenic-
biologist based at the U.S. Geological Survey media to confirm there were no phosphorus laden hydrothermal vent–like environments
in Menlo Park, California, and two colleagues contaminants. She and her colleagues then of early Earth, where some researchers think
argued that arsenic could have stood in for began to subject the microbes to sophisticated life first arose, and that later organisms may
phosphorus in ancient living systems. Phos- analyses to see if arsenic had been utilized have adapted to using phosphorus. Others say
phorus, in the form of the compound phos- by the bacteria. “I held my breath with every they’ll refrain from such speculation until they
phate, forms the backbone of strands of DNA one,” says Wolfe-Simon. see more evidence of GFAJ-1’s taste for arse-
and RNA, as well as ATP and NAD, two mol- One form of mass spectrometry showed nic and understand how the DNA and other
ecules key to energy transfer in a cell. Arse- that the arsenic was inside the bacterial cells biomolecules can still function with the ele-
nic, Wolfe-Simon pointed out, sits just below and not some impurity sticking to the outside ment incorporated. “As in this type of game
CREDITS: HENRY BORTMAN
phosphorus on the periodic table and has sim- of the cell. When the researchers added radio- changer, some people will rightly want more
ilar chemical properties. Indeed, its toxicity actively labeled arsenate to the bacteria’s cul- proof,” says microbiologist Robert Gunsalus
to people and most forms of life arises when ture, they were later able to discern its presence of the University of California, Los Angeles.
cells try to use arsenic in lieu of phosphorus. in the protein, lipid, nucleic acid, and metab- “There is much to do in order to firmly put this
Despite that, Wolfe-Simon speculated that olite fractions of the cells, suggesting that microbe on the biological map.”
some microbes might be able to adapt to using arsenic had been incorporated in molecules –ELIZABETH PENNISI

Published by AAAS
NEWS OF THE WEEK
ScienceNOW
From Science’s Online Daily News Site
Meet the Squidworm

Deep in the Pacific Ocean, scientists have discovered a remarkable
new creature that they have dubbed the “squidworm.” Karen
Osborn, then at the Scripps Institution of Oceanography in San
Diego, California, and colleagues used a remotely operated vehicle
to find Teuthidodrilus samae at the bottom of the Celebes Sea off the
eastern coast of Borneo. As the team reports online in Biology Letters,
the segmented worm sports a series of 10 large appendages near its
head that give it a squidlike appearance. http://scim.ag/squidworm

Black Hole May Offer Clues team has used genetic engineering to accom- 6 months—mice usually live for 3 years. Then
To Extra Dimensions plish something similarly curious in mice. the team created a new batch of mice with
String theory posits that space has more than Scientists don’t fully understand what the same infirmity but with a telomerase gene
three dimensions, curled up in loops triggers aging, but many suspect the gradual activated by a certain drug. The researchers let
so small that they could prob- shrinking of telomeres, the protective DNA these mice prematurely age, then at 6 months
ably be probed only in high- caps on the end of chromosomes. The they switched on the telomerase gene.
energy particle collisions. telomeres shrink as cells divide, and The burst of telomerase production
Now one theorist suggests telomerase, the enzyme that main- spurred almost total recovery, the researchers
these hypothesized extra tains the caps, isn’t typically active in report online in Nature. This indicates that
dimensions might reveal adult tissues. the cells that divide to replenish tissues don’t
themselves in the stars. To investigate the connection, simply die when their telomere clock expires,
According to Einstein’s molecular biologist Ronald DePinho but can be revived, says DePinho. Other
theory of gravity, matter of the Belfer Institute at the Dana-Farber researchers point out that these mice were
warps spacetime, changing the Cancer Institute of Harvard Medical School and genetically modified, so it still remains to
paths of free-falling objects. It even affects colleagues first genetically engineered mice to be seen if aging can be delayed in a normal
light; when light from a star passes near a lack a working copy of the telomerase gene. The mouse—or in a human.
CREDITS (TOP TO BOTTOM): LAURENCE MADIN/WOODS HOLE OCEANOGRAPHIC INSTITUTION; NASA; NASA; THINKSTOCK
galaxy, its path bends, changing where the animals aged prematurely and died at about http://scim.ag/aging-mouse
star appears in the sky—an effect called
gravitational lensing.
Now, physicist Amitai Bin-Nun of the Giant Eruption Cut Down to Size
University of Pennsylvania argues in Physical Just how devastating was the eruption of
Review D that gravitational lensing around the Indonesian “supervolcano” Toba 74,000
the supermassive black hole thought to be at years ago? Some researchers have suggested
the center of the Milky Way might provide a that it locked the world in a deep freeze,
way to search for extra dimensions. Bin-Nun whereas others have put the cooling at only
used numerical simulations to show that, in 3˚ to 5˚C. Similarly, some archeologists
a world with extra dimensions compared with have proposed that the explosion impacted
one without, a star known as S2 could be as human evolution, creating a genetic “bottle-
much as 44% brighter when it reaches its neck” where only the survivors’ genes got
peak brightness in 2018. passed on; others have argued that people Toba’s crater, now
But researchers say the hypothesis will be would have survived the eruption handily. a lake (inset).
hard to validate with telescopes, and if the Now researchers led by climate modeler
black hole is rotating, it would produce effects Claudia Timmreck of the Max Planck Institute for Meteorology in
that could easily be confused with Bin-Nun’s Hamburg, Germany, have weighed in with a climate model that
predictions. focused specifically on the way the blast’s sulfate aerosol particles cooled the atmosphere
http://scim.ag/extra-dimensions by reflecting the sun’s rays. Assuming that Toba pumped out about 850 million metric tons
of sulfur, the number used in previous models and supported by ice core data, tempera-
The Curious Case of the tures would have dipped only 3˚ to 5˚C across the globe, the team reports online in
Backwardly Aging Mouse Geophysical Research Letters. Overall, Toba didn’t wipe out flora and fauna, Timmreck
In F. Scott Fitzgerald’s short story, “The Curi- says, but it would have made life harder for a few years. http://scim.ag/toba-impact
ous Case of Benjamin Button,” an old man
gets younger with each passing day. Now a Read the full postings, comments, and more at http://news.sciencemag.org/sciencenow.

Published by AAAS
NEWS OF THE WEEK
U.S. RESEARCH UNIVERSITIES
Panel Explores New Funding Pact With Washington

The leaders of U.S. higher education have inherent conservatism within academia. His how those rules put an undue burden on uni-
complained for a decade or longer that the telephoned comments struck a chord with versities and force them to make spending
unwritten arrangement between the federal the panel, which also welcomed Alexander’s choices they shouldn’t have to make.”
government and the universities, struck in the suggestion that they compile separate rank- The gloomy fiscal outlook led panel mem-
1950s, is badly strained. According to that ings of “10 things that cost money and 10 ber James Duderstadt to propose a bold quid
arrangement, the government would pay the things that don’t.” pro quo in line with Alexander’s description
full cost of university research it funds and, “If you assume that the amount of research of the problem. What if, he said, the com-
in return, universities would educate the next funding is fixed, then the only question is how munity abandoned its perennial request
generation of scientists. Over time, however, it’s divvied up,” says Chad Holliday, chair of for more—on the grounds that investing in
a growing number of federal regulations and the panel. “So I hope that we can come up research is a public good that will pay large
a cap on reimbursement rates have led to bil- with a robust set of recommendations, some dividends—and accepted flat funding in
lions in university research costs that aren’t of which do not require more money.” return for the government allowing universi-
reimbursed. Rising federal spending has The panel’s charge deliberately mimics ties to be reimbursed fully for what they con-

helped keep the relationship intact, but now a the 2005 request from Congress that led to sider the true cost of hosting federally funded
fiscal crisis threatens to rip it apart. Rising Above the Gathering Storm, the acad- research? He also suggested an easing of
That was the backdrop to an intense meet- emies’ improbably influential report on how the regulatory burden, which he speculated
ing last week at the National Academies in the overall U.S. scientific enterprise could might save enough in administrative costs to
Washington, D.C. A blue-ribbon panel asked strengthen the economy. The higher edu- nearly eliminate the shortfall in paying for the
by Congress to recommend how the coun- cation community, which planted the seed so-called indirect costs of research: adminis-
for the congressio- tration, buildings, utilities, etc.
nal request, hopes Duderstadt says his idea is just a trial bal-
the new report will loon to address a problem “that we’ve spent
address what it sees 40 years debating and haven’t made a dent
as glaring flaws in in solving.” But Robert Berdahl, president
the system that gov- of the Association of American Universities,
erns the $32 billion an organization of 63 research powerhouses,
a year that the fed- joined many in the room in voicing support
eral government now for it. “I don’t think we’ll see an increase in
spends on academic the federal investment in research in the fore-
research. seeable future so, yes, I think we’ll have to
At the top of the redistribute the money in a different way,”
list, university lead- Berdahl said. “I would support an increase in
ers want changes in funding at the inflation rate,” he added, a rise
the formula used to much flatter than the 7- to 10-year doubling
reimburse universi- that the community has long sought.
ties for the ancillary At the same time, Duderstadt acknowl-
Cut-rate science? Federal grants don’t cover the true cost of academic research, costs of conduct- edged that increasing reimbursement for
say university administrators. ing federally funded overhead costs, in a flat budget, would leave
research on campus. agencies with less money for research grants.
try’s research universities can bolster the The negotiated reimbursement rate is too low That would increase competition among fac-
nation’s well-being (Science, 9 July, p. 126) and allows too many exceptions, they say. As ulty members already bemoaning low success
spent a day and a half in public discussion a result, universities must dip into their own rates on their applications. “Would your fac-
with several dozen leaders in higher educa- coffers or curtail other educational activities. ulty support that approach?” wondered pan-
tion, followed by a day of private delibera- “We fail to recover $600 million a year,” says elist Walter Massey, president of Morehouse
tions aimed at producing a final report next Steven Beckwith, vice president for research College in Atlanta and a former director of
summer. Overshadowing the discussions was for the 10-campus University of California the National Science Foundation. “They want
growing concern that federal investment in system, tallying up the shortfall caused by a a grant with lower, not higher, indirect costs.
academic research may shrink as the govern- cap in so-called indirect cost recovery rates Could you bring them around?”
CREDIT: RICHARD SLATTERY/GETTY IMAGES
ment struggles to reduce the federal deficit, and by various exceptions to those rates. Several university administrators thought
even as states are cutting support to their flag- University leaders also want the gov- they could, provided the changes were
ship public research institutions in an attempt ernment to ease some of the administrative explained properly. “The challenge is to be
to balance their recession-battered budgets. requirements that come with federal research consistent and understandable,” said Kim
Senator Lamar Alexander (R–TN), one dollars, saying they drive up costs without Wilcox, provost and vice president for aca-
of four science-savvy legislators from both providing any significant societal benefits demic affairs at Michigan State University in
parties who requested the study, summed or protections. “When I began I wasn’t sure East Lansing. “If we can do that, then it’s up
up the community’s predicament this way: about that,” says Holliday, a former DuPont to us to explain it to the faculty.”
too many rules, not enough money, and an CEO. “But after our discussion, I now see –JEFFREY MERVIS

Published by AAAS
NEWS OF THE WEEK
U.S. ENERGY POLICY
With Money Tight, White House Panel From the Science

Policy Blog
Offers New Path to Energy Research The Howard Hughes Medical Institute
In a period of avowed austerity in Washington, is holding a competition to support the
D.C., can the government spark a techno- research programs of early-career scien-
logical revolution in energy without raiding tists trained in the United States who
the U.S. Treasury? A new report embraced have now returned home. Up to 35 scien-
by the White House says yes. Small sur- tists in 18 countries will receive $650,000
charges on energy production and use could over 5 years. The deadline is 23 February.
raise the billions of dollars needed for addi- http://scim.ag/Hughes-international
tional research and development, it says,
and a quadrennial energy review and other Two car bombings in Tehran killed one
administrative changes would help spend Iranian nuclear scientist and wounded a
the money wisely. But even fans of the plan,

second in circumstances similar to the slay-
which echoes other recent high-level reports ing of a theoretical physicist in January.
on the topic, say it faces sizable political and Government leaders accuse the West of try-
logistical challenges. ing to dismantle Iran’s nuclear program.
“American economic competitiveness, http://scim.ag/iranian-bombings
environmental stewardship, and enhanced
security depend on picking up the pace of President Barack Obama has asked his
energy technology innovation,” says the bioethics council to conduct a “thorough
report, Accelerating the Pace of Change in review” of U.S. rules protecting human
Energy Technologies Through an Integrated Fill ‘er up. A 2-cent-per-gallon fee on subjects to ensure that they “protect
Federal Energy Policy, from the President’s liquid fuels could help fund a tripling of people from harm or unethical treatment,
Council of Advisors on Science and Tech- spending on energy R&D, a study says. domestically as well as internationally.”
nology (PCAST). It notes that “extraordi- http://scim.ag/human-subjects
nary actions” by the government are needed
to do so. the report was written. The review could be a An expert panel says the Dutch government
The report says that current federal useful process for defining and coordinating failed to mount a robust response to the
energy R&D spending should be tripled, to energy policy, agreed David Goldston of the world’s worst outbreak of Q fever, a disease
$16 billion a year. Its innovative approach Natural Resources Defense Council in Wash- transmitted from farm animals to humans
to obtaining the needed funds is likely ington, D.C., as long as it can avoid reaching that has killed 14 people and sickened
to make waves. Running for president in bland conclusions or drawing a “map as big as thousands more since the outbreak began in
2008, Barack Obama proposed a 10-year, the territory” it is examining. 2007. http://scim.ag/Q-fever-response
$150 billion fund for energy funded mostly At the same time, warned Goldston, a
by revenue from a national greenhouse gas former staff director of the House of Repre- University officials say it’s not clear that
emissions trading scheme. Even months sentatives science committee, collecting “rev- new immigration policies in the United
before that scheme was declared dead in enue streams” for innovation from various Kingdom will do more to allow the free flow
Washington, PCAST member Maxine energy sources could create a “patchwork of of foreign scientists into the country.
Savitz told Science, members looked at other separate energy policies.” Political or legal http://scim.ag/uk-visa-policies
sources, in particular surcharges on energy entanglements can stymie surcharge systems,
transmission or partnerships with industry. Simon added, pointing to a $23 billion fund The Croatian government has backtracked
A one-tenth of a cent surcharge for every for nuclear waste storage, funded by small on a plan to store all of the country’s low-
1 kWh of electricity used, for example, could charges on electricity produced by nuclear and medium-level nuclear waste at a
generate $4 billion, the report says, as could power, that’s never been spent on storing fuel. major research institute in the heart of its
a 2-cent-a-gallon charge on liquid fuel. Despite those challenges, the report’s capital. http://scim.ag/zagreb-waste-reversal
But the government needs a “more coordi- backers hope that the message in its 39 pages
nated and robust” effort to spend that money will find a receptive audience among those Germany’s high court has upheld a law
well, the report declares. Toward that end, who feel that action is urgently needed. By governing genetically modified crops
PCAST called for a “Quadrennial Energy coincidence, Energy Secretary Steven Chu, that requires a buffer zone and a pub-
Review (QER),” led by the Department of who requested the report, spoke about the lic database of planting, and that holds
Energy and involving all relevant federal need for energy innovation during a previ- researchers who plant GM crops liable for
CREDIT: ISTOCKPHOTO.COM
agencies, industry, and the public. The QER ously scheduled talk to the national media any pollen that escapes.
“could reduce bureaucracy” by getting federal only 2 hours after the report’s release. “Amer- http://scim.ag/german-gm-law-upheld
agencies “reading from the same memo,” says ica still has the opportunity to lead in a world
Robert Simon, staff director for the Senate that needs a new energy revolution,” he said. For more science policy news, visit http://
Energy and Natural Resources Committee, “But I think time is running out.” news.sciencemag.org/scienceinsider.
who gave input to PCAST members before –ELI KINTISCH

Published by AAAS
NEWSFOCUS
Brazilian Science:

Riding a Gusher
A fast-growing economy and oil discoveries are propelling Brazil’s
research to new heights. But scientific leaders must overcome a
weak education system and a low-impact track record
NATAL—Miguel Nicolelis stands with his Brazil’s poorest regions. Nicolelis, who now place is going to create the next generation
arms spread, pointing out a rectangular pit spends part of the year in Brazil, is eager to of Brazilian leaders.”
carved into dry earth on the outskirts of the offer a visitor “categorical proof ” of success. Some continue to think Nicolelis’s idea
Brazilian beach city of Natal. “That is where He’s built two hands-on science schools for is eccentric. But his timing couldn’t have
the supercomputer will go,” he says. And, children and a maternity clinic, been better. Over the past 8 years,
pointing to an area still thick with shrubbery,
“that is the sports complex.”
and recruited 11 Ph.D. neuro-
scientists who run labs in a tem-
Online
sciencemag.org
Latin America’s largest nation
has begun to boom. Its economy
Nicolelis is Brazil’s best-known scientist. A porary headquarters. Within a few Podcast interview
is growing fast, and it has become
neurobiologist at Duke University in Durham, months, he says, $25 million in with author a player in world affairs, reveling
North Carolina, he is renowned for spectacu- Brazilian federal money should Antonio Regalado. in an unprecedented bout of self-
lar experiments that use signals tapped from begin pouring into his sandy acre- confidence. It will host the World
the brains of monkeys to make robots walk. age, creating the sprawling neuroscience com- Cup in 2014 and the Olympics 2 years later.
But when Nicolelis launched plans in 2003 for plex Nicolelis calls his “Campus of the Brain.” The good times are lifting science, too.
a neuroscience institute in Brazil’s backward “In Brazil we need science to build a Between 1997 and 2007 the number of Bra-
northeast districts, few thought it could work country,” says Nicolelis, an energetic nation- zilian papers in indexed, peer-reviewed jour-
(Science, 20 February 2004, p. 1131). alist whose passions include wearing a nals more than doubled to 19,000 a year. Bra-
CREDITS (TOP TO BOTTOM): DANNY LEHMAN/CORBIS; A. REGALADO/SCIENCE

The notion was to pair cutting-edge sci- green Palmeiras soccer club hat and down- zil now ranks 13th in publications, accord-
ence with a social mission: developing one of ing pitchers of yellow maracujá juice. “This ing to Thomson Reuters, having surpassed
the Netherlands, Israel, and Switzerland.
Brazil’s universities awarded twice as many
Ph.D.s this year as they did in 2001, and thou-
sands of new academic jobs have opened up
on 134 new federal campuses.
It’s a reversal of fortune for a nation that
during the 1990s was beset by dire economic
problems. Back then, researchers scrounged
for funds; Brazil even saw its flag removed
from the logo of the International Space Sta-
tion after it failed to come up with funding
Proud moment. Neuroscientist Miguel Nicolelis

at the foundations of his “Campus of the Brain” in
northeastern Brazil.

Published by AAAS
SCIENCE IN BRAZIL NEWSFOCUS
Science hub. Brazil’s richest city, São Paulo, and

eponymous state are home to most of the nation’s
research.
Manaus
to build six components. “We kept thinking Amazon
smaller and smaller,” says Sérgio Rezende, Natal
science minister for the past 5 years. “If we
couldn’t solve small problems, how could we
solve big ones? Now we are in a position to
think big again.”
The fuel that propels science in Brazil is an
R&D tax on big industries; it has swelled the
budget of Rezende’s ministry to $4 billion, up Brasília
from $600 million a decade ago. The national
oil company, Petrobras, is the largest contrib-
utor (see sidebar, p. 1308). Brazil restarted
its nuclear research program in 2008, after a

20-year lull, and in October, a delegation trav- BRAZIL
São Paulo
eled to Geneva to negotiate associate mem- Rio de Janeiro
bership in CERN. With Brazil’s economy
growing at a pace of 7% this year, the country
can afford the dues of $14 million per year.
Scientists here say their arguments in favor
of more education, innovation, and technol- Highlights
ogy have been heard in Brasília, the capi-
tal, and they expect budgets to keep grow- Amazon: Half as large as Europe, the Brazilian Amazon is home to fewer than 3000
ing under Brazil’s president-elect, Dilma Ph.D. scientists. Government incentives seek to increase Brazil’s scientific presence in
Rousseff, the first woman to hold that post. the forest region.
By 2020, say officials of the Brazilian Soci-
ety for the Advancement of Science, Brazil
Brasília: Brazil’s rapid economic growth means rising budgets at the Ministry of
should again redouble or triple output of stu- Science and Technology. It now spends about $4 billion a year.
dents, papers, and spending and become a
“formidable” force in science. Government
Natal: Construction is beginning on a $25 million “Campus of the Brain” conceived
by Miguel Nicolelis, a neuroscientist at Duke University.
officials want to see Brazil among the top 10
science nations. Rio de Janeiro: A massive new R&D facility built by oil company Petrobras is billed
But Brazil is not formidable yet. Like as the largest laboratory in the Southern Hemisphere. Workers will explore
Nicolelis’s institute—where construction is deep-water oil deposits.
several years behind schedule—Brazil’s sci-
entific output trails its ambitions. The coun- São Paulo: The wealthiest state in Brazil is also its science leader. São Paulo
try produces few high-impact papers and researchers publish half of all Brazilian papers.
Brazilian Science Begins to Boom

Brazilian Papers R&D Spending as % of GDP U.S. Patents for Selected Countries
SOURCE: MCT/Thomson Reuters 32,100 SOURCE: IADB SOURCE: USPTO
54.4%
2.7% 3.44% JAPAN
35 3.5
Number of papers U.S.A.
U.S.A.: 82,382
30 % Brazil of Latin America 3.0 CHINA
% Brazil of World 2.68% JAPAN: 35,501
U.S. Patents, 2009
RUSSIA
2.5 GERMANY: 9,000
(Thousands)
BRAZIL
GRAPHIC CREDIT: N. KEVITIYAGALA/SCIENCE
25
CHILE SOUTH KOREA: 8,762
17,714
(%)
2.0 CHINA: 1,655

20 47.6% MEXICO
1.8% 1.49% COSTA RICA INDIA: 679
1.5
15 1.13% 1.11% COLOMBIA RUSSIAN FEDERATION: 196
6,038 1.0 BRAZIL: 103
39.1% 10,521 0.67%
10 0.8% 0.46% MEXICO: 60
42.9% 0.5 0.32% ARGENTINA: 45
1.4% 0.16%
5 0.0
1995 2000 2005 2009 2007 0 20,000 40,000 60,000 80,000 100,000
2009 figure elevated due to increase in number of indexed journals Based on country of residence of first listed inventor

Published by AAAS
NEWSFOCUS
only a trickle of patents. Its pri- can countries, Brazil’s research

mary and secondary public edu- base is heavily skewed toward
cation system is in shambles, agriculture, ecology, and infec-
leaving the nation of 195 mil- tious diseases—it is first in the
lion chronically short of tech- world in publications related to
nical workers. “We need to be sugar, coffee, and orange juice.
lucid and not fall into a victory Brazil’s cattle industry produces
discourse,” cautions Sidarta 33% of the world’s cow embryos.
Ribeiro, a neuroscientist trained Once a sideshow, such research
at Rockefeller University in is increasingly well placed to
New York City and co-founder address global preoccupations
of Nicolelis’s brain institute. over food production, climate
“In terms of impact we are mar- change, and conservation.
ginal. The external discourse for Nicolelis says he sees an
the world should be that we are “emerging tropical way of doing
interested in science and we are science” driven by research on
growing. The discourse inter- renewable energy, agriculture,

Tweaking nature. Researcher in a government research lab in Brasília inspects
nally should be, ‘Let’s improve. a transgenic cotton plant. water, and animal and plant
Let’s focus on merit.’ ” genetics. “These are the issues
tina took only 3 years ago and which neighbor defining the planet, and, believe it or not,
Boom times Bolivia is still debating. “Brazil is the only the players are down here,” says Nicolelis.
Brazil is clearly breaking away from the pack example in Latin America where 1% of GDP Biological research is a hot area of growth.
in Latin America, indicators show. Brazil now goes into R&D and the science minister is a The Empresa Brasileira de Pesquisa Agro-
accounts for over 60% of all research spend- physicist that still publishes. So Brazil is the pecuária, the government-owned agricul-
ing in Latin America, and Brazilian scien- beacon,” says Juan Asenjo, president of the tural research company known as Embrapa,
tists write half of the papers. Brazil’s science Chilean Academy of Sciences. plans to hire 700 new researchers this year.
bureaucracy is influential, too, having had its Globalization of markets is also working Embrapa is considered one of the world’s
own ministry since 1985. That’s a step Argen- in Brazil’s favor. Like other Latin Ameri- premier agricultural units, and its budget
October, Petrobras inaugurated a sprawling new $700 million research center

Tapping a Deep, ‘Pre-Salt’ Bounty in Rio de Janeiro. At the event, da Silva, a former union leader with a fourth-
Three years ago, a drill bit struck immense oil deposits deep off the coast of grade education, left no doubt what the vast R&D complex represents to him:
Brazil. Petrobras, the national oil company, tapped undersea fields now esti- “Brazil will never have to lower its head to anyone again,” he roared to a bois-
mated to hold about 80 billion barrels of oil and natural gas—about three terous crowd of oil workers.
times the size of the reservoir under Prudhoe Bay, Alaska. It brought the prom- Deep-water petroleum exploration is Brazil’s largest technology project,
ise of new wealth and expectations that Brazil will climb to the world’s top rung and Petrobras’s money is pouring into research labs throughout the coun-
of achievement in science and technology. try. In order to retrieve the oil, which lies a daunting 7 kilometers below the
Brazilian President Luiz Inácio Lula da Silva once termed the oil strike “a ocean surface, Petrobras has opened a fire hose of funding that is “chang-
second independence for Brazil” and promised to use the oil revenue for edu- ing the face of science in Brazil,” says Angela Uller, dean for research at the
cation and public health. But Brazil’s R&D sector has been first to benefit. This Federal University of Rio de Janeiro, whose campus on an island outside the
city also houses Petrobras’s R&D Center,
known as Cenpes.
Petrobras now spends about $1 billion
CREDITS (TOP TO BOTTOM): DEVA RODRIGUES/EMBRAPA; PETROBRAS

a year on R&D, including some $225 mil-
lion that goes directly to universities, for
which Petrobras has been rushing to outfit
laboratories, erect new geophysics centers,
and train a new generation of engineers.
“We want to transform the technological
capabilities of Brazil and help build univer-
sity labs equal to any in the world,” says
Oil money. National Carlos Tadeu da Costa Fraga, head of Petro-
oil company Petrobras bras R&D operations.
inaugurated a $700 Rio’s engineering school, known as
million research center COPPE, is the biggest single beneficiary of
in Rio de Janeiro the oil gusher. Petrobras has paid for the
in October. construction of numerous laboratories on
campus, including the world’s deepest wave

Published by AAAS
SCIENCE IN BRAZIL NEWSFOCUS
of $1 billion is now the same size as that of unit. Countless shiploads of soybeans that we needed help, but where could we work
the U.S. Department of Agriculture’s Agri- embark for Asia every day from Brazilian together,” de França says.
cultural Research Service. “I’ve never seen ports could instead, he says, power domestic
so many resources for science as in the last industries in lipochemistry and plastics that Private money
5 years,” says Maria Fátima Grossi de Sá, a produce “value-added products.” The most important goal right now, by
plant geneticist who recently received $1.5 The project represents an important shift Rezende’s reckoning, “is for science to make
million to develop a transgenic cotton plant. in Brazilian thinking: namely, that science a difference in the productivity of industry.
De Sá works at Embrapa’s research sta- can transform the nation’s economy, cur- I’d have to say that is our great challenge.”
tion in Brasília, which is also finishing tests rently dominated by commodities like soy, Other goals are to increase the number of
on a herbicide-resistant soybean that will be beef, sugar cane, iron ore, and petroleum. scientists, invest in strategic areas, and solve
the first genetically modified crop designed “The new Brazil will be a natural knowledge key social problems.
by Brazilian scientists to reach the market. economy,” says Gilberto Câmara, head of The disconnect between science and busi-
Demand for Ph.D. scientists is running so Brazil’s space agency. ness is almost total in Brazil, researchers say.
high that de Sá says it’s difficult to find people With more money and an emerging In the United States, about 80% of research
to take postdoc positions. “We’ve gone pretty green-science mission, Brazilian research- personnel work in industry, according to
rapidly from having trouble placing Ph.D.s to ers say they’re being taken more seriously. OECD data, whereas in Brazil, that figure
having stipends that don’t have takers.” Most of Embrapa’s senior scientists were hovers near 25%. Brazil produces hardly any

Embrapa has nearly completed a four- trained in the United States, like Executive patents—just 103 U.S. patents were issued to
story, $15 million agro-energy center that Director José Geraldo Eugênio de França, inventors in Brazil in 2009—and Brazilian
will employ 100 researchers on the campus in who in 1987 traveled to Texas A&M Univer- companies spend half of what European ones
Brasília. One goal is to turn Brazil’s 22 mil- sity to study sorghum genetics. De França do on R&D. When they do spend, it’s often
lion hectares of soybeans into more valuable says he noticed a change during a mission to to import technology rather than develop it.
products, such as biodiesel. “We capture solar Washington, D.C., last November, where he Researchers say Brazil’s 20-year dic-
energy and turn it into other forms of energy. met U.S. science adviser John Holdren and tatorship, which ended in 1984, is partly
We think we can move very quickly from other officials. “For the first time in history, to blame for the lag. Universities became
agriculture for food to agriculture for energy. we had a recognition that something was redoubts of political opposition and Marx-
We can be a player,” says Frederico Ozanan changing in Brazil. They didn’t ask us how ist reading lists, where patents were viewed
Machado Durães, general director of the new many postdocs we needed to send, or where as oppressive. “We isolated ourselves from
pool, used to test automobile-sized models of oil platforms. “It’s starting to look
like Dubai around here,” says Segen Farid Estefen, a director of COPPE, which
gets about $60 million a year from Petrobras. He says the industry-academic
complex on the island is the “largest offshore oil research cluster in the world.”
Petrobras, founded in 1947, began to follow the scent of oil offshore in
the mid-1970s, investing in R&D to extend its reach. Brazil was importing
equipment from the North Sea and the Gulf of Mexico and adapting it to
tropical conditions. But Brazil’s decision to pump its own oil demanded grow-
ing investment in R&D.
“You cannot simply cut and paste,” says Martin Landrø, deputy chair
of petroleum engineering and applied geophysics at the Norges Teknisk-
Naturvitenskapelige Universitet in Trondheim, Norway. “You have to build
up competence, and the easiest way to do that is to build up research. You
have to bite the apple, so to speak.”
Landrø, who has visited Brazil three times to give courses to Petrobras
Petro power. With workers in 2008, President Luiz Inácio Lula da Silva celebrates
CREDIT: HO NEW/REUTERS; (BACKGROUND IMAGE) GOOGLE MAPS
geophysicists, says he’s noticed an accelerating change in Brazil. “They have the “second independence” oil discoveries will give Brazil.
maneuvered from the position of being not so competent to being on the cut-
ting edge in 10 years,” says Landrø. and later encased beneath thick layers of salt. These are “a new kind of geo-
Petrobras, the world’s largest deep-water oil producer, is reaching logic play. They are new types of reservoirs and there are lots of things being
depths where experience is scarce or nonexistent. At the Laboratory for learned,” says William Fisher, a geologist at the University of Texas, Austin. One
Non-Destructive Testing, Corrosion and Soldering, for instance, four COPPE critical difficulty is spotting the oil reservoirs beneath the salt domes, frequently
professors work alongside 30 Petrobras engineers to submit steel to corro- over a kilometer thick; seismic signals are hard to interpret. “As far as the poten-
sive hydrogen sulfide gas at extreme pressures. “At 7000 meters [below sea tial discoveries—what is the potential volume of oil and gas—well, you can
level], we don’t have any information about how materials perform, or how hazard all kinds of guesses, but it’s going to be big,” says Fisher.
long they can last,” says Oscar Rosa Mattos, director of the lab, which Petro- Estefen hopes that Brazil’s exploration of the ocean does not stop at oil.
bras paid $30 million to build in 2008. “My foreign visitors are surprised He says the country could use its deep-water expertise to be at the forefront
when they encounter a facility like this in Brazil.” of wave energy and undersea communications, too. “The analogy I use is that
The superdeep petroleum deposits now being discovered are in the “pre- deep-sea exploration can do in Brazil what the space race did for the United
salt” zone, an area where organic matter was deposited 125 million years ago States,” Estefen says. “If Brazil only pumps oil, it would be a big loss.” –A.R.

Published by AAAS
NEWSFOCUS
the big industries, which supported the mili- ogy transfer offices, which many are doing
tary. They couldn’t come in the university. for the first time. At the Federal University
The university became closed, hermetic, and of Minas Gerais, the number of patent appli-
now we have to change that,” says Maria cations has reached 356, including one for a
Bernardete Cordeiro da Sousa, dean for canine vaccine against leishmaniasis, now on
research at the Federal University of Rio the market. “All these things are leading to
Grande do Norte. resonance in the system,” says Ado Jorio, the
Officials have been trying to close the professor who coordinates the university’s
innovation gap. In 2004 and 2005, Bra- patent efforts. “There has been an explosion
zil passed laws giving R&D tax breaks to in publications, and this is also going to hap-
companies and began allowing the Minis- pen in innovation.”
try of Science and Technology to give com-
panies grants, even pay salaries of indus- Share the wealth
trial researchers. In August, the ministry Brazilian science suffers another imbalance,
announced a major industrial R&D program, between the wealthy south and the poor
offering $294 million in grants to back inno- northern regions, that officials have put a
vation projects inside companies in “strate- priority on trying to correct. Most science

gic areas” including electric cars, pacemak- P.h.D.-in-chief. Physicist Sérgio Rezende has kept still occurs in just three southern states, with
ers, and genetically engineered crops. publishing while running Brazil’s science ministry. the University of São Paulo alone account-
It’s too early to say if the government ing for nearly a quarter of all scientific publi-
incentives are working; only a small number he wanted the MIT [Massachusetts Insti- cations. “One of the great questions we face
of companies signed up for the tax breaks. tute of Technology] of Vale,” recalls Mello. is that Brazilian asymmetry, the inequality
But U.S.-style, risk-taking innovation, once “I was being invited to lead something that of the regions,” says Lucia Melo, head of the
viewed as alien, is increasingly seen in favor- would be a new Bell Labs or Xerox PARC.” Center for Strategic Studies and Manage-
able terms. Venture capitalists have begun Mello traveled this fall to Silicon Valley ment in Science, Technology and Innova-
setting up shop in Brazil, and in 2010 both to get ideas. Although Vale’s business is low- tion, a government science policy think tank
IBM and General Electric announced plans tech, the commodity company, which ships in Brasília.
for research centers in the country. vast amounts of ore to China and Europe, To push science out into Brazil’s neglected
“We lack a culture of innovation and wants to spend heavily on research partly hinterlands, Brazil’s government has gone on
entrepreneurship. There is a long path ahead because it faces a sharp shortage of skilled a binge of university construction and ear-
to change that,” says Luiz Mello, a physi- labor, increasing pressure from environ- marked 30% of research funds for poor north-
cian who last year was tapped by Brazil’s mentalists, and competition from global ern and western states. Under a 2009 program
second largest company, the iron-ore miner companies. Vale’s three labs will work on called “Stipends for Everyone,” officials in
Vale S.A., to spend $180 million establishing biodiversity, renewable energy, and mining Brasília said they would give study grants
three new corporate science institutes. Mello technology. “This is the biggest spontaneous to all graduate students in distant regions,
says he was hired after approaching Vale’s investment in R&D that I know of in Brazil,” regardless of academic merit.
CEO, Roger Agnelli, to raise money for an says Mello. The idea flows from Brazil’s governing
engineering program. “It turned out to be a The new laws also encourage Brazilian Workers’ Party, which has made improving
meeting for him to say what he wanted. And universities to file patents and set up technol- conditions in poor areas a priority. A greatly
expanded welfare program has helped move
several million Brazilians out of poverty. It
has given researchers breathing room, too.
“Before, we’d face the question, ‘Why are
you giving a monkey food and milk when
there is a hungry child next door?’ ” says
Cordeiro da Sousa, who is also a primate
researcher. But she sees a tradeoff: Research-
ers feel increasing pressure to spend time
solving local problems. She’s considering
CREDITS (TOP TO BOTTOM): GE GLOBAL RESEARCH; MCT
creating a salt institute to support the local

salt-mining industry. “You have to have a
vocation, because in the future we could be
called to answer in a big way.”
Nowhere is the dearth of Brazilian-led sci-
ence of greater concern than in the Amazon,
the tropical forest that accounts for about 49%
Tropical innovation. An architect’s rendering of Brazil’s territory but is home to only about
of the General Electric global research center 3000 Ph.D. researchers, very few of whom do
near Rio de Janeiro. The lab, when built, will bench science. “Imagine what a totally irrele-
be GE’s fifth international R&D center.
vant number that represents for this immense
region,” says Odenildo Teixeira Sena, secre-

Published by AAAS
tary of science and technology in the state of
Amazonas. Although larger than France and
Spain combined, Amazonas has only a single
resident Ph.D. archaeologist, and despite its
vast river systems, no naval engineers, Teix-
eira says.
Increasing scientific labor power in the
region could help find alternatives to slash-
and-burn agriculture. But national anxiet-
ies figure into the calculation as well. “The
majority of publications on the Amazon don’t
have a Brazilian author. That is a worry for
us,” says Jorge Guimarães, the Ministry of
Education official who oversees higher edu-
cation in Brazil. “We need more Brazilians
participating.”
Brazil has never felt secure in its control

over the vast region, which Spain ceded to
Portugal under the 1750 Treaty of Madrid.
With the Amazon a focus of international Talented But Underfunded: Brazil’s Future Scientists
maneuvering on carbon credits, Brazil’s
dependence on foreign knowledge produc- It’s an unusual prelab routine. Wake up in a below-ground apartment where five people sleep
tion has become a “very delicate question,” (four on the floor), grab your books, then head out past the machine gun–toting gangsters who
says Adalberto Val, director of the Insti- guard your street against police vehicles. That’s been reality for 25-year-old Reinaldo Sousa dos
tuto Nacional de Pesquisas da Amazônia in Santos, a Ph.D. candidate in biochemistry at the Federal University of Rio de Janeiro (UFRJ) and a
Manaus. During a national science and tech- resident of Parque União, a crowded favela where residents live under the thumb of a drug gang.
nology conference last May, Val called for Dos Santos owes his journey from shantytown to lab bench to his mentor, Leopoldo de Meis, a
Brazilian “informational hegemony” over the 72-year-old professor of biochemistry at UFRJ. In 1985, de Meis began offering a hands-on sci-
forest biome. “There is a question of national ence course for low-income adolescents called Young Talents. Dos Santos enrolled when he was
sovereignty,” he says. 14, a year after his father died, leaving him orphaned.
Such nationalist tones may sound harsh “I don’t want people to think I am the poor little kid from the favela,” says Dos Santos, a
outside Brazil, but they play well at home. fast, concise talker who is studying the metabolic response of goldfish to low temperatures.
Physicist Luiz Davidovich, who chaired the Although he may sleep some nights on the laboratory couch, “intellectually I don’t think I am
May conference, says Brazil’s scientific com- below anyone.”
munity needs to raise “big flags” to rally the Brazil must write thousands more stories like Dos Santos’s if it is to overcome deep social
country around. “ ‘The Amazon is Ours’ is divisions and achieve its dream of becoming a major player in scientific research. Many say the
one of those,” he says. task must begin with improving public schools, where poorly paid teachers offer rote lessons.
Even some foreign experts have Brazil’s math and science scores vie for the worst among 57 countries ranked on the so-called
responded to the call. Daniel Nepstad, a PISA scale by OECD, barely edging out Tunisia. College is mainly for the elite. Prestigious federal
prominent American tropical forest ecolo- universities in Brazil offer free education, but it’s difficult to pass tough entrance exams unless
gist, dropped his job in October at Woods families have paid for private secondary schools. “We have had a perverse system of social apart-
Hole Research Center in Massachusetts heid, where the poor don’t have access to higher education,” says Luiz Davidovich, a physicist at
to become a Brazilian resident and full- the university who sits on the board of the Brazilian Academy of Sciences. Only 14% of 18- to
time employee of the Instituto de Pesquisa 24-year-olds are in college, not enough to meet Brazil’s growing demand for researchers and
Ambiental da Amazônia, a nonprofit he engineers. “We’re working with just a sliver of the population,” Davidovich says.
co-founded, based in the city of Belém. In recent years, the government has tried to broaden access to education. Scores of state
Nepstad says his U.S. affiliation “was inter- technical schools have opened; since 1979, Brazil has hosted what is now billed as the world’s
CREDIT: FLÁVIA MARTINS; (BACKGROUND IMAGE) GOOGLE MAPS
preted that I am less committed to the scien- largest math olympiad. Another national program, ProUni, since 2005 has paid for 748,000
tific agenda in Brazil.” Brazil’s forest policy lower income students who can’t get into federal schools to attend private, for-profit universi-
is evolving rapidly and, Nepstad says, “as ties. Davidovich says the efforts fall short: “Brazil needs a revolution in education at all levels,
long as the science is led by Northerners, especially at the most basic levels.”
we are missing the opportunity to get really Dos Santos recognizes that his life is exceptional. “Most Brazilians don’t know any scientists
good information into policy decisions.” and don’t know what a scientist does,” he says. For him, science has proved both an intellectual
and financial life belt. His state stipend of $1000 a month (he gets another $180 for supplies)
Making it real makes him his family’s top earner, and this summer he helped his grandmother, uncle, and
Despite its growing ambitions, Brazil has cousins move into a larger house. Now that he has his own room, he hopes to decorate it with a
yet to prove it can do world-class basic metabolic chart like one he saw at a conference.
research. The impact scores of its scien- “I like to see the molecule in vivo doing something,” says Dos Santos of his career choice.
tific papers are modest, about two-thirds the “The lab is really a psychological escape from all the situations I live through. It’s my ideal place.”
world average, and have slid in some areas. –A.R.
No Brazilian has won a Nobel Prize in sci-

Published by AAAS
NEWSFOCUS
Brazilian officials have instead focused

on beating another problem: the insecurity
of research funds. In 2008, in its largest-
ever funding round for basic research, Bra-
zil’s Ministry of Science and Technology
offered $350 million over 3 years to fund 122
national institutes to tackle subjects from
quantum computing and stem cells to an
upgraded Antarctic research station. “They
saw that we needed long-term programs with
stability,” says Davidovich, who co-leads the
program on quantum computing.
Other scientists privately express doubts
about the grandiosely named institutes, not-
Missed opportunity? Many ing that in reality they are virtual networks
researchers have published studies
with an average of 20 university investigators
involving the Amazon region; most
each and money spread too thin to achieve

are not Brazilian.
much. In position papers, the Brazilian Soci-
ety for the Advancement of Science has said
ence or medicine, whereas regional rival has been trapped in customs for 4 months. that Brazil needs to focus on creating more
Argentina has three. Researchers blame “You can have the best head in the world pure research jobs outside of the university
structural problems at Brazil’s state-run and you will never be competitive because system. It wants a new, heavily staffed state
universities. Critics say they discourage the government works against us,” says institute to study the oceans, and another for
competition, for example, with automatic Bertolini. “When we begin thinking that way, the Amazon, modeled on the agricultural
tenure after 3 years on the job and evalu- we want to go back.” agency Embrapa—in this case with funding
ations that reward Portuguese-language Some say the prospects will remain bleak to match the grandiose vision.
publication. “The attitude for many years until such problems are solved. “I know of In the city of Natal, Nicolelis’s neuro-
was to avoid competition, keep your head no extraordinary science in Brazil,” says science institute, currently housed in a con-
low, and choose a marginal subject,” says Andrew J. G. Simpson, scientific director of verted hotel, has also yet to produce a Bra-
Ribeiro. Instead of competing head-to-head the Ludwig Institute for Cancer Research in zilian breakthrough. But it’s increasingly
on hot topics with big labs overseas, he New York City. A naturalized Brazilian cit- well-positioned to do so. It has reasonably
says, Brazilian researchers have sometimes izen, he lived in São Paulo for 7 years and equipped laboratories, a primate facility, and
been content to study local questions. “The coordinated one of Brazil’s memorable tri- a crowd of young professors with promising
thinking was, ‘The anteater is yours so don’t umphs, the sequencing of the plant patho- track records who have signed on, includ-
worry about the gringos.’ ” gen Xylella fastidiosa, which landed on the ing two recruited from a Max Planck center
Brazilian researchers returning from over- cover of Nature in 2000. But when Simpson in Germany. In August, the École Polytech-
seas, drawn by jobs and start-up funds, com- returned this year for a 10th-year celebration nique Fédérale de Lausanne in Switzerland
plain that there are still many obstacles that of the feat, he noticed that, at least in the field donated an IBM Blue Gene/L supercom-
make producing world-class science nearly of genomics, “there was never again a big puter, which Nicolelis says will be the fastest
impossible. After 11 years in the United impact paper. There was no upwards process. in South America.
States, biologist Luciana Relly Bertolini It was a blip.” Ribeiro, the Brazilian who returned from
returned to Brazil in 2006 with her a postdoc at Rockefeller to be the institute’s
husband, Marcelo, to start a labo- scientific director, says the year of science
ratory that aims to clone trans- he expected to lose while organizing the
genic goats. Although the effort center has dragged into three, as he faced
is adequately funded, Relly Ber- down customs officials and coped with
tolini says a heavy teaching load large numbers of poorly trained students. CREDIT (TOP TO BOTTOM): WORLD WIND/NASA; PAULO BRANDO/IPAM
required of professors and lack of “Now I’m finally starting to fight reviewers
trained staff means “it’s science by again instead of bureaucrats, which is a sign
persistence here.” the plan worked,” says Ribeiro, whose work
Also notorious are Brazil’s includes experiments to look at the effect of
Kafkaesque importation regula- sleep and dreaming on motor and percep-
tions. Even simple reagents can tual skill retention.
take months to arrive, with radio- The dirt road outside his building that
active or biological samples often leads to a nearby shantytown, he says,
in doubtful condition. Relly Ber- reminds him of a photograph he saw of Rock-
tolini says a cell-fusion instru- efeller’s Founder’s Hall after it was built in
ment she ordered from Hungary 1906 and still surrounded by muddy fields
Returnee. Tropical ecologist Dan Neps- and horse-drawn carriages: “They didn’t start
tad studies a controlled fire in Tanguro as the best place to do science either.”
Forest, Mato Grosso, Brazil, in August. –ANTONIO REGALADO

Published by AAAS
COMMENTARY
Rosalind Franklin’s image GE Essay Prize Winner
1326 1334
LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES
LETTERS
edited by Jennifer Sills
Fishing for Data in the Ross Sea Zealand. 17Ecology Department, Montana State University,
Bozeman, MT 59717, USA. 18Moss Landing Marine Labo-
ratories, Moss Landing, CA 95062, USA. 19Biodiversité et
WE ARE AMONG THE SCIENTISTS OBJECTING TO THE ECO-CERTIFICATION OF ROSS SEA ANTARCTIC gestion des territoires, Université de Rennes 1, UMR 7204,
toothfish (Dissostichus mawsoni), as described by E. Stokstad in his News Focus story “Behind Museum National d’Histoire Naturelle, 35042 Rennes
the eco-label, a debate over Antarctic toothfish” (24 September, p. 1596). The public perceives Cedex, France. 20Sustainability Office, University of Wis-
consin, Oshkosh, 650 Witzel Avenue, Oshkosh, WI 54902,
a certification by the Marine Stewardship Council (MSC) to mean an environmentally friendly USA. 21School of Biological Sciences and Gateway Antarc-
fishery, not one characterized by the “dearth of key data” as indicated in the article. tica, University of Canterbury, Christchurch, New Zealand.
Significant data deficiencies lead us to conclude that an eco-friendly label for this fishery 22
Department of Environmental Sciences, Applied Ecol-
ogy, University of Siena, 53100 Siena, Italy. 23Department
is scientifically indefensible. Credible life history data are missing: Spawning areas, eggs, and of Geosciences, University of Arizona, Tucson, AZ 85721,
larvae have never been found, spawning intervals are unknown, and most density-dependent USA. 24Department of Ecology, Evolution, and Behavioral
aspects of ecological relation- Biology, University of Minnesota, St. Paul, MN 55108, USA.
ships are undetermined (1, 2).
25
Virginia Institute of Marine Sciences, College of William &
Mary, Gloucester Point, VA 23062, USA. 26Hubbs-SeaWorld
Stock assessment is problematic Research Institute, San Diego, CA 92109, USA. 27Department
because severe Antarctic pack of Biological Sciences, University of Alberta, Edmonton, AB
ice conditions for more than 9 T6G 2E9, Canada. 28HR Wallingford, Ltd., Oxfordshire OX10
8BA, UK. 2917 Modena Crescent, Auckland, New Zealand.
months a year prevent scientists 30
School of Zoology, University of Tasmania, Sandy Bay, Tas-
from effectively using standard mania 7005, Australia.
models, which require random *To whom correspondence should be addressed. E-mail:
tagging over time, space, and age lkblight@interchange.ubc.ca
classes (3). The number of fish
harvested by illegal, unregulated, References and Notes
1. A. L. DeVries, J. T. Eastman, “Brief review of the biology
and unreported fisheries is likely
of Dissostichus mawsoni” (CCAMLR Doc WG-FSA-98/49,
substantial (4, 5). Finally, ecosystem effects of removing 50% of spawning biomass [the fish- CCAMLR, Hobart, Australia, 1998).
ery’s stated management goal (6, 7)] of this slow-to-mature species are unlikely to be neutral: 2. S. M. Hanchet, G. J. Rickard, J. M. Fenaughty, A. Dunn,
The large, adult toothfish targeted by the fishery are a key structural link in the food web of the M. J. H. Williams, CCAMLR Sci. 15, 35 (2008).
3. C. M. Brooks, J. R. Ashford, “Spatial distribution and age
Ross Sea (8 –11), currently the most pristine marine area on Earth (12). structure of the Antarctic toothfish (Dissostichus
As with MSC-certified fisheries elsewhere, toothfish certification requires that industry mawsoni) in the Ross Sea, Antarctica” (CCAMLR
eventually provide missing biological data (13, 14). However, the harsh Antarctic environment WG-FSA-08-18, CCAMLR, Hobart, Australia, 2008).
4. H. Österblom, U. R. Sumaila, Ö. Bodin, H. J. Sundberg,
makes data collection painstaking and often prohibitively expensive. Thus, such expectations A. J. Press, PLoS ONE 5, e12832 (2010).
are unrealistic for a commercially viable fishery. Instead of a certification that lacks proper 5. TRAFFIC, “Australia confiscates 130 km long deepwater
data, a moratorium should be placed on further Ross Sea fishing until the quality of science at gillnet, Press Release 6, November” (Traffic International,
least equals that of certified fisheries elsewhere (13). Cambridge, 2009).
6. A. J. Constable, W. K. de la Mare, D. J. Agnew, I. Everson,
LOUISE K. BLIGHT,1 DAVID G. AINLEY,2 STEPHEN F. ACKLEY,3 GRANT BALLARD,4 TOSCA BALLERINI,5 ROBERT D. Miller, ICES J. Mar. Sci. 57, 778 (2000).
L. BROWNELL JR.,6 C.-H. CHRISTINA CHENG,7 MARIACHIARA CHIANTORE,8 DANIEL COSTA,9 MALCOLM C. 7. M. Pinkerton, S. Hanchet, J. Bradford-Grieve, Water
COULTER,10 PAUL DAYTON,11 ARTHUR L. DEVRIES,7 ROBERT DUNBAR,12 SYLVIA EARLE,13 JOSEPH T. EAST- Atmos. 15, 20 (2007).
MAN,14 STEVEN D. EMSLIE,15 CLIVE W. EVANS,16 ROBERT A. GARROTT,17 STACY KIM,18 GERALD KOOYMAN,11 8. D. G. Ainley, G. Ballard, S. Olmastroni, Aquatic Mam. 35,
335 (2009).
AMÉLIE LESCROËL,19 MICHAEL LIZOTTE,20 MELANIE MASSARO,21 SILVIA OLMASTRONI,22 PAUL J. PONGA- 9. D. G. Ainley, D. B. Siniff, Antarctic Sci. 21, 317 (2009).
CREDIT: JORIS VAN OSTAEYEN/ISTOCKPHOTO.COM
NIS,11 JOELLEN RUSSELL,23 DONALD B. SINIFF,24 WALKER O. SMITH JR.,25 BRENT S. STEWART,26 IAN STIR- 10. J. T. Eastman, Antarctic Fish Biology (Academic Press,
LING,27 JAY WILLIS,28 PETER WILSON,29 ERIC J. WOEHLER30 San Diego, CA, 1993).
11. W. O. Smith Jr., D. G. Ainley, R. Cattaneo-Vietti, Philos.
1
Centre for Applied Conservation Research, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. 2H. T. Harvey Trans. R. Soc. London Ser. B 362, 95 (2007).
and Associates, Los Gatos, CA 95032, USA. 3Geological Sciences Department, University of Texas San Antonio, San Anto- 12. B. S. Halpern et al., Science 319, 948 (2008).
nio, TX 78249, USA. 4PRBO Conservation Science, Petaluma, CA 94954, USA. 5Center for Coastal Physical Oceanography, 13. Marine Stewardship Council, “’Net benefits’ report”
Old Dominion University, Norfolk, VA 23529, USA. 6NOAA Fisheries, Southwest Fisheries Science Center, Pacific Grove, CA (September 2009); www.msc.org/documents/fisheries-
93950, USA. 7Department of Animal Biology, University of Illinois, Urbana, IL 61801, USA. 8Universita di Genova, Corso factsheets/net-benefits-report/.
Europa, 26, 16132, Genoa, Italy. 9Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA 14. Marine Stewardship Council, “Ross Sea toothfish long-
95060, USA. 10Specialist Group on Storks, Ibises, and Spoonbills, Chocorua, NH 03817, USA. 11Scripps Institution of Ocean- line” (October 2010); www.msc.org/track-a-fishery/in-
ography, University of California, La Jolla, CA 92093, USA. 12Department of Environmental Earth Systems Science, Stanford assessment/southern-ocean/ross-sea-toothfish-longline.
University, Stanford, CA 94305, USA. 13National Geographic Society, Washington, DC 20090, USA. 14Department of Biomed- 15. The opinions of R.L.B. Jr. do not represent an official
ical Sciences, Ohio University, Athens, OH 45701, USA. 15Department of Biology and Marine Biology, University of North position or endorsement of third-party certification
Carolina, Wilmington, NC 28403, USA. 16School of Biological Sciences, University of Auckland, PB 92019 Auckland, New schemes for fisheries by NOAA and the U.S. Government.

Published by AAAS
LETTERS
Assisted Colonization: and Invasive Species Specialist Groups to

review and update its 1998 guidelines to
Move Ahead with Models explicitly accommodate these issues sur-
rounding assisted colonization. The task
MOVING SPECIES OUTSIDE THEIR NATURAL force will report to the World Conservation
ranges has long been recognized as risky Congress in 2012.
(“Home, home outside the range?”, R. Stone, MARK R. STANLEY PRICE
News Focus, 24 September, p. 1592). Current Wildlife Conservation Research Unit, University of Oxford,
accepted procedure allows for translocations Zoology Department, Oxford OX13 5QL, UK. E-mail: mark.
stanleyprice@zoo.ox.ac.uk
outside the historic range only reactively—
when there is no suitable habitat left in that References
1. IUCN/SSC Re-introduction Specialist Group, “Guidelines
range (1). For example, flightless birds such for re-introductions” (IUCN, Gland, Switzerland, 1998).
as kakapo and takahe have been introduced to 2. A. Ricciardi, D. Simberloff, Trends Ecol. Evol. 24, 248
offshore islands because exotic mammalian (2009).
3. S. G. Willis et al., Conserv. Lett. 2, 45 (2009).
predators had rendered them unable to persist 4. CBD, Conference of the Parties, “Biodiversity and climate
on the New Zealand mainland. Soon, we may change” (UNEP/CBD/COP/10/L.36, 2010).
have to move species proactively as a means
to save them from anticipated shifts in habitat
due to climate change. Proactive assisted col-
Assisted Colonization:
onization is understandably contentious. The Facilitate Migration First
best way forward involves careful modeling
and collaboration. IN HIS NEWS FOCUS STORY “HOME, HOME
There are many cautionary experiences outside the range?” (24 September, p. 1592),
from invasive species and biological control R. Stone explores the risks and current data
releases (2). However, tools such as struc- gaps associated with the practice of assisted
tured decision-making enable us to make colonization—actively translocating spe-
decisions in the face of uncertainty about cies from degrading ecosystems to locations
ecological roles and relationships that we more favorable to long-term survival. As
will have least cause to regret. We can con- Stone discussed, many scientists assert that
struct models around the fate of species if we the practice, also known as assisted migra-
leave them to face climate change either by tion, should play a substantial role in com-
adapting, by moving, or by dying out. We can bating the effects of climate change on the
explore a deliberately moved species’ pros- survival of species that cannot successfully
pects of (i) dying out at its human-selected migrate or adapt. In addition to its risks and
destination, (ii) establishing and becoming a uncertainties, however, assisted colonization
pest, or (iii) settling down within desired pop- presents many ethical and legal issues (1),
ulation limits. and may be more appropriate as the choice
The test case in the News story of the of last resort. An intermediate strategy
two butterflies in the United Kingdom (3) between doing nothing and active transloca-
is an example of the low-risk and poten- tion is facilitated migration, which involves
tially reversible type of experiment that we securing the conditions necessary for suc-
should be starting now. With assisted migra- cessful species migration to and eventual
tion recognized as one means to reduce the settlement in more hospitable environments
impacts of climate change on biodiversity (2). Facilitated migration might include, for
(4), we need international guidelines on the example, conserving migratory corridors
conditions under which it may be an accept- and areas believed likely to transition into
able solution. Consequently, the IUCN Spe- habitat suitable for a species’ new range.
cies Survival Commission has established a Although it may not work for all climate-
task force from within its Re-introduction threatened species, such as those stranded on
mountaintops by rising temperatures, facili-
Letters to the Editor tated migration presents none of the thorny
ethical issues of assisted colonization and, if
Letters (~300 words) discuss material published
carried out properly, is perfectly legal under
in Science in the previous 3 months or issues of
general interest. They can be submitted through
laws such as the Endangered Species Act. It
the Web (www.submit2science.org) or by regular also helps scientists focus on forward-looking
mail (1200 New York Ave., NW, Washington, DC planning and conservation to ensure that both
20005, USA). Letters are not acknowledged upon the habitat of a species’ future and a way to
receipt, nor are authors generally consulted before get there are in place when needed. Facilitated
publication. Whether published in full or in part, migration should be developed in science and
letters are subject to editing for clarity and space. in policy as an important option for counter-
ing the effects of climate change on species

Published by AAAS
LETTERS
survival and, where possible, should be used risks of introducing maladapted genes and
before turning to assisted colonization. invasive populations are inherent to this type
J. B. RUHL of strategy. Some have even proposed intro-
Florida State University College of Law, Tallahassee, FL ducing subtropical species from the southern
32306, USA. E-mail: jruhl@law.fsu.edu hemisphere in northern temperate countries
References because of the species’ suitability to future
1. A. E. Camacho, Yale J. Reg. 27, 171 (2010). warmer climates (2). As a result, exotic trees
2. J. B. Ruhl, Natl. Wetlands Newsl. 32, 26 (July–August, could be introduced legally into rural land-
2010).
scapes, thereby modifying terrestrial ecosys-
tems for centuries in the name of responding
Assisted Colonization: to climate change.
Protect Managed Forests We agree with Stone’s conclusion that sci-
entists should closely advise programs con-
IN HIS NEWS FOCUS STORY “HOME, HOME sidering assisted colonization, and we add a
outside the range?” (24 September, p. 1592), similar plea for managed forests programs.
R. Stone presents a lucid view of the strengths The attention paid to the ecological, ethi-
and weaknesses of assisted colonization of cal, and legal issues of assisted colonization
endangered species. Unfortunately, the focus of endangered species should not eclipse the
on assisted colonization is overshadowing risk assessment of natural and managed for-
far-reaching climate change adaptation pro- est adaptation strategies.
grams targeting forests managed for produc- JUAN F. FERNÁNDEZ-MANJARRÉS1,2,3*
ing timber, producing nontimber products, or AND LOUISE TSCHANZ4
stocking biomass to capture CO2. 1
Université Paris-Sud 11, UMR 8079, Orsay F-91405,
In an effort to help managed forests France. 2CNRS, Orsay F-91405, France. 3AgroParisTech,
Paris F-75231, France. 4Faculté de Droit Jean Monnet,
respond to the effects of changes in climate, Université Paris-Sud, Bagneux F-92220, France.
some propose the intentional translocation
of tree species outside of their ranges. For- *To whom correspondence should be addressed. E-mail:
juan.fernandez@u-psud.fr
est managers seek to increase forest resil-
ience by introducing new genotypes and new References
species (1). Social pressure to adjust man- 1. E. Marris, Nature 459, 906 (2009).
2. D. J. Read et al., Eds., Combating Climate Change—
aged forests in response to climate change A Role for UK Forests: An Assessment of the Potential of the
should not be underestimated; managers are UK’s Trees and Woodlands to Mitigate and Adapt to Cli-
pushed to make decisions immediately, and mate Change (The Stationery Office, Edinburgh, 2009).
CORRECTIONS AND CLARIFICATIONS

News Focus: “Killer bots are getting human” by J. Bohannon (1 October, p. 30). The game referred to as “Ultimate
Tournament 2004” is actually named “Unreal Tournament 2004.”
News of the Week: “New type of cosmic dust tells of galaxy’s violent history” by Y. Bhattacharjee (24 September,
p. 1590). The reference to “unprecedented images of the cloudshine feature” should have read “unprecedented images
of the coreshine feature.”
News Focus: “Has China outgrown the one-child policy?” by M. Hvistendahl (17 September, p. 1458). In the graph
“Having fewer babies anyway” (p. 1459), the top label on the y axis should have been 5, not 5%. The graph has been
corrected in the HTML version online.
Letters: “Archaeology augments Tibet’s genetic history” by P. J. Brantingham et al. (17 September, p. 1467). The
Letter referred to both the Report by T. S. Simonson et al. and the Report by X. Yi et al. Only the Report by X. Yi et al.
should have been cited; T. S. Simonson et al. did not estimate a divergence time for high-altitude Tibetans.
Policy Forum: “Achieving scientific eminence within Asia” by A. S. Huang and C. Y. H. Tan (17 September, p. 1471). Refer-
ences 12 and 13 were incomplete. Reference 12 should be “Y. Shi, Y. Rao, Science 329, 1128 (2010).” Reference 13 should
be “S. Tole, R. D. Vale, Science 329, 1441 (2010).” The references have been corrected in the HTML version online.
Brevia: “Island biogeography reveals the deep history of SIV” by M. Worobey et al. (17 September, p. 1487). A grant was
omitted from the acknowledgment. The study was supported in part by Public Health Service grant RR000164.
News Focus: “The dour Frenchman on malaria’s frontier” by M. Enserink (3 September, p. 1142). The profile stated that
combining an artemisinin-derived drug with another antimalarial was a novel concept in the early 1990s. In fact, others
had explored that idea before. Researchers from the Guangzhou College of Traditional Chinese Medicine in Guangdong,
China and the Roche Far East Research Foundation Hong Kong described the first clinical trial in which artemisinin was
combined with other drugs: G. Q. Li, K. Arnold, X. B. Guo, H. X. Jian, L. C. Fu, Lancet 2, 1360 (1984).
News of the Week: “NSF misfires on plan to revamp minority programs” by J. Mervis (23 July, p. 376). Stephen Cox was
identified incorrectly. He is project director for the greater Philadelphia region Louis Stokes Alliance for Minority Partici-
pation program.
Review: “Development of monocytes, macrophages, and dendritic cells” by F. Geissmann et al. (5 February, p. 656).
Reference 71 was incorrect. It should be A. Aziz et al., Science 326, 867 (2009).

Published by AAAS
BOOKS ET AL.
NEUROSCIENCE gap between some finding of sex differences
in the brain and the relation of such distinc-
How Neuromythologies Support tions to complex behaviors.
Although these books are
Sex Role Stereotypes Brain Storm
The Flaws in the Science
more similar than different,
each author has a particular
Diane F. Halpern of Sex Differences slant to her message. Fine
coined the term “neurosex-
I
by Rebecca M. Jordan-Young
n a televised newscast this September, a test what Fine (a psycholo- Harvard University Press, ism” to describe the use of
medical correspondent for CBS News, gist at Macquarie Univer- Cambridge, MA, 2010. neuroscience to support a
Jennifer Ashton, M.D., explained that sity) calls “brain scams”: 408 pp. $35, £25.95, €31.50. sexist agenda that proclaims
“men have six and a half times more gray the irresponsible use of find- ISBN 9780674057302. “girls are like this” and “boys
matter than women do. Gray matter is partly ings from the brain sciences are like that” because their
responsible for information processing, so to declare that females and Delusions of Gender brains work in different ways
[this] may explain in general [why] men males are essentially differ- How Our Minds, Society, (4). It can aptly describe the
tend to be better in math” (1). Ashton went ent and the assumption that and Neurosexism Create work of some sloppy scien-
on to explain that women “have as much as we can use brain morphology tists and nonscientist popu-

Difference
10 times as much white matter,” which could as an explanation for sex-role larizers who claim that they
by Cordelia Fine
“contribute to why women are such good stereotypes. Both authors rail can explain on the basis of
Norton, New York, 2010.
multi-taskers.” You don’t need to be a brain against the claim that men are the neuroanatomy of male
368 pp. $25.95, C$32.50.
scientist to question these data, and you don’t good at math and women are ISBN 9780393068382. and female human brains
need to know anything about sex differences good at interpersonal tasks why men don’t ask for direc-
in brain structures to recognize the long leap because they are hard-wired tions or why women gossip.
from neurons to math achievement or the that way—a distinction that psychologist The erudite term, however, does not apply to
ability to share attention among tasks. Care- Simon Baron-Cohen has labeled synthesiz- everyone who studies sex differences in the
fully researched and reasoned, Rebecca Jor- ing (male-typical) and empathizing (female- brain. Many of the scientists who study neu-
dan-Young’s Brain Storm and Cordelia Fine’s typical) brains (2). In addition to poking holes roanatomical sex differences are responsible
Delusions of Gender offer antidotes to neuro- in the distinction between these two hypoth- in the way they conduct research and cautious
fallacies such as these. esized brain types, Fine and Jordan-Young about the conclusions they make.
The ease with which a popular media also enjoy critiquing extreme examples from Despite the large amount of junk science
spokesperson can generalize from gray and the writings of psychiatrist Louann Brizen- on the topic that is reported in the popular
white matter (which correspond to, respec- dine, who promotes the idea that the differ- media and in some academic outlets, there
tively, cell bodies and myelinated axons) to ences in male and female brains can be seen are also consistent findings of sex differences
why one sex might be better in math or in in our everyday interactions (3). Both authors that hold up across studies, across species,
tasks that require shared attention is a per- point out biases in the work of “essentializ- and across cultures. Most of these are ignored
fect example of what Jordan-Young describes ers” (people who believe that females and by Fine. This may, in part, reflect the fact that
as an “ ‘infomercial’ for cherished beliefs.” males are “essentially” and immutably dif- whereas sex differences are reliably found in
The physician–news reporter got just about ferent), and both emphasize how gender is several areas of research, none of the differ-
everything wrong. Females and males have, learned, even at a young age. They take aim at ences support essentialist claims that girls
on average, comparable ability in math researchers who seem blind to the cavernous and boys need separate educations based on
(although there are more males than females their brain types, that one sex is better suited
in both the low and high ends of the abil- to become engineers, or that one sex is inher-
ity distribution), and we can all get better ently more intelligent—to name just a few
at doing multiple tasks simultaneously of the ideas being promoted under the guise
with practice. Perhaps the most glaring of “science.” For example, in a recent set of
flaw in her news report is what she left studies that collected data from over
out: our brains change in response to expe- 200,000 men and women via a BBC
rience, so any purported brain differences Web site, a test of estimating the orienta-
between males and females could have been tion of lines and other visuospatial tasks
CREDIT: DESIGN, KELLY BLAIR/COURTESY W. W. NORTON
caused by (and not the cause of) different life found medium to large sex differences
experiences. Even though this media sound favoring males across 53 countries (5). On
bite fails on every dimension, it leaves view- the other hand, training studies have found
ers with the message that modern neurosci- that everyone can improve on visuospatial
ence can explain common stereotypes about tasks (6), so any explanation must consider
the differences between females and males. the various ways in which biological and psy-
Similar in many ways, both books pro- chosocial factors affect one another.
Consider a biopsychosocial model in
which individuals are predisposed by their
The reviewer, the author of Sex Differences in Cognitive
Abilities, is at the Department of Psychology, Claremont
biology to learn certain skills more readily
McKenna College, 850 Columbia Avenue, Claremont, CA than others while everyone selects experiences
91711, USA. E-mail: diane.halpern@claremontmckenna.edu that are biased by prior learning histories,

Published by AAAS
BOOKS ET AL.
opportunities afforded in their environments, recessive disorders that impair the synthesis
and beliefs about appropriate behaviors for of cortisol in the adrenal glands. Most cases
females and males. Experiences change neu- involve an enzyme deficiency that causes the
ral structures, which in turn alter how individ- adrenal glands to excrete excessive levels of
uals respond, and so on. As many stereotypes masculinizing hormones throughout gesta-
about sex differences reflect group differ- tion. Jordan-Young notes the contradictory
ences between males and females, by learning findings from this atypical population, and
and endorsing them, individuals may also be she suggests that the unique experiences of
selecting environments and experiences that these girls could underlie their unusual gen-
increase or reduce these differences. der responses. She also assails the notorious
A relatively recent paradigm shows just case of a child who was raised as a girl after
how complicated sex differences can be. Sev- an accident destroyed his penis during routine
eral different researchers examined the way circumcision when he was seven months old.
sex differences vary as a function of the gen- Her criticisms are well-founded, but she also
der equality across societies. Consider the ignores much relevant high-quality research
finding that, in more gender-equal societies, by scientists who take care in their work and
females perform as well as males in mathe- in the scope of their claims. For some exam-

matics (7), much better than males in reading ples: Jay Giedd (National Institute of Men-
(7), and much worse than males in visuospa- tal Health) has found sex differences in nor-
tial tasks (5). No simple theory, such as the mal brains at every stage of development
hypothesis that sex differences reflect soci- (8). Larry Cahill (University of California,
etal norms or that gender-equal societies will Irvine) has shown that sex and brain lateral-
reduce all sex differences, can explain this ization are important influences on emotion
pattern of results. and memory (9). Bruce McEwen (Rocke-
Not all claims of biological influences feller University) has spent decades carefully
are wrong or wrong-headed. On the simplest documenting estrogen and other hormonal
level, there is a genetic basis for some types effects on neural development (10).
of mental retardation that are more common Cleverly written with engaging prose,
among males than among females. There are Delusions of Gender and Brain Storm con-
numerous gonadal hormone receptors in the tain enough citations and end notes to signal
brain, and such hormones do influence some that they are also serious academic books.
sex-typed behaviors, both prenatally and, to a Fine and Jordan-Young ferret out exagger-
lesser extent, after puberty. Both books lack ated, unreplicated claims and other silliness
a more nuanced approach that might teach regarding research on sex differences. The
readers how to distinguish solid research on books are strongest in exposing research
hormonal and genetic influences of male and conclusions that are closer to fiction than
female behavior from overhyped and simple- science. They are weakest in failing to also
minded claims. It is much more difficult to point out differences that are supported by
delve into the sometimes messy details that a body of carefully conducted and well-
good research must address than it is to take replicated research. The question is not
a one-sided approach that highlights only the whether female and male brains are similar or
bad work. Unfortunately, it is also less enter- different, because they are both. The questions
taining to read about a balanced approach to we need to answer are: How can we under-
complex questions. Nonetheless, the more stand the ways in which we are similar and dif-
balanced view more accurately reflects the ferent? And how can we use that knowledge to
current state of research on sex differences. help everyone achieve their fullest potential?
The subtitle of Brain Storm characterizes
its focus on “the flaws in the science of sex References
1. www.cbsnews.com/stories/2010/09/22/earlyshow/health/
differences.” There are many, but like Fine, main6890474.shtml?tag=cbsnewsSectionContent.9;
Jordan-Young (a sociomedical scientist at updated 22 September 2010.
Barnard College) sometimes fails to distin- 2. S. Baron-Cohen, The Essential Difference: Men, Women,
and the Extreme Male Brain (Allen Lane, London, 2003).
guish between valid research claims and psy- 3. L. Brizendine, The Female Brain (Bantam, London, 2007).
chobabble. Throughout the book, the author 4. C. Fine, Neuroethics 1, 69 (2008).
explores three main themes: sex, gender, and 5. R. A. Lippa et al., Behav. 39, 997 (2010).
sexuality. She wisely reminds us that context 6. D. Tzuriel, G. Egozi, Child Dev. 81, 1417 (2010).
7. L. Guiso et al., Science 320, 1164 (2008).
is a critical variable in understanding these 8. http://intramural.nimh.nih.gov/research/pi/pi_giedd_j.
three strands of human experience, and like html.
Fine, she emphasizes socialization. 9. http://cahill.bio.uci.edu/.
10. http://rockefeller.edu/research/faculty/abstract.
She takes particular aim at research find- php?id=109.
ings from girls afflicted with congenital
adrenal hyperplasia, a family of autosomal 10.1126/science.1198057

Published by AAAS
BOOKS ET AL.
THEATER: HISTORY OF SCIENCE and Franklin scholar Lynne

Osman Elkin (3) shook her
Shades of Gray in head in disagreement, vehe-
mently arguing that Franklin
DNA Drama would “not hand unpublished
data to a competitor. Period.”
Barbara Juncosa The panel moderator,
neuroscientist Stuart Fires-
K
ing’s College, London, 1952: Rosa- tein, posited that the histori-
lind Franklin develops x-ray dif- cal narrative might have been
fraction photograph number 51. affected by glaring differ-
Illuminated by her light box, a crisp striped ences in the tone and quality
“X” stares back at her from the center of the of the two groups’ scientific
photo. In this brief moment, Franklin reveals communiqués. “The Watson
the helical nature of DNA and cements a leg- and Crick paper is, I have to
acy doomed to controversy. say, a work of scientific art,”
Anna Ziegler’s play Photograph 51 dra- he commented. “You see

matizes the race to determine the structure of immediately what they saw
DNA—the Nobel Prize–winning work that … It lets you into the beauty
produced one of the most important scientific of the whole molecule. And
discoveries of the 20th century. The narra- Kristen Bush in the role of Rosalind Franklin. Franklin’s paper is extremely
tive centers on Rosalind Franklin, who is pre- informative, but if you’re not
sented as a prickly biophysicist obsessed with 38, her research remains the priority. The play a crystallographer, you’re not going to get
capturing the perfect image needed to resolve culminates in a stirring confrontation with anywhere through this paper.”
the molecule’s structure. Wilkins in which he—confessing his love for Although the controversy over Franklin’s
The play opens as Franklin arrives at Franklin—laments the multitude of missed rightful credit will continue to be debated,
King’s College, where she faces anti-Semitic opportunities between the two scientists. the panelists agreed that photograph 51 pro-
and sexist sentiments from fellow research- Photograph 51 provides an emotional vided key data for Watson and Crick’s DNA
ers. But Franklin’s combative temperament journey into the complex realities of labo- model. Despite the inequalities between male
and secrecy soon become the focus of the ratory science. Ziegler portrays scientists and female scientists at King’s College, the
story. Although Franklin ultimately suc- who—far from the rational, white-coated panelists contended that the men involved in
ceeds in capturing the elusive structure on automatons of popular culture—strug- the DNA race did not discriminate against her
film, she refuses to share her gle with personal ambitions, because she was a woman or Jewish. Rather,
work with colleague Maurice Photograph 51 intense competition, and rocky they suggested that her severe disposition and
Wilkins or even construct a relationships as they strive to impossible relationship with Wilkins likely
by Anna Ziegler;
model before completing her directed by Linsay Firman
define the material of hered- stymied her progress.
detailed calculations. ity. The small stage provides a In the end, Photograph 51 provides a com-
Ensemble Studio Theatre,
Alienating Wilkins with New York. 22 October–21
dynamic environment for the pelling perspective on Franklin in which even
her repellent disposition, November 2010. A produc- characters, who often observe the complex science is engaging and acces-
Franklin inadvertently drives tion of the EST/Alfred P. Sloan Franklin’s progress from the sible. Whether she was a wronged heroine or
him to secretly disclose pho- Foundation Science and sidelines, arguing their view- quarrelsome perfectionist, her contributions
tograph 51 to James Watson. Technology Project. www. points directly to the audience. to the human understanding of life’s heredi-
Realizing the significance ensemblestudiotheatre.org/ The play’s director, Linsay Fir- tary material are difficult to understate. She
of Franklin’s image, Watson photograph-51 man, makes use of the cramped passed away before the Nobel Prize was
returns to the laboratory of space to create striking juxta- awarded to Crick, Watson, and Wilkins in
Francis Crick, where the two complete the positions between the theorists Watson and 1962. But even had she survived until then,
CREDIT: GERRY GOODSTEIN/COURTESY THE ENSEMBLE STUDIO THEATRE
model of double-stranded DNA presented in Crick fiddling with their models and the one wonders whether the scientific commu-
their 1953 Nature paper. Franklin’s contribu- staunch experimentalist Franklin scribbling nity would have recognized her substantial
tion, however, goes largely unacknowledged calculations in her notebook. role in the revolutionary breakthrough.
by her male colleagues. The play raises a number of important
Despite the harsh portrayal by playwright issues, from the role of women in science References and Notes
1. Those seeking a nonfictional portrait of Franklin should
Anna Ziegler (1), Franklin’s unwavering ded- to the importance of scientific collabora- turn to Brenda Maddox’s nuanced biography (4).
ication to the pursuit of truth makes her an tion. To discuss these themes, the Ensemble 2. A two-part podcast of the panel discussion is avail-
admirable character. Rather than dwell on her Studio Theatre hosted a lively panel discus- able at www.scientificamerican.com/podcast/episode.
cfm?id=photograph-51-rosalind-franklin-and-10-11-03
defeat, Franklin celebrates humanity’s victory sion 2 November at the Julia Miles Theater and www.scientificamerican.com/podcast/episode.
over the mysteries of life. Even as she fights a in New York (2). Although James Watson cfm?id=photograph-51-rosalind-franklin-and-10-11-05.
losing battle with ovarian cancer at the age of could not attend as planned, tense moments 3. L. O. Elkin, Phys. Today 56, 42 (2003).
still emerged following the assertion by 4. B. Maddox, Rosalind Franklin: The Dark Lady of DNA
(HarperCollins, London, 2002); reviewed in (5).
Nicholas Wade, the New York Times sci- 5. A. Fausto-Sterling, Science 298, 1177 (2002).
The reviewer is at the Laboratory of Bacterial Pathogenesis
and Immunology, Rockefeller University, 1230 York Avenue, ence reporter, that “the idea that [Franklin]
New York, NY 10065, USA. E-mail: bjuncosa@gmail.com was robbed of credit is incorrect.” Biologist 10.1126/science.1199778

Published by AAAS
POLICYFORUM
PUBLIC HEALTH
Developing Health Workforce In Africa, programs to improve health will

require major investments in institutions that
Capacity in Africa
can train and retain health professionals.
Francis S. Collins,1 Roger I. Glass,2* Jack Whitescarver,3 Mary Wakefield,4 Eric P. Goosby5
A
s we mark World AIDS Day, it is The MEPI awards were announced in posals), 64% plan to have offerings in pub-
important to assess the relationship October 2010 and will invest about $130 mil- lic health (to include biostatistics, epidemi-
between the challenges of AIDS pre- lion over 5 years to directly support African ology, and the like), 45% propose strategies
vention and control and the huge gaps in the institutions in 12 countries, creating a net- to increase the number of women clinicians
health workforce needed to address these work of 30 regional partners and more than and researchers, and 36% of programmatic
and other critical shortages. Although Africa 20 U.S. collaborating institutions (7) (see the awards are intended to include maternal-child
bears 24% of the global disease burden, it has table). For example, in South Africa, the two health training and research in the context of
only 3% of the world’s health workforce and proposals are directed at two of the hardest HIV/AIDS (including training of midwives).

less than 1% of the world’s financial resources hit areas: Stellenbosch University is work- This venture is aligned with the principles
for health (1, 2). Sub-Saharan Africa as a ing on the training of physicians to work in of President Obama’s Global Health Initiative
whole has 18 physicians per 100,000 popu- rural areas with the least resources by recruit- (8). MEPI is among several programs to sup-
lation, ranging from 60 in South Africa to 2 ing students from these underserved areas. port the congressional mandate that PEPFAR
in Mozambique. Meanwhile, Africa strug- The University of KwaZulu-Natal is located help partner countries train and support the
gles to retain its precious health workers. in a region with the highest rates of HIV and retention of at least 140,000 new health care
Approximately 65,000 African-born physi- tuberculosis in South Africa and has been a workers at all levels to strengthen the capacity
cians, or about one-fifth of those trained, have training ground and research center for the in developing countries, particularly in sub-
migrated to high-income countries within 5 region. Researchers there are conducting Saharan Africa (9). Finally, this will allow an
years of completing their training (3). More- large-scale interventions (e.g., circumcision) explicit intervention priority—HIV/AIDS—
over, one-third of medical school faculty and have had a history of supporting research to drive overall improvements in the local
posts are vacant, and 30% of faculty must training for physicians from surrounding health systems and provide care for a broader
supplement their income to continue teaching countries and former homelands, where non- range of conditions, as recommended by the
(4). It is vital to increase the quality and quan- whites were segregated during apartheid. Institute of Medicine (10).
tity of local health care workers, particularly The MEPI program was developed after PEPFAR’s initial emergency approach—
in remote rural areas. Scientific expertise consultation with African leaders and inter- while necessary early on—has had both posi-
must be enhanced to determine the best pre- national donors, and the sites were selected tive and negative impacts on country-level
vention and treatment strategies for the local based on competitive peer review by NIH. health systems and budgets. In moving for-
context, as well as improve target countries’ The Office of the Global AIDS Coordinator ward to quickly establish a service capac-
(5) ability to monitor and evaluate outcomes. (OGAC) will provide about $105 million for ity, implementation did not always take into
The U.S. President’s Emergency Plan for AIDS-related training; the Office of AIDS account existing national health care struc-
AIDS Relief (PEPFAR) has recently part- Research at NIH will provide an additional tures or plans. Nor did the focus on HIV/AIDS
nered with the National Institutes of Health $10 million; and the NIH Director’s Common carefully translate to broader service and
(NIH) and the Health Resources and Services Fund will provide $15 million to extend train- delivery needs such as training and providing
Administration (HRSA) in an initiative to ing into other critical areas such as maternal, health workers to peripheral areas or family
transform medical education at select medical neonatal, and child health and noncommu- planning and child health. This partnership,
schools in Africa (6). The Medical Education nicable diseases. By engaging national min- with its emphasis on long-term response rep-
Partnership Initiative (MEPI) includes col- istries of health and education, participating resents a new direction for PEPFAR (11).
laboration with the U.S. Agency for Interna- institutions will leverage MEPI to strengthen Many participants will devote funds to
tional Development, the Centers for Disease national plans to improve medical education, strengthen mentoring, peer-to-peer training,
Control and Prevention, and the Department expand the quality and scope of instruction, and hands-on clinical research experience.
of Defense and is intended to provide a focus and scale up the existing cadre of health care Through MEPI, medical schools in sub-Saha-
for U.S. agencies to support local health min- workers. In addition to increasing the qual- ran Africa will be able to develop and expand
istries and academic centers to improve the ity and quantity of health care workers, MEPI novel models of education, recruit and retain
quantity, quality, and retention of their gradu- will develop the in-country scientific exper- qualified faculty, and increase the capacity
ates and improve their research capabilities. tise needed to conduct research on imple- for locally driven, multidisciplinary research.
menting programs and identify novel solu- They will also be able to broaden their curri-
tions to the most pressing problems to bridge cula to develop and expand training focused
1
National Institutes of Health (NIH), Bethesda, MD 20892,
USA. 2Fogarty International Center, NIH, Bethesda, MD
gaps in the delivery of care. Of the current on maternal and child health, emergency med-
20892, USA. 3Office of AIDS Research, NIH, Bethesda, MD grants, 80% plan to incorporate commu- icine, cardiovascular disease, mental health,
20892, USA. 4Health Resources and Services Administra- nity-based education and/or rural training, and HIV-related cancers. Because Africans
tion, Rockville, MD 20857, USA. 5Office of Global AIDS 73% target nurses and nursing students for with HIV/AIDS are now living longer, thanks
Coordinator, Washington, DC 20522, USA.
enhanced training (schools of pharmacy and to antiretroviral drugs, they are developing
*Author for correspondence. E-mail: glassr@mail.nih.gov dentistry are included in some of the pro- chronic diseases in growing numbers, yet

Published by AAAS
POLICYFORUM
MEPI Partners and education on a country by country basis.

The ultimate goal is to create long-term
Country and Institutions U.S. Universities and Other Partners capacity at Africa’s educational institu-
Botswana Harvard University
tions to produce the quantity and quality of
University of Botswana* University of Pennsylvania
Emory University
health care workers and scientists required
Ethiopia
Addis Ababa University* Johns Hopkins University and broaden the training provided to cover
Hawassa and Haremaya Universities University of Wisconsin not only HIV-related disease but also issues
Defense Forces Medical Colleges University of California, San Diego (UCSD of maternal and child health, noncommuni-
Kenya University of Maryland, Baltimore cable diseases, and other national priorities.
University of Nairobi* University of Washington, Seattle
Measurable outcomes should be a substantial
Mozambique UCSD and World Health Organization
University of Eduardo Mondlane*
increase in the numbers and quality of phy-
Canadian Network for International Surgery
Universities of Lurio and Zambeze American College of Surgeons sicians and health professionals trained and
Nigeria Northwestern University retained in country (many working in under-
University of Ibadan* Harvard University served areas), more robust residency train-
Universities of Jos, Nigeria, Maiduguri, Ahmadu Bello, and Lagos ing programs, and removal of silos between
AIDS Prevention Initiative Nigeria, Ltd.
AIDS and non-AIDS care. The only means to
South Africa Columbia University
bring our efforts to effective scale is through

University of KwaZulu-Natal*
South Africa
creative partnerships with other governments,
Johns Hopkins University
Stellenbosch University* philanthropies, international organizations,
University of Cape Town and industry. With the launch of MEPI, we
Makerere University (Uganda) welcome cooperation to build synergies and
Tanzania Duke University accelerate our shared objectives and we will
Kilimanjaro Christian Medical Centre*
be reaching out to prospective partners. A
Uganda Johns Hopkins University
Makerere University* Case Western Reserve University
generation of skilled African professionals
Mbarara, Kampala International, Busitema, and Gulu Universities Yale University trained to apply evidence-based solutions for
Medical Research Council improved health would be a lasting legacy.
Zambia Vanderbilt University
University of Zambia* University of Alabama, Birmingham References
1. World Health Organization, Report on the WHO/PEPFAR
Zimbabwe University of Colorado, Denver
Planning Meeting on Scaling Up Nursing and Medical
University of Zimbabwe* Stanford University
Education, Geneva, 13 to 14 October 2009; www.who.int/
University of Cape Town (South Africa) University College and King‘s College, London
hrh/resources/scaling-up/en/index.html.
Ghana University of Michigan 2. Global Health Workforce Alliance, Scaling Up: Saving
Kwame Nkrumah University of Science and Technology* Lives (World Health Organization, Geneva, 2008); www.
Ghana Ministry of Health and Komfo Anokye Teaching Hospital who.int/workforcealliance/documents/Global_Health%20
Ghana College of Physicians and Surgeons and Ghana Ambulance Service FINAL%20REPORT.pdf.
Malawi University of North Carolina 3. International Organization for Migration, World Migra-
University of Malawi College of Medicine* Johns Hopkins University tion 2005 (International Organization for Migration,
University of Cape Town (South Africa) Geneva, 2005).
4. F. Mullan et al., The Sub-Saharan African Medical School
Coordinating Center George Washington University* Study: Data, Observation, and Opportunity (SAMSS,
The African Center for Global Health and Social Transformation (Uganda) Washington, DC, 2010).
5. www.pepfar.gov.
*Principal grantee.
6. National Institutes of Health, News release: HHS Partners
With PEPFAR to Transform African Medical Education with
$130M Investment, October 2010; www.fic.nih.gov/news/
there are very few African health care work- This initiative will enable participat- publications/global_health_matters/2010/1010_
ers trained in these specialties. Deaths from ing institutions to strengthen their informa- medical-education-africa.htm.
chronic diseases are rising quickly in Africa. tion technology infrastructure to enhance 7. National Institutes of Health, Directory of Awards and
Collaborating Partners for the Medical Education Partner-
The World Health Organization is forecasting educational and research activities, includ- ship Initiative (MEPI), October 2010; www.fic.nih.gov/
a 27% increase over the next decade (12). ing increasing bandwidth and computer programs/training_grants/mepi/awards.htm.
MEPI will also support establishment of access, introducing intranets and other shared 8. Implementation of the Global Health Initiative: Con-
a network (via a Web-based platform) to link resources, and encouraging mobile technol- sultation Document (1 February 2010); www.usaid.
gov/our_work/global_health/home/Publications/docs/
African sites and U.S. collaborators, lever- ogy platforms for information delivery and ghi_consultation_document.pdf.
age resources, and provide technical exper- data collection for clinical research. MEPI 9. Tom Lantos and Henry J. Hyde United States Global Lead-
tise. Initially, the network will be coordi- will also support distance education and data ership Against HIV/AIDS, Tuberculosis, and Malaria Reau-
thorization Act of 2008, Pub. L. No. 110-293 (2008).
nated by the George Washington University sharing that will encourage much-needed 10. Institute of Medicine, PEPFAR Implementation: Progress
School of Public Health, in partnership with clinical registries to inform health care deci- and Promise (National Academies Press, Washington, DC,
HRSA, NIH, and OGAC, and will moni- sions and research needs on national levels. 2007); www.nap.edu/catalog.php?record_id=11905.
11. The U.S. President’s Emergency Plan for AIDS Relief:
tor progress and identify best practices. In This initiative is not without challenges.
Five-Year Strategy (2009); www.pepfar.gov/strategy/
5 years, many of these functions will be trans- Given current faculty shortages and infra- document/index.htm.
ferred to the Center for Global Health and structural deficits at many schools, assuring 12. World Health Organization, 2008–2013 Action Plan for
Social Transformation in Kampala, Uganda. quantity and quality scale-up and retention the Global Strategy for the Prevention and Control of
Noncommunicable Diseases (WHO, Geneva, 2008);
Our goal is to empower the sites to pursue strategies will require disciplined efforts on www.who.int/nmh/Actionplan-PC-NCD-2008.pdf.
their own priorities and network within the the part of all involved. Success will neces-
program and their own countries. sitate coordination with ministries of health 10.1126/science.1199930

Published by AAAS
PERSPECTIVES
OCEANS
Analysis of the bacterial communities in
Cryptic Links in the Ocean oxygen-depleted water columns reveals

a new strategy how microbes link the nitrogen
and sulfur cycles in the ocean.
Andreas Teske
O
xygen depletion in the ocean
water column, in stratified Biomass Biomass
basins such as the Santa _ _ _
Barbara and Cariaco basins, and in 4 NO2 + SO4 + 2 H
2 +
NH4+ + SO42
_ _
enclosed anoxic oceans such as the 4 NO3 + H2S NO3 + H2S + H2O
Black Sea provide vivid examples of CO2 CO2
a type of marine habitat where mul-
ticellular life gradually disappears OMZ water column: SUP05 bacteria Benthic sediment: Thioploca
with depth as oxygen is consumed by
respiration and not replenished. Only

bacteria and archaea with anaero-
bic metabolisms persist, controlling
the microbial cycling of nitrogen,
sulfur, metals, and carbon. Genu-
ine fondness for these environments
and their anaerobic microbial inhab-
itants, although possibly an acquired
taste, is widespread among marine
microbiologists and geochemists 10 µm 50 µm
who are tracing the path of key ele-
ments and the role of microbes in the Ocean biochemistry. Simplified scheme of sulfur and nitrogen cycle linkages for pelagic SUP05 bacteria, contrasted
ocean’s biogeochemical economy. On with benthic filaments of Thioploca. Lower left, epifluorescence in situ hybridization image of SUP05 cells, appearing
page 1375 of this issue, Canfield et in yellow, from the Suiyo Seamount hydrothermal plume; lower right, bright-field image of sheathed benthic Thioploca
al. (1) analyze the microbial cycling filaments from shelf sediments underlying oxygen-depleted water near Concepción, Chile.
of nitrogen and sulfur in the oxygen
minimum zone offshore of northern Chile. filamentous bacteria, the sulfide-oxidizing, ined the OMZ bacteria by high-throughput
They show that our working hypotheses on nitrate-reducing Thioploca species (2, 3). In sequence sampling of all detectable micro-
microbial interactions and processes of strati- conjunction with a highly active microbial bial genomes in the water column. The domi-
fied marine water columns have overlooked iron cycle in the upper sediments, the Thiop- nant sulfur-oxidizing bacterial lineage was
a critical component: the contribution of the loca mats reoxidize sulfide so efficiently that identified as the SUP05 cluster within the
“cryptic” microbial sulfur cycle. the amount of sulfide actually remains near γ-proteobacteria, a cosmopolitan cluster of
Why cryptic? In principle, microbial pro- the detection limit within the mat, despite yet-uncultured bacteria that is consistently
cesses should reveal themselves by the reac- an extremely high sulfate reduction rate in associated with anaerobic marine water col-
tion partners that are produced and con- the immediately surrounding sediment (4). umns (6). The acronym SUP refers to the
sumed. For example, the dominant anaerobic The water column of northern Chile contains original discovery of these bacteria in the
respiration pathway in the marine environ- an oxygen minimum zone (OMZ) in which hydrothermal plume of Suiyo Seamount, a
ment, sulfate reduction—the use of the sea- sulfate reduction accounts for a surprisingly submarine volcano near Japan (7). SUP05
water anion sulfate for respiration by special- large proportion of total carbon remineraliza- bacteria from this location appear as microm-
ized groups of bacteria and archaea—gener- tion. However, the resulting sulfide is reoxi- eter-sized coccoid to slightly oval individual
ates the end product sulfide. The gradual dis- dized so efficiently that it does not accumu- free-swimming cells, unattached to particles
appearance of sulfate, concomitant with sul- late, effectively camouflaging sulfate-reduc- (see the figure). The Chilean results link the
fide production, would be a reliable indicator ing activity in the water column. In both SUP05 lineage with cryptic sulfur cycling
for microbial sulfate reduction. Yet studies of habitats, sediment and water column, cryptic and answer the question of why SUP05 sul-
CREDIT: (LOWER LEFT) A. MARUYAMA/AIST, JAPAN
marine sediments have already shown that sulfur cycling does not mean that the process fide oxidizers are also found in anaerobic, but
these microbial processes are easily camou- is environmentally irrelevant. On the con- apparently sulfide-free, waters.
flaged; sulfide accumulation is often held in trary, preventing the buildup of free sulfide The results also imply that cryptic sulfur
check by specialized sulfide-oxidizing bacte- by instant reoxidation is essential to counter cycling and its microbial catalysts are more
ria, as long as suitable oxidants are available. sulfide toxicity. widespread than currently documented. For
For example, the continental shelf of Chile Canfield et al. link cryptic, nitrate-driven example, the SUP05 cluster constituted the
and Peru harbors extensive microbial mats of sulfur cycling to characteristic sulfur-oxi- most frequently recovered bacterial group
dizing microbial communities of the Chil- in hydrothermal vent plumes at the Mid-
Department of Marine Sciences, University of North Car-
ean OMZ. Previously, this bacterial com- Cayman Rise, the deepest currently known
olina, Chapel Hill, NC 27599, USA. E-mail: teske@email. munity had been identified by 16S rRNA hydrothermal vents at 5000 m depth (8).
unc.edu gene sequencing (5); Canfield et al. reexam- How resilient is this microbial ecosystem

Published by AAAS
PERSPECTIVES
in the Chilean OMZ? In sediments with a coping with sulfide limitation. relevant; in the near future, we will certainly
high input of easily degradable biomass that As a caveat, such scenarios oversimplify hear more about these resilient microbial
fuel sulfate reduction, microbial sulfide pro- the very complex nitrogen cycle; they neglect engines of the changing world oceans.
duction can overwhelm the nitrate pool and the highly active microbial cycling of ammo-
lead to sulfide poisoning of life in the sea- nia in the oxycline (11). They also do not take References
1. D. E. Canfield, A. N. Glazer, P. G. Falkowski, Science 330,
bed; sulfide-blackened dead Thioploca mats into account anaerobic ammonia-oxidizing 1375 (2010); 10.1126/science.1196889.
(nicknamed “Thioploca nigra”) are a fre- bacteria in the water column, which combine 2. H. Fossing et al., Nature 374, 713 (1995).
quent occurrence on the Chilean shelf sedi- nitrite and ammonia to nitrogen gas (5). To 3. V. A. Gallardo, Nature 268, 331 (1977).
ments (9). In the OMZ water column, the trace the dynamics of sulfide oxidation and 4. J. Zopfi, M. E. Bottcher, B. B. Jorgensen, Geochim.
Cosmochim. Acta 72, 827 (2008).
chemistry balance is tilted toward sulfide lim- nitrate reduction processes and their micro- 5. H. Stevens, O. Ulloa, Environ. Microbiol. 10, 1244
itation. Comparison of the redox stoichiome- bial populations, Canfield et al. call for sys- (2008).
tries of the pelagic SUP05 group and benthic tematic seasonal studies. 6. D. A. Walsh et al., Science 326, 578 (2009).
7. M. Sunamura, Y. Higashi, C. Miyako, J. Ishibashi, A.
Thioploca lends plausibility to this scenario. The Chilean OMZ and continental shelf Maruyama, Appl. Environ. Microbiol. 70, 1190 (2004).
Benthic Thioploca reduce nitrate to ammo- provide two examples for microbial sulfur 8. C. R. German et al., Proc. Natl. Acad. Sci. U.S.A. 107,
nia in order to oxidize sulfide as effectively and nitrogen cycling ecosystems that thrive in 14020 (2010).
9. V. A. Gallardo, Gayana Oceanol. 1, 27 (1992).
as possible in a 1:1 ratio of nitrate and sulfide a highly dynamic balance and persist through
10. S. Otte et al., Appl. Environ. Microbiol. 65, 3148 (1999).
(10). The SUP05 populations favor a more annual or interannual oscillations in redox

11. V. Molina et al., Mar. Ecol. Prog. Ser. 288, 35 (2005).
parsimonious mode of sulfide oxidation by regime and water column stratification (12, 12. H. N. Schulz, B. Strotmann, V. A. Gallardo, B. B. Jør-
the reduction of nitrate to nitrite, with a sul- 13). With ocean temperatures and anthro- gensen, Mar. Ecol. Prog. Ser. 200, 117 (2000).
13. C. E. Morales, S. E. Hormazábal, J. Blanco, J. Mar. Res.
fide/nitrate stoichiometry of 1:4 (see the fig- pogenic water column anoxia on the rise, 57, 909 (1999).
ure). The dominant OMZ bacteria may have nitrate-dependent microbial controls on sul-
additional, as yet unexplored, mechanisms of fide concentrations will become increasingly 10.1126/science.1198400
GENETICS
The DNA Damage Road Map Comparing maps of gene interactions offers
insight into how yeast cells repair DNA
damage.
Nir Friedman1,2 and Maya Schuldiner3
I
f you were on your way to a new coun- a cell. These maps are created by measuring for a diverse array of cellular processes,
try, you would pack a map to help you genetic interactions, specifically the effect including the early secretory pathway, chro-
find the major cities, roads, and interest- that a mutation in one gene has on the phe- mosome function, signaling pathways, and
ing places to explore. Recently, research- notype of a mutation in a second gene (see RNA processing (3, 4). These E-MAPs,
ers have created similar maps to help them the figure). Using novel genetic tools for however, have all have been collected from
start unraveling the complex architecture of studying budding yeast (1) and automated cells grown under the same condition: in a
technology, investigators can now system- rich growth medium. But just as a snowstorm
1
School of Computer Science and Engineering, The Hebrew atically and rapidly measure these genetic can block some roads and force changes in
University of Jerusalem, Jerusalem, Israel. 2Institute of Life interactions (epistasis) for all pairs in gene traffic, changing environmental conditions
Sciences, The Hebrew University of Jerusalem, Jerusalem, subsets of interest (about 400 to 800 genes). can cause cells to rewire their genetic net-
Israel. 3Department of Molecular Genetics, Weizmann Insti-
tute of Science, Rehovot, Israel. E-mail: nir@cs.huji.ac.il; The resulting E-MAPs (epistasis miniarray works, necessitating the drawing of a new
maya.schuldiner@weizmann.ac.il profiles) (2) have helped chart interactions map. On page 1385 of this issue, Bandyo-
A B MMS E-MAP C
Genotype CBF Cell cycle
Untreated E-MAP
WT X∆ Y∆ X∆Y∆ Tel1
dE-MAP
G2 M
Fitness
P
Cbf1 Cbf1
S G1
None Neg Pos
Interaction Pph3
MMS
Neg Pos Dec Inc
Compare and contrast. (A) E-MAPs are created by measuring the effect of gene than expected (yellow bar). (B) By comparing E-MAPs of yeast cells grown under
mutations on a phenotype of interest such as fitness. By comparing wild-type treated (MMS) and untreated conditions, researchers created a dE-MAP illustrat-
(WT) strains to single mutations (X∆, Y∆) and their combinations (X∆Y∆), inves- ing the change in interactions (increased or decreased) between the two con-
tigators can identify mutation pairs that have no effect (black bar, right), a nega- ditions. (C) The dE-MAP highlighted the role of Tel1 and Pph3 in regulating Cbf1
tive effect that is greater than expected (blue bar), or a positive effect that is less activity in cell cycle checkpoints (right).

Published by AAAS
PERSPECTIVES
padhyay et al. (5) describe the creation of in dealing with stress. By highlighting these E-MAPs for many gene subsets and in many
just such a condition-specific E-MAP and a condition-dependent interactions, the dE- conditions. In fact, we are quickly nearing
novel method for analyzing it. MAP allows the scientist interested in DNA the day when all genetic interactions in yeast
Yeast cells can grow under a myriad of damage to zoom in on the regulatory inter- will be collected (7). The central challenge
conditions by changing their cellular wir- actions between proteins that play a spe- has become distilling biological insights
ing through the regulation of transcription, cific and major role in fighting the effects from this rich data. This challenge can be met
translation, protein modification, and degra- of MMS. There are hundreds of such sto- computationally (8–11) or by using careful
dation. To study an example of this changing ries in the data. One example that demon- experimental designs, as shown by Bandyo-
landscape, Bandyopadhyay et al. (5) chose strates the power of the approach involves padhyay et al. More broadly, the dE-MAP
a subset of 418 yeast genes that includes a novel function that the authors found for approach provides an important reminder
all known signal transduction components the transcription factor CBF1 (centromere that most high-throughput interaction data
and transcription factors, as well as DNA binding factor) in repressing cell cycle provides just a snapshot representing a spe-
damage proteins and chromatin remodel- checkpoints (which ensure the fidelity of cific state. The new frontier is probing the
ers. Then, they created two E-MAPs show- cell division). One of the advantages of dynamic interactions that enable cells to sur-
ing how these genes interacted both under having systematic interaction data are that, vive and thrive in varying environmental and
standard conditions (“untreated”) and when instead of examining only specific interac- genetic contexts. Experiments and analysis
treated with methyl methanesulfonate tions, E-MAPs allow scientists to examine at this next, dynamic, level will give biolo-

(MMS), a DNA-damaging agent (see the the “interaction profile” of a gene, or its pat- gists truer insights into the ever-changing
figure). A key idea underlying their analysis tern of interactions with all other genes in environment of a living cell.
was to compare and contrast the two maps, the map. This profile provides a rich finger-
thereby isolating interactions that are condi- print of the gene’s function. Two genes with References
1. A. H. Tong et al., Science 294, 2364 (2001).
tion specific (see the figure). They used the similar or related functions will have simi-
2. M. Schuldiner et al., Cell 123, 507 (2005).
differences in these interactions to create a lar interaction profiles. In the past, this con- 3. P. Beltrao, G. Cagney, N. J. Krogan, Cell 141, 739
third map, which they named the dE-MAP cept allowed the identification of several key (2010).
(differential epistasis mapping). The dE- gene complexes (6). The authors used this 4. S. J. Dixon, M. Costanzo, A. Baryshnikova, B. Andrews,
C. Boone, Annu. Rev. Genet. 43, 601 (2009).
MAP uncovers novel information on how tool to reveal that CBF1’s genetic interaction 5. S. Bandyopadhyay et al., Science 330, 1385 (2010).
a yeast cell deals with DNA damage. For profile changed between the untreated and 6. M. Breker, M. Schuldiner, Mol. Biosyst. 5, 1473
example, two genes that encode proteins MMS growth conditions. Only in the MMS (2009).
that repair DNA damage might not show E-MAP is CBF1’s activity highly correlated 7. M. Costanzo et al., Science 327, 425 (2010).
8. A. Battle, M. C. Jonikas, P. Walter, J. S. Weissman,
any interaction in untreated cells but might with the activity of two other genes, TEL1 D. Koller, Mol. Syst. Biol. 6, 379 (2010).
show crucial interactions in yeast treated (encoding a kinase) and PPH3 (encoding a 9. A. Beyer, S. Bandyopadhyay, T. Ideker, Nat. Rev. Genet. 8,
with MMS. phosphatase). Previous work had shown that 699 (2007).
A bird’s-eye view of the dE-MAP reveals Tel1 phosphorylates Cbf1, and the authors 10. I. Ulitsky, T. Shlomi, M. Kupiec, R. Shamir, Mol. Syst. Biol.
4, 209 (2008).
that protein complexes tended to remain show that Pph3 regulates CBF1 by dephos- 11. A. Jaimovich, R. Rinott, M. Schuldiner, H. Margalit,
stable across the two conditions. The rela- phorylating it (see the figure). N. Friedman, Bioinformatics 26, i228 (2010).
tionships between these complexes, how- With advances in automation and tech-
ever, were reprogrammed to assist the cell nology, researchers can now easily collect 10.1126/science.1199862
PLANT SCIENCE
Dynamic Metabolons The assembly and disassembly of enzymes

complexes may differentiate plant defense
responses to insect attack and fungal infection.
Birger Lindberg Møller
T
he metabolic activities of higher membrane anchoring. A metabolon allows called “functioning dependent structures,”
organisms are highly coordinated. At the direct passage of a product from one assembling in response to specific metabolic
the cellular level, compartmentaliza- enzymatic reaction to a consecutive enzyme demands or abiotic and biotic challenges (3).
tion into organelles and substructures thereof in a metabolic pathway. Such channeling of For example, in humans, the six enzymes
optimizes the concentration of substrates tar- intermediates limits their diffusion into the that constitute the purine nucleotide biosyn-
geted by enzymes. At the molecular level, surrounding milieu, maintains separate pools thetic complex assemble upon depletion of
further substrate concentration is gained by of intermediates, facilitates fast turnover of purines. Phosphorylation of the metabolon
the formation of multienzyme complexes— labile or toxic intermediates, and may prevent by the CK2 kinase promotes its dissociation,
so-called metabolons. The protein constitu- undesired crosstalk between different meta- whereas assembly can be initiated by phos-
ents of a metabolon are held together by non- bolic pathways (1, 2). phatases or kinase inhibitors (4). The tran-
covalent interactions and often stabilized by Coordinating metabolic pathways may sient complex of glycolytic enzymes that
involve dynamic shifts between the assembly forms at the outer mitochondrial membrane
Plant Biochemistry Laboratory, Department of Plant Biology and disassembly of metabolons or their inter- in response to the respiratory demand for
and Biotechnology, University of Copenhagen, DK-1871
Frederiksberg C, Copenhagen, Denmark. E-mail: blm@life. actions with proteins that modulate their out- pyruvate is another example (5).
ku.dk put function. Such transient metabolons are In plants, biosynthetic pathways that gen-

Published by AAAS
PERSPECTIVES
Sorghum produces the cyanogenic glu-

UGT coside dhurrin.When sorghum microsomes
85 Metabolon UGT
85 harboring just the two cytochrome P450
Assembly
CPR CYP CYP CPR CYP CYP enzymes of the metabolon are isolated in the
79 71 79 71
absence of a strong reductant, an oxime is
Disassembly the final product (17). It is not clear what sig-
nal would induce dissociation of the metab-
olon in vivo, but one possibility might be the
oxygen burst that accompanies most fungal
infections. Conversion of oximes to highly
reactive nitroso compounds may also give
Oximes Amides Cyanogenic
glucosides rise to the formation of diverse protein con-
jugation products that could signal an innate
immune response.
Mitochondrial Amines Cellular Experimental studies are needed to deter-
dysfunction disruption mine whether oxime production is facilitated
by dissociation of the metabolons involved

in cyanogenic glucoside and glucosinolate
production or by inhibition of the CYP71E
Reactive oxygen Detoxification Hydrogen
species cyanide enzyme, or whether specific activation of
oxime-producing CYP79 enzymes occurs
during fungal infection. In all cases, targeted
synthesis of highly reactive compounds to
Fungal defense Insect defense combat fungal pathogens would be achieved.
Thus, detonation of an “oxime bomb” to
Defense reactions. Coordinated assembly and disassembly of metabolic complexes, such as the cyano- combat fungal infection would supple-
genic glucoside and glucosinolate metabolons in plants, may optimize the production of chemical constitu- ment the ability of cyanogenic glucoside- or
ents that combat pathogens and insects. CPR, cytochrome P450 reductase. glucosinolate-containing plants to detonate a
“cyanide bomb” or “mustard oil bomb” when
erate bioactive natural products also may be plants must therefore rely on other defense attacked by chewing insects (18).
organized as metabolons. This applies to mechanisms. Recently, a broad-spectrum
cyanogenic glucosides and glucosinolates antifungal defense and innate immune References and Notes
1. K. Jørgensen et al., Curr. Opin. Plant Biol. 8, 280
(1). Following tissue damage by herbivorous response in plants were proposed to be (2005).
insects, these two classes of phytoanticipins triggered by the turnover of glucosinolates 2. R. J. Conrado, J. D. Varner, M. P. DeLisa, Curr. Opin. Bio-
are cleaved by β-glucosidases to release (11, 12). In this scenario, small amounts of technol. 19, 492 (2008).
toxic hydrogen cyanide and isothiocyanates, amines were generated, but their route of 3. M. Thellier, G. Legent, P. Amar, V. Norris, C. Ripoll, FEBS
J. 273, 4287 (2006).
respectively, thereby protecting against gen- formation and possible involvement in the 4. S. An, M. Kyoung, J. J. Allen, K. M. Shokat, S. J. Benkovic,
eralist insects (6). In both biosynthetic path- innate immune response were not clarified J. Biol. Chem. 285, 11093 (2010).
ways, the initial steps convert amino acids into (12). The structures of the amines corre- 5. I. Brandina et al., Bba-Bioenergetics 1757, 1217
(2006).
corresponding oximes, a reaction sequence spond to those obtained following a Beck- 6. B. L. Møller, Curr. Opin. Plant Biol. 13, 337 (2010).
catalyzed by distinct but homologous cyto- mann rearrang-ement of the oxime interme- 7. O. Sibbesen, B. Koch, B. A. Halkier, B. L. Møller, J. Biol.
chrome P450 enzymes of the CYP79 family diates in glucosinolate biosynthesis. One Chem. 270, 3506 (1995).
8. U. Wittstock, B. A. Halkier, J. Biol. Chem. 275, 14659
(7, 8) (see the figure). In cyanogenic glucoside possibility is that amine formation reflects
(2000).
synthesis, the resulting oxime is converted detoxification of oximes that have diffused 9. S. Bak, R. A. Kahn, H. L. Nielsen, B. L. Moller, B. A.
into a labile cyanohydrin by a cytochrome into noninfected plant cells. Oximes are Halkier, Plant Mol. Biol. 36, 393 (1998).
P450 enzyme of the CYP71E subfamily (9), generally toxic to fungi and are frequently 10. P. R. Jones, B. L. Moller, P. B. Hoj, J. Biol. Chem. 274,
35483 (1999).
and finally into a stable cyanogenic gluco- used as chemical fungicides (13, 14). They 11. N. K. Clay, A. M. Adio, C. Denoux, G. Jander, F. M. Aus-
side by a UGT85 family glucosyltransferase inhibit mitochondrial oxidases and thereby ubel, Science 323, 95 (2009).
(10). In glucosinolate biosynthesis, the oxime promote lipid peroxidation and the produc- 12. P. Bednarek et al., Science 323, 101 (2009).
13. L. Bartosova, K. Kuca, G. Kunesova, D. Jun, Neurotox.
is converted into the final glucosinolate struc- tion of toxic reactive oxygen species (15, Res. 9, 291 (2006).
ture via aci-nitro compounds or nitrile oxides, 16). The cationic properties of indol-type 14. J. E. Drumm et al., Acs. Sym. Ser. 584, 396 (1995).
S-alkylthiohydroximates, thiohydroximic oximes may increase their toxicity and ren- 15. K. Sakurada et al., Toxicol. Lett. 189, 110 (2009).
acids, and desulfoglucosinolates. The inter- der them particularly active in launching an 16. G. M. Rusch, A. Tveit, I. D. Waalkens-Berendsen, A. P.
Wolterbeek, G. Armour, Drug Chem. Toxicol. 32, 381
mediates in both synthetic pathways are labile innate immune response. Oximes could be (2009).
and chemically highly reactive. Metabolon formed in plants that produce cyanogenic 17. B. L. Møller, E. E. Conn, J. Biol. Chem. 255, 3049
formation may thus facilitate swift conversion glucosides and glucosinolates if the respec- (1980).
18. A. V. Morant et al., Phytochemistry 69, 1795 (2008).
and prevent undesired interference with gen- tive metabolons were to dissociate in a func- 19. Supported from the Villum Kann Rasmussen research
eral metabolism. tioning dependent manner at the precise center “Pro-Active Plants”, the UNIK Synthetic Biology
Numerous pathogenic fungi are not site of infection. Oximes produced by the Program, and the Danish Research Council for Indepen-
dent Research.
deterred by, or rapidly develop resistance to, released CYP79 enzyme might thus enable
hydrogen cyanide and isothiocyanates, and preferential combat of fungal infection. 10.1126/science.1194971

Published by AAAS
PERSPECTIVES
CELL BIOLOGY
The assembly and activation of a mitochondrial
Opening the Cellular Poison Cabinet channel is triggered by direct interaction with
signaling molecules that promote cell death.
Seamus J. Martin
I
n complex multicellular
organisms, millions of cells
BH3 direct Apoptosis Neutralization BH3 sensitizer Bcl-2 proteins
die each day as a result of
activators of prosurvival (Hrk, Noxa, prosurvival
stress, injury, or infection, and as (Puma, Bid, Bim) proteins Bad, Bik, Bmf)
part of the natural cell turnover
process that is essential to opti-
mal tissue functioning. Remark-
ably, cell death in most of these
situations is orchestrated by the
dying cell itself, a form of cel-

lular suicide called apoptosis
Outer membrane
(1). On page 1390 in this issue,
Ren et al. (2) help to resolve how
cells execute this process. Bax-Bak
A cell must commit suicide Oligomerization Blocks
at the appropriate time, other- oligomerization
wise malfunctioning or damaged Cytochrome c
cells could accumulate and lead
to tumor development and other
pathological conditions. Thus, Death decisions. Bax-Bak–dependent mitochondrial outer membrane permeabilization is positively regulated through the
sensors are needed to monitor actions of BH3-only sensors and negatively regulated through prosurvival Bcl-2 family proteins.
the integrity of cellular functions
and relay this information to the channel effectively constitutes the decision (Bid, Bim, and Puma) directly activates Bax
cell death machinery. A battery of such sen- to commit cellular suicide. and Bak, whereas the remaining BH3-only
sors have been identified—so-called BH3- Because of the deadly consequences of proteins neutralize prosurvival Bcl-2 pro-
only proteins (named for the Bcl-2 homol- mitochondrial outer membrane permeabili- teins (5).
ogy domain 3 they possess)—that detect zation, this major checkpoint on the road to The direct activation model predicts that
cellular stress or damage through transcrip- cell death is heavily policed. Opening of the loss of the direct activators (Bid, Bim, and
tional or posttranslational mechanisms (3). Bax-Bak channel is opposed by the prosur- Puma) renders the Bax-Bak channel inca-
Upon activation, BH3-only proteins pro- vival members of the Bcl-2 family (Bcl-2, pable of activation, thereby producing cells
voke permeabilization of the mitochondrial Bcl-x, Mcl-1, A1, Bcl-w, and Bcl-b). These that are resistant to apoptotic signals. By
outer membrane, allowing release of cyto- proteins either bind to Bax and Bak and pre- contrast, the sensitizer model predicts that
chrome c to the cytosol. This efflux trig- vent oligomerization, or are “neutralized” cells lacking this subset of BH3-only pro-
gers assembly of the apoptosome, a struc- when they are directly bound by BH3-only teins can still undergo mitochondrial per-
ture that sets in motion a proteolytic cascade proteins. Thus, BH3-only proteins and pro- meabilization through the remaining BH3-
that coordinates cell death through destruc- survival Bcl-2 proteins represent opposing only proteins. Ren et al. support the direct
tion of hundreds of proteins (see the figure). forces in the struggle to control the channel, activation model by demonstrating that
Because of their ability to unlock this cellu- the outcome of which dictates whether a cell deletion of the Bim, Bid, and Puma genes
lar poison cabinet, BH3-only proteins have will live or die. This has led to two alterna- in mice produces defects very similar to
come under intensive scrutiny. How do these tive models, not necessarily mutually exclu- those displayed by mice lacking the Bax
proteins provoke cytochrome c efflux? sive, to explain how BH3-only proteins pro- and Bak genes, rendering cells refractory to
Bax and Bak, proteins that either reside mote mitochondrial outer membrane perme- Bax-Bak activation in response to apoptotic
in or insert into mitochondrial membranes, abilization (5, 6). stimuli. Mice deficient in the three putative
constitute the pore or channel that permea- The “sensitizer model” argues that BH3- direct activators (Bid, Bim, and Puma) dis-
bilizes mitochondria. Loss of Bax and Bak only proteins act indirectly to promote cell played developmental defects (interdigital
renders cells resistant to permeabilization death by binding to prosurvival Bcl-2 pro- webs, imperforate vaginas, and enlarged
(and subsequent apoptosis) caused by BH3- teins. This interaction liberates Bax and Bak lymphocyte populations) and were born at
CREDIT: Y. HAMMOND/SCIENCE
only proteins (4). Upon activation, BH3- from their prosurvival captors and allows 50% of the expected Mendelian frequency,
only proteins promote oligomerization of oligomerization and channel formation (6). indicating partial embryonic lethality. Bio-
Bax and Bak within the mitochondrial outer The “direct activation” model argues that at chemical evidence also suggests that cer-
membrane. Thus, opening of the resultant least some BH3-only proteins directly bind tain BH3-only proteins act as direct Bax-
to Bax and Bak and promote the conforma- Bak activators (7, 8), although these inter-
Molecular Cell Biology Laboratory, Department of Genetics, tional changes that allow oligomerization. actions are transient and hard to record with
Trinity College, Dublin 2, Ireland. E-mail: martinsj@tcd.ie Indeed, only a subset of BH3-only proteins conventional techniques. Thus, accumulat-

Published by AAAS
PERSPECTIVES
ing evidence argues that a subset of BH3- pies aimed at selectively engaging the Bax- delay period, simultaneously present the
only proteins function as direct activators of Bak channel in cell populations that resist rat with two test objects: the now “famil-
Bax and Bak, and indeed, may be indispens- death due to disturbances among the ranks iar” one and a new “novel” object. Typi-
able for this. The work of Ren et al. provides of BH3-only proteins and their prosurvival cally, rats that do not have brain damage
strong support for the idea that Bim, Bid, Bcl-2 family antagonists (9). In the case of spend less time exploring the familiar object
and Puma are the central Bax-Bak activa- tumors that adopt the strategy of survival by (demonstrating memory). Rats with peri-
tors, with the remaining BH3-only proteins avoiding cell death, direct activator BH3- rhinal cortex damage spend about the same
functioning primarily as sensitizers. The only mimetic drugs may prove highly effec- amount of time exploring both objects, sug-
major distinction between these two cat- tive antidotes. gesting that they have forgotten the famil-
egories of cell death sensors may be their iar object. In McTighe et al.’s variation, they
relative affinities for Bax-Bak compared to References presented the familiar and novel test objects
1. R. C. Taylor, S. P. Cullen, S. J. Martin, Nat. Rev. Mol. Cell
those of the prosurvival Bcl-2 proteins. The Biol. 9, 231 (2008).
separately, instead of simultaneously. They
emerging view is that a combination model 2. D. Ren et al., Science 330, 1390 (2010). showed that animals with perirhinal damage
of activator-sensitizer appears to most accu- 3. R. J. Youle, A. Strasser, Nat. Rev. Mol. Cell Biol. 9, 47 did not forget the familiar object but rather
rately reflect BH3-only protein function. (2008). treated the novel object as familiar, a kind
4. T. Lindsten et al., Mol. Cell 6, 1389 (2000).
The precise composition and structure of 5. T. Kuwana et al., Mol. Cell 17, 525 (2005). of false memory. On the basis of this and
the Bax-Bak channel have yet to be eluci- previous findings, the authors argue that the

6. S. N. Willis et al., Science 315, 856 (2007).
dated, and how BH3-only proteins sense the 7. P. F. Cartron et al., Mol. Cell 16, 807 (2004). perirhinal cortex’s normal role in recogni-
8. J. F. Lovell et al., Cell 135, 1074 (2008).
multitude of signals that trigger apoptosis is 9. T. Oltersdorf et al., Nature 435, 677 (2005).
tion is to configure, or bind, elemental fea-
not yet fully understood. However, there is tures of stimuli already processed earlier in
progress toward developing targeted thera- 10.1126/science.1199461 the perceptual pathway. If the perirhinal cor-
tex is damaged, however, object memories
exist only as fragmented representations of
these featural elements formed at the ear-
NEUROSCIENCE
lier stages. These fragmented memories are
Dedicated to Memory? susceptible to interference from a constant

natural stream of subsequent perceptual
input, leading to the false memory for test
Howard Eichenbaum items that share features with the interfering
inputs. Consistent with this idea, McTighe et
Rats with a damaged perirhinal cortex exhibit false memory, raising questions about al. were able to eliminate false memories in
brain organization. brain-damaged rats by placing the animals
into a dark chamber during the delay, thus
E
arly observations of individuals with back to the dedicated area view, or move reducing new perceptual interference.
circumscribed damage to the cere- it forward in understanding how the MTL How do these findings affect the dedi-
bral cortex led to a consensus that memory system is organized? cated memory system view? McTighe et al.
memory is not localized to any particular McTighe et al. employed a clever vari- support a return to the idea that brain areas
brain area. Rather, neuroscientists believed ation of a popular memory-research para- contribute to memory only as a by-product
that memories were incorporated within the digm. In the usual procedure, researchers of their specialized information processing
information processing functions of many initially present a rat with a novel object for functions, in this case the perirhinal cor-
specialized brain areas. In 1957, this view a few minutes and then, after a significant tex’s perceptual binding function. However,
changed dramatically after Scoville and Mil-
ner (1) described a patient, known as H.M., Functional organization of a
in whom damage to the medial temporal memory system. Information
about specific objects and events
lobe (MTL; including the hippocampus and
arrives through the “what” corti-
surrounding cortex) resulted in global mem- cal stream into perirhinal cortex
ory impairment but spared perceptual and PFC (PRC), while information about the
cognitive functions. A principal interpreta- spatial-temporal context in which
tion of these findings was that the MTL is events occurred arrives through
a dedicated memory system, and this per- the “where” cortical stream into
spective dominated subsequent research on “Where” parahippocampal cortex (PHC).
memory (2). Recent studies have called this These two streams of information
idea into question, however, and McTighe et PHC combine within the hippocampus
H “What”
al. (3) add another twist on page 1408 of this (H), which represents relation-
PRC ships between objects and events
issue. They claim that one part of the MTL,
and their context. When cued for a
the perirhinal cortex, has a specific informa-
CREDIT: P. HUEY/SCIENCE
memory, feedback pathways from

tion processing function not directly related the hippocampus to the cortical
to memory. Does this finding turn the clock areas generate representations of
object and context memories that
Center for Memory and Brain, Boston University, Boston, are tested for a match in prefron-
MA 02215, USA. E-mail: hbe@bu.edu tal cortex (PFC).

Published by AAAS
PERSPECTIVES
other recent studies that distinguish between a parallel “where” stream in the cortex, end- study, McTighe et al. offer a partial answer:
forgetting and false memories due to dam- ing in the MTL within the parahippocampal The MTL’s perirhinal cortex binds featural
age in other parts of the MTL system offer area, which represents the spatiotemporal elements into cohesive configural memories,
a broader perspective. In these experiments, contexts in which objects have been experi- and this function is supported by known plas-
rats initially study items chosen from a large enced. These streams merge within the hip- ticity mechanisms (8). The hippocampus and
list each day, and then investigators mea- pocampus, which represents relationships prefrontal cortex, as well as other key brain
sure their memory performance in terms between objects and between objects and areas of this system, also contribute directly
of “hits” (correct identifications of stim- their context. In this model, the role of the to memory through plasticity in their par-
uli that were on that study list) and “false perirhinal cortex is to bind perceptual fea- ticular information processing tasks. These
alarms” (errors where subjects incorrectly tures into representations of whole objects, findings, then, support the view that there is
judge new stimuli as appearing on the study and make these representations resistant to a dedicated MTL memory system. They also
list). Damage to the hippocampus results in perceptual interference—just as McTighe further our understanding of that system as a
a decrease in hits with no effect on the false et al. describe. The hippocampus normally set of specialized areas that interact to coor-
alarm rate (4), indicating that the deficit is encodes and retrieves representations of the dinate the information processing functions
due to forgetting rather than false memo- objects and context when cued, and damage required for successful memory.
ries—the opposite of the pattern observed to the hippocampus prevents this retrieval,
by McTighe et al. In contrast, damage to the resulting in forgetting. The prefrontal cor- References

1. W. B. Scoville, B. Milner, J. Neurol. Neurosurg. Psychiatry
prefrontal cortex results in an increase in tex normally receives that information from 20, 11 (1957).
false alarms and no effect on the hit rate (5), feedback pathways through the MTL, and 2. H. Eichenbaum, N. J. Cohen, From Conditioning to
similar to the pattern observed by McTighe monitors the match between object and con- Conscious Recollection: Memory Systems of the Brain
et al. These studies suggest that distinguish- text memories (“Is this object from today’s (Oxford Univ. Press, New York, 2001).
3. S. M. McTighe, R. A. Cowell, B. D. Winters, T. J. Bussey,
ing forgetting from false memory provides list?”). Lacking that monitor, the ani- L. M. Saksida, Science 330, 1408 (2010).
researchers with a powerful tool for identify- mals cannot tell whether an object is from 4. N. J. Fortin, S. P. Wright, H. Eichenbaum, Nature 431,
ing the contributions of different brain areas today’s list or a previous list—leading to the 188 (2004).
5. A. Farovik, L. M. Dupont, M. Arce, H. Eichenbaum,
to memory. increase in false memories following pre- J. Neurosci. 28, 13428 (2008).
Furthermore, these diverse findings can frontal damage. 6. H. Eichenbaum, A. P. Yonelinas, C. Ranganath, Annu. Rev.
be integrated into a model of the anatomical A decade ago (2), available evidence Neurosci. 30, 123 (2007).
7. L. G. Underleider, M. Mishkin, in Two Cortical Visual
pathways of MTL system and its interactions led to the conclusion that different forms of
Systems. Analysis of Visual Behavior, M. A. Ingle, M. I.
with cortical areas (see the figure) (6). The memory should be viewed as the outcome Goodale, R. J. W. Masfield, Eds. (MIT Press, Cambridge,
perirhinal cortex is the end point of the so- of plasticity within systems organized to MA, 1982), pp 549–586.
called “what” stream, a cortical hierarchy of perform particular information processing 8. S. Griffiths et al., Neuron 58, 186 (2008).
perceptual processing areas that represents functions. The MTL’s information process-
information about objects (7). There is also ing functions, however, were unclear. In their 10.1126/science.1199462
MATERIALS SCIENCE
High-Temperature Rubber Made A mixture of carbon nanotubes creates a

material that can recover its shape after
from Carbon Nanotubes deformation over a wide temperature range.
Yury Gogotsi
C
arbon nanotubes have been among (think honey) and reversibly deform through strain behavior. For example, the amount
the most studied materials for the elasticity (think rubber band). Polymer foam of stress needed to maintain the same level
past two decades ( 1); they dis- earplugs are a typical viscoelastic material; of strain will drop over time, and for a given
play several remarkable properties, such as they conform to any shape of ear channel stress, the material will continue to deform.
extremely high tensile strength and electri- but fully recover to the original form after The material reported by Xu et al. is a
cal conductivity. On page 1364 of this issue, being pulled out. Viscoelasticity is exhibited special case of a viscoelastic material; it
Xu et al. (2) report another case of extreme by a large number of materials (3), including behaves like rubber under moderate defor-
mechanical performance of a carbon mate- amorphous and semicrystalline polymers, mations. Rather than store energy in perma-
rial—viscoelastic behavior of nanotubes in a biomaterials, crystalline materials experi- nent deformation, like a bent metal part, a
wide temperature range—that no other solid encing reversible phase transformations, and rubber releases the energy when the applied
has shown so far. some metallic alloys. force is removed. Viscoelastic behavior of
Viscoelasticity is the ability of a material Viscoelastic behavior is determined by nanotubes has been observed for vertically
to dissipate energy through viscous behavior measuring stress-strain curves: The mate- aligned brushes and foams of tubes (highly
rial is pushed on or pulled at a given force intertwined random networks) tested at
(stressed), and deformation (strain) is mea- room temperature ( 4– 7). The groups of
Department of Materials Science and Engineering, Drexel
University, Philadelphia, PA 19104, USA. E-mail: gogotsi@ sured. A viscoelastic material exhibits Gogotsi and Greer independently observed
drexel.edu “memory” (hysteresis) effects in its stress- buckling and irreversible compressibility

Published by AAAS
PERSPECTIVES

B
Nanotube arrangements leading to rubbery mechanism governing viscoelasticity, is a Many high-temperature applications of
materials. Images are from atomistic models of the thermally activated process. Xu et al. sug- materials expose them to oxidizing condi-
displacement of entangled nanotubes relative to gest that, unlike polymers, thermal stabil- tions. Most small-diameter nanotubes burn
each other or temporary collapse (flattening) of thin ity in their material stems from energy dis- at temperatures close to 400°C, so very-
tubes. (A) Three nanotubes (single-walled, double- sipation through the zipping and unzipping high-temperature applications may only be
walled, and triple-walled) are shown in contact with
of carbon nanotubes upon contact (see the possible in a vacuum or protective (reduc-
each other and interacting only by van der Waals
forces. (B) The dumbbell shape of the collapsed figure, panel A). An additional mechanism ing) atmosphere. Incorporating these nano-
nanotube decreases the distance between the walls of energy dissipation is the flattening and tube rubbers into commercial products
to the interplanar spacing of graphite and temporar- recovery of nanotubes (see the figure, panel will depend in part on lowering of material
ily creates van der Waals bonding in the middle area B). For the tubes with inner diameters of 3 costs, but note that the cost and production
of the tube. (C) A hypothetical material consisting of to 5.5 nm, the collapsed state is metastable volume issues have been largely resolved
interlocking nanotube rings that would be expected and is separated from the energetically favor- for multiwalled tubes that are at least 5 nm
to display viscoelastic behavior. able cylindrical shape by an energy barrier in diameter. With further developments,
(8). The barrier decreases with the diameter nanotube materials may find use not only
of various nanotube architectures at large and disappears when the single-walled tube in space vehicles but also in down-to-earth
strains (4, 5). The material developed by Xu diameter is smaller than 3 nm. applications, such as wrinkle-free textiles
et al. is a random network of long, intercon- The entangled nanotube material is a kind or viscoelastic shoe insoles that reduce
nected and entangled carbon nanotubes (a of versatile rubber that could be used in cold mechanical shocks.
mix of single-, double-, and triple-walled interstellar space or inside a high-temper-
tubes) that exhibits rubberlike behavior over ature vacuum furnace. It may not be easy References and Notes
a very wide temperature range. They tested to find a material with viscoelastic proper- 1. R. H. Baughman, A. A. Zakhidov, W. A. de Heer, Science
297, 787 (2002).
their samples from –196°C to 1000°C, but ties similar or superior to those reported by 2. M. Xu, D. N. Futaba, T. Yamada, M. Yumura, K. Hata,
the carbon-carbon bonds in graphitic walls Xu et al. Thin-walled carbon nanocapsules Science 330, 1364 (2010).
of nanotubes are stable above 1500°C. Thus, (onions or giant multishell fullerenes) may 3. R. S. Lakes, Viscoelastic Materials (Cambridge Univ.
an even broader temperature range can be offer reversible compressibility (9) similar to Press, Cambridge, 2009).
4. S. Pathak, Z. G. Cambaz, S. R. Kalidindi, J. G. Swadener,
expected for viscoelastic response, at least nanotubes (see the figure, panel B), but only Y. Gogotsi, Carbon 47, 1969 (2009).
in a non-oxidizing environment. The dem- fibrous materials can offer rubberlike behav- 5. S. B. Hutchens, L. J. Hall, J. R. Greer, Adv. Funct. Mater.
onstration of viscoelastic behavior and ior and such a large reversible deformation 20, 2338 (2010).
large reversible deformation for a wide tem- under tension. Graphene foam would lack 6. A. Cao, P. L. Dickrell, W. G. Sawyer, M. N. Ghasemi-
Nejhad, P. M. Ajayan, Science 310, 1307 (2005).
perature range makes the random, entan- elastic recovery because there would not be
IMAGE: VADYM MOCHALIN/DREXEL UNIVERSITY
7. Q. Zhang et al., J. Phys. D 43, 315401 (2010).

gled nanotube network described by Xu et a driving force necessary to accommodate it; 8. S. Rotkin, Y. Gogotsi, Mater. Res. Innov. 5, 191 (2002).
al. a versatile viscoelastic material. It can moreover, all carbon foams and porous car- 9. K. Asaka, K. Miyazawa, T. Kizuka, Nanotechnology 20,
dampen vibrations and absorb impact at bons—even those that show plasticity and 385705 (2009).
10. Y. Gogotsi, Carbon Nanomaterials (CRC Press, Boca
extremely low or very high temperatures, elastic recovery at the nano- and microscale Raton, FL, 2006).
where neither a perfectly elastic rubber nor a (10)—fail in brittle manner when subjected 11. I thank V. Mochalin for Material Studio simulation of
metallic felt or wool would work. to macroscopic deformation. Nanotube rings viscoelastic nanotube structures, J. R. Greer for helpful
The viscoelastic behavior of the most may form an incompressible material show- comments, and the U.S. Department of Energy, Office of
Basic Energy Sciences, for research funding.
typical viscoelastic materials, such as poly- ing rubberlike behavior, but such an arrange-
mers, is temperature-dependent; the molec- ment of carbon rings (see the figure, panel C)
ular rearrangement of polymer chains, a remains hypothetical. 10.1126/science.1198982

Published by AAAS
ESSAY
GE PRIZE-WINNING ESSAY
A New Approach to Precise localization of switchable fluorescent

molecules facilitates nanoscale biological
imaging.
Fluorescence Microscopy
Mark Bates GE Healthcare and Science are
T
pleased to present the prize-
he fluorescence microscope is a pow- cability to the life sciences. winning essay by Mark Bates, appears as a diffraction-limited
erful tool for the study of molecular Early in my doctoral a regional winner from North spot several hundred nanome-
and cell biology, enabling research- research, I made a sur- America, who is the Grand Prize ters in size. By measuring the
ers to peer into cells and living organisms to prising observation about winner of the GE & Science centroid of this spot, the posi-
understand their spatial organization. Meth- a commonly used red flu- Prize for Young Life Scientists. tion of the fluorophore can be
ods for tagging specific biomolecules with orophore, Cy5: Its fluo- determined with a high degree of
fluorescent labels, such as green fluores- rescence emission can be precision, often on the nanome-
cent protein (GFP), provide a toolbox for the switched on and off using ter scale (6, 7). When the active
observation of protein organization, interac- pulses of light. We stud- fluorophores have been switched

tions, and dynamics (1). This enables the ied this “optical switching” off, a new subset is stochastically
visualization of a host of biological phenom- effect and found that dur- activated by a pulse of activation
ena, such as enzymatic processes, the devel- ing each excitation, Cy5 light, and the process is repeated.
opment of cells and organisms, and the struc- emits thousands of pho- When a sufficient number of
tural dynamics of individual biomolecules. tons before going dark. A fluorophore positions have been
Despite its benefits, a significant draw- brief pulse of ultraviolet obtained, a plot of their coordi-
back of fluorescence microscopy is its spatial light will then efficiently reactivate the mol- nates reconstructs an image of the sample.
resolution. Beyond a certain magnification, ecule to its fluorescent state, and this process The spatial resolution of the resulting image
the fine structure of a specimen is obscured can be repeated for hundreds of cycles (4, 5). is not diffraction-limited. Rather, it is lim-
when viewed through the optical microscope. We realized that the switchable fluorescence ited only by the precision of each fluoro-
As described by Abbe in the late 19th century, exhibited by Cy5 is a strongly nonlinear pro- phore localization and the number of local-
the diffraction of light imposes a resolution cess, and we reasoned that this nonlinearity izations present. We called this method sto-
limit, which makes it impossible to resolve could be used to overcome the diffraction chastic optical reconstruction microscopy,
spatial features smaller than about 250 nm limit of resolution. or STORM (8). This concept for imaging
using visible light. This leaves much of the Inspired by this idea, we found a novel was simultaneously developed by several
architecture of cells and their inner workings method to obtain sub–diffraction limit fluo- groups, and it is also known as PALM and
inaccessible to study. The electron micro- rescence images (see the figure, parts A to fPALM (9, 10).
scope allows researchers to obtain images D). If a biological sample is labeled with Multicolor fluorescence imaging enables
with higher resolution, but in this case the photoswitchable fluorophores, the experi- the study of the relative organization and
sample preparation is not compatible with ment can be controlled so that only one fluo- interactions of cellular components, and we
live cell imaging, and it is difficult to achieve rophore, or a sparse subset of fluorophores, developed a strategy for multicolor STORM
the molecular specificity and multicolor are in the fluorescent state at any given time. using Cy5 as the photoswitchable fluoro-
capability offered by fluorescent labeling. The image of each isolated fluorophore phore. We found that when Cy5 is paired
My graduate thesis focused on the devel-
opment of an imaging modality that would A B C D
combine the advantages of both electron
microscopy and fluorescence imaging. In
the past several years, a number of innovative
approaches have been conceived to capture
fluorescence images with nanometer-scale
spatial resolution (2, 3). These techniques
take advantage of nonlinearities inherent in E F G
molecular fluorescence, such as saturation or
depletion of the excited state, to obtain opti-
cal resolutions an order of magnitude beyond
the classical diffraction limit. We invented a
new approach that is based on single-mole-
cule detection and localization, using a stan- 500 nm 500 nm 200 nm
dard fluorescence microscope and readily
available fluorescent labels, for wide appli- STORM imaging. (A to D) A cell labeled with switchable fluorophores (A) is imaged by activating only a sub-
set of fluorophores at a time (B and C). The locations of each fluorophore are determined computationally
The author is in the Department of NanoBiophotonics, Max (crosses) and a plot of their coordinates yields a super-resolution image which is not limited by diffraction
Planck Institute for Biophysical Chemistry, 37077 Gottin- (D) (17). A comparison of conventional epifluorescence imaging (E) and the corresponding STORM image
gen, Germany. E-mail: mbates@ggweg.de. (F) of clathrin-coated pits (red) and microtubules (green). The boxed region in (F) is magnified in (G).

Published by AAAS
ESSAY
with a second fluorophore (such as Cy2 or Although imaging techniques based on References and Notes
1. B. N. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien,
Cy3), its activation wavelength is strongly this concept are relatively recent, they have Science 312, 217 (2006).
influenced by the spectral characteristics of already been used to understand mecha- 2. S. W. Hell, Science 316, 1153 (2007).
its neighbor (11). We took advantage of this nisms in biology. Greenfield et al. (13) have 3. M. G. L. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 102,
13081 (2005).
property to selectively activate specific pop- used these techniques to develop a model of 4. M. Bates, T. R. Blosser, X. Zhuang, Phys. Rev. Lett. 94,
ulations of dye pairs, enabling us to capture how chemotaxis receptors in Escherichia 108101 (2005).
images of multiple molecular targets within coli organize in growing cells. Biteen et al. 5. G. T. Dempsey et al., J. Am. Chem. Soc. 131, 18192
(2009).
the same sample. Using antibodies labeled (14) have visualized the nanoscale structure 6. R. E. Thompson, D. R. Larson, W. W. Webb, Biophys. J.
with either Cy3 and Cy5, or Cy2 and Cy5, we of MreB in live Caulobacter crescentus, tak- 82, 2775 (2002).
imaged microtubules and clathrin-coated pits ing advantage of the photoswitchable fluores- 7. A. Yildiz et al., Science 300, 2061 (2003).
8. M. J. Rust, M. Bates, X. Zhuang, Nat. Methods 3, 793
in cultured mammalian cells (see the figure, cence of enhanced yellow fluorescent protein (2006).
parts E to G). In comparison with the conven- (EYFP). Also, Hess et al. (15) have obtained 9. E. Betzig et al., Science 313, 1642 (2006).
tional fluorescence image, the STORM image high-resolution images and dynamic infor- 10. S. T. Hess, T. P. Girirajan, M. D. Mason, Biophys. J. 91,
reveals previously unseen detail due to its mation from influenza hemagglutinin, a 4258 (2006).
11. M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science
high spatial resolution (~25 nm) and clearly clustered membrane protein, to differentiate 317, 1749 (2007).
resolves the rounded structure of clathrin pits between membrane organization models in 12. B. Huang, W. Wang, M. Bates, X. Zhuang, Science 319,
only 150 to 200 nm in diameter (11). fibroblast cells. 810 (2008).
13. D. Greenfield et al., PLoS Biol 7, e1000137 (2009).
Many cellular structures have a com- The development of sub–diffraction limit

14. J. S. Biteen et al., Nat. Methods 5, 947 (2008).
plex three-dimensional (3D) shape, requir- fluorescence microscopy has created new 15. S. T. Hess et al., Proc. Natl. Acad. Sci. U.S.A. 104, 17370
ing a 3D imaging method for study. Tak- possibilities for the observation of biological (2007).
16. M. Fernandez-Suarez, A. Y. Ting, Nat. Rev. Mol. Cell Biol.
CREDITS: (LEFT TO RIGHT) PETER GOLDMANN/MAX PLANCK INSTITUTE FOR BIOPHYSICAL CHEMISTRY; COURTESY OF ATAMAN SENDOEL; TAKUYA YAMAMOTO; COURTESY OF MELISSA FULLWOOD
ing advantage of a technique for 3D particle processes, and a new assay for the organiza- 9, 929 (2008).
localization, we used astigmatic imaging to tion and composition of biomolecular com- 17. Figure panels A to D are adapted from Current Opinion in
generate 3D STORM images with 25-nm plexes. With continued advances in fluores- Chemical Biology, vol. 12, “Super-resolution microscopy
by nanoscale localization of photo-switchable fluorescent
lateral resolution and 50-nm axial resolu- cent labels and labeling methods (16), it will probes,” M. Bates, B. Huang, and X. Zhuang, pages 505–
tion. With this strategy, we were able to be exciting to see how these techniques are 514, Copyright 2008, with permission from Elsevier.
obtain images of the full spherical structure applied to bring about insights into life at the
of clathrin-coated pits (12). nanometer scale. 10.1126/science.1200252
2010 Grand Prize Winner

Japan: Sakiko Honjoh for her essay “Is
Mark Bates was born in Toronto, Canada. Aging Necessary?” Dr. Honjoh was born
He received a B.Sc. degree in engineering in Yokohama, Japan. Inspired by a high-
physics from Queen’s University and an school biology teacher, she decided to
M.Sc. degree in physics from McGill Uni- major in molecular biology and entered
versity. He conducted his doctoral research Kyoto University. Continuing on this
at Harvard University, working under the track, Dr. Honjoh completed her Ph.D.
guidance of Xiaowei Zhuang, where he in the laboratory of Eisuke Nishida at the Graduate School of
studied the properties of photoswitchable Biostudies, Kyoto University, working on the signal transduction
fluorescent molecules and applied these networks that regulate life span. She is continuing her work in the
results to develop a new method for high- same lab, still trying to elucidate the molecular changes that occur
resolution optical imaging. Dr. Bates is during aging.
now a postdoctoral fellow in the laboratory of Stefan Hell in Göttin-
gen, Germany, where he is applying super-resolution fluorescence All Other Countries: Melissa Fullwood for her
microscopy to study prokaryotic cell biology. essay “Genome-Wide Chromatin Loops Regu-
late Transcription.” Dr. Fullwood, born and
Regional Winners raised in Singapore, graduated from Stanford
Europe: Ataman Sendoel for his essay University in 2005 and completed her Ph.D.
“Is Death Without Oxygen as Sweet as in 2009 at the Genome Institute of Singa-
Apoptosis?” Dr. Sendoel was born in pore under the auspices of the National Uni-
Zurich, Switzerland. He studied med- versity of Singapore where she was super-
icine at the Universities of Zurich vised by Yijun Ruan. In 2009, she was selected
and Lausanne. After finishing medi- for the inaugural L’Oreal for Women in Science National Fel-
cal school, he joined the M.D.-Ph.D. lowships in Singapore. She is currently a Lee Kuan Yew Post-
program of the University of Zurich. Doctoral Fellow at the Duke-NUS Graduate Medical School Singa-
He conducted his Ph.D. work in the pore under the supervision of Shirish Shenolikar.
laboratory of Michael Hengartner, where he studied mecha-
nisms of controlling programmed cell death in Caenorhabditis For the full text of essays by the regional winners and for informa-
elegans. Dr. Sendoel is currently a postdoctoral fellow and contin- tion about applying for next year’s awards, see Science Online at
ues to work on hypoxia responses in C. elegans. www.sciencemag.org/feature/data/prizes/ge/index.dtl.

Published by AAAS
SPECIALSECTION
INTRODUCTION
Metabolism
Is Not Boring

SO INVESTIGATOR STEVEN MCKNIGHT ADMONISHED HIS AUDIENCE LAST SPRING
after a presentation at a conference (on metabolism and cancer, no less). He may
have been “preaching to the choir” in this case, but what led him to issue such a
blunt reminder? It seems that for a generation of biological scientists, metabolism
was an area of biochemistry to be mastered and then put aside. Metabolism was
always there in the background, providing the cell with the energy and resources to
do what was required, but was rarely recognized to determinedly influence, and be Metabolism
influenced by, the physiological state of the cell.
This special issue of Science celebrates a resurgence of interest in metabolism CONTENTS
and an appreciation of its central role in disparate areas of cell biology, physiol-
ogy, medicine, and synthetic biology (www.sciencemag.org/special/metabolism/). Perspective
McKnight’s Perspective (p. 1338) takes a broad look at the field and justifies his 1338 On Getting There from Here
excitement that recognizing the reciprocal regulation of metabolism and other cel- S. L. McKnight
lular processes promises to advance our understanding of complex physiology.
There is renewed interest in the metabolism of cancer cells and its potential as Reviews
a therapeutic target. Levine and Puzio-Kuter (p. 1340) review metabolic changes 1340 The Control of the Metabolic Switch
in cancer cells, as well as the recent suggestion that alterations in a metabolic in Cancers by Oncogenes and Tumor
enzyme can lead to the production of an “oncometabolite” that supports cancer Suppressor Genes
cell growth. When energy sources are limited, cells use a process known as autoph- A. J. Levine and A. M. Puzio-Kuter
agy for breaking down cellular components to provide substrates for metabolism. 1344 Autophagy and Metabolism
Rabinowitz and White’s Review (p. 1344) discusses roles of autophagy in metabo- J. D. Rabinowitz and E. White
lism, its regulation, and its implications for cancer and degenerative diseases. 1349 Circadian Integration of Metabolism
Of course not all fields have neglected metabolism over the years, and an enor- and Energetics
CREDIT: PAINTING: DAVID S. GOODSELL/SCIENTIFIC DESIGN: ZHOU DU AND DANIEL J. KLIONSKY
mous literature describes the role of insulin and related hormones in controlling J. Bass and J. S. Takahashi
cellular metabolism. In a Focus Issue of Science Signaling, a Research Article and 1355 Manufacturing Molecules Through
Perspective describe an unexpected signal from the insulin receptor that confers Metabolic Engineering
sensitivity to cell death when insulin is not present. Also highlighted are a role for J. D. Keasling
lipids as cellular sensors of glucose metabolism and a mechanism by which cells See also Science Translational Medicine and Science
can survive oxidative stress by shifting the activity of the cell’s protein degradation Signaling at www.sciencemag.org/special/metabolism/.
complex, the proteasome.
In Science Translational Medicine, Vallerie and Hotamisligil review how in a
strategy to combat obesity and insulin resistance, a therapeutic inhibitor of cell
signaling pathways must have coordinated actions in multiple cell types. Back in
Science, Bass and Takahashi (p. 1349) review new insights into the interaction of
metabolism with circadian clocks. Displacement of eating and periods of activity
away from the normal light-dark cycle, as experienced in jet lag or shift work, have
marked effects on metabolic diseases.
Cellular metabolic pathways, particularly in yeast or bacteria, can be exploited
to synthesize compounds that are difficult or expensive to produce by other means.
Keasling (p. 1355) reviews advances in metabolic engineering and looks forward
to a future in which customized microbes made by computer-aided design can
efficiently produce desired chemicals, ranging from fuels to pharmaceuticals.
See? It’s not boring at all! – L. BRYAN RAY

Published by AAAS
Metabolism
giving away exceptional energetic value stored
PERSPECTIVE in the lactate hydrocarbon. Any cell that wants to
grow—and there is nothing cancer cells care
On Getting There from Here more about than growth—would be crazy to

waste hydrocarbon; this would be akin to a
motorist driving down the New Jersey Turnpike
Steven L. McKnight throwing away gasoline. The spendthrift waste of
lactate likewise deprives the cancer cell of huge
Studies on a variety of interesting biological problems, ranging from circadian rhythm to amounts of acetyl-CoA to be used for the syn-
cancer cell growth to longevity, have begun to give evidence that the physiological state of cells thesis of lipids, sterols, and other cellular building
and tissues reflects both the cell’s regulatory systems and its state of intermediary metabolism. It is blocks. Despite progress, attention, and plenty
appreciated that the regulatory state of a cell or tissue, as driven by transcription factors and of hype, it is safe to conclude that the famous
signaling pathways, can impose itself upon the dynamics of metabolic state. It follows that the Warburg effect remains a mystery.
reciprocal must also be the case, that metabolic state will feed back to impose itself on regulatory Cancer researchers now recognize that reg-
state. An appreciation and understanding of this reciprocity may be required to crack open ulatory proteins, such as the hypoxia-inducible
problems in biological research that have heretofore been insoluble. transcription factors, can directly regulate the ex-

pression of genes encoding glycolytic enzymes
or the past 30 years, research in the bi- would these cells choose to dispose of the ter- (3). They now pay attention to how their favorite
F ological sciences has been dominated by

molecular biology. The successes of this
approach have shaped our understanding of
minal product of glycolysis? Instead of allowing
pyruvate to be converted into acetyl–coenzyme
A (CoA) via the spectacularly beautiful pyruvate
regulatory proteins, including the Myc and p53
transcription factors, help set the metabolic state
of cells. The fact that these masterful transcription
innumerable domains of biology. But any field dehydrogenase enzyme complex within mito- factors participate in dictating the metabolic state
that becomes sufficiently muscular can over- chondria, cancer cells instead convert pyruvate of a cell is now beginning to be accepted. The
shadow other credible approaches to scientific into lactate through the lactate dehydrogenase equally compelling corollary, however, remains
inquiry. One field etiolated by the cloud of mo- enzyme, and then simply excrete it—blindly largely unappreciated. That is, if regulatory state
lecular biology has been metabolism (Fig. 1).
The vast majority of discoveries made by molec-
ular biologists over the past several decades
required no attention to the metabolic state of a
cell. Molecular biologists needed no distracting
thoughts about the metabolic state of a cell to
discover microRNAs, the reprogramming of
somatic cells into pluripotent stem cells, or gene
rearrangement as the underlying basis for the
generation of antibody diversity.
One simple way of looking at things is to
consider that 9 questions out of 10 could be
solved without thinking about metabolism at all,
but the 10th question is simply intractable. As the
saying goes, you simply “can’t get there from
here” to answer this 10th question if you are
ignorant about the dynamics of metabolism. This,
I propose, is where we are now finding pregnant
opportunities in the field of experimental biology.
The low-lying fruit that could be picked by mo-
lecular biologists without having to consider the
metabolic state of a cell, tissue, or organism is
largely gone. The more sticky problems that re-
quired attention to the dynamics of metabolism
and that were pushed aside for decades now loom
as interesting and important challenges.
Consider a prime example of how molecular
biologists have begun to embrace the importance
of metabolic regulation. Cancer researchers have
long known of the enigmatic ability of tumor
cells to undertake aerobic glycolysis, the so-
called Warburg effect (1, 2). It makes sense that
cancer cells would be highly glycolytic, yet why
Department of Biochemistry, University of Texas South-

western Medical Center, 5323 Harry Hines Boulevard,
Dallas, TX 75390–9152, USA. E-mail: steven.mcknight@ Fig. 1. All eyes were focused on metabolism back in 1953 when Hans Krebs received the Nobel
utsouthwestern.edu Prize for his discovery of the citric acid cycle.

SPECIALSECTION
(transcription factors, signaling pathways, etc.) is and negatively regulate the HIF-1a (hypoxia- tional fertilizer imaginable. The growth environ-
accepted to control metabolic state, is it not also inducible factor 1a) and HIF-2a transcription fac- ment of cancer cells within tumors, especially
unconditionally certain that metabolic state will tors in oxygenated cells (7, 8). Partial elimination solid tumors, could hardly be more different than
reciprocally control the regulatory state itself? of these dioxygenase enzymes could be interpreted that of cells being grown under standard, tissue
Understanding this reciprocity, and digging to the to lead to the activation of the hypoxia response culture conditions. It is reasonable to suspect that
bottom of it, is where the future lies. Perhaps fit- pathway, thereby accounting for the activation of cancer cells weave their way through exception-
tingly, this research will require the sophistication transcription of genes encoding glycolytic en- ally selective oncogenetic gymnastics to achieve
of scientists having genuine skills in the study of zymes just as happens in the absence of the Von a growth-permissive metabolic state. If so, what
enzymology and intermediary metabolism. Hippel–Lindau (VHL) tumor suppressor gene features of this metabolic state can be anticipated
Whole-genome sequencing efforts of individ- (9, 10). This interpretation is very likely over- to be preserved and studied when cells are prac-
ual tumors, now numbering in the thousands— simplistic. Attenuation of a-ketoglutarate concen- tically grown in Karo syrup or alongside floating
yet soon to be numbered in the hundreds of trations and accumulation of increased amounts logs of Snickers Bar candy? Returning to the
thousands—are providing unbiased views of the of 2-hydroxyglutarate almost certainly lead to “you can’t get there from here” theme, it is pre-
myriad of “oncogenotypes” that underlie human the inhibition of other dioxygenase enzymes, of dictable that we will have to find more biolog-
cancer. Instead of ignoring mutations that happen which mammalian cells have scores of isoforms. ically sound ways to grow cancer cells in culture
to fall in the genes encoding metabolic enzymes, Intriguingly, some of these additional dioxygen- in order to favorably use them in efforts to dis-

scientists seem more keen than ever to find and ase enzymes have been implicated in the control cover therapeutics that might exploit their unique
study such mutations. This change may reflect of histone demethylation (11–13), leaving open metabolic state.
the recognition by cancer researchers that muta- the possibility that changes in metabolic state The resurrection of research involving or
tions that alter the function of metabolic enzymes might impose alterations in the epigenetic state of including metabolism is clearly upon us—that
might help to resolve the enigmatic, aerobic gly- cancer cells. is the good news. The bad news is that the field
colytic state of certain cancer cells. It is equally The drift of this thinking is concordant with was sufficiently snuffed over the past several dec-
likely that an understanding of how cancer cells the recent discovery of mutations found in renal ades that we have precious few scientists who
veer away from normality with respect to inter- cell cancers in the mitochondrial enzyme fuma- have been trained to genuinely understand inter-
mediary metabolism might lead to the concep- rate hydratase that converts fumarate to malate mediary metabolism. Just because we can now
tualization of new and inventive strategies for (14). These mutations are more rare and of a re- pronounce the names of the metabolic enzymes
therapeutic intervention. cessive nature—wherein both alleles of the gene whose encoding genes and mRNAs show up on
For example, a set of recurrent genetic lesions encoding fumarate hydratase must be inactivated. our ChIP-Seq (chromatin immunoprecipitation–
believed to influence the metabolic state of glio- Because fumarate is known to be an inhibitor of sequencing) and DNA microarrays lists does not
blastoma cancer cells have been identified in the the aforementioned family of dioxygenase en- necessarily mean that we can put two and two
genes encoding either of the two isoforms of isozymes, it is conceptually logical to hypothesize together. Despite the handicap of not being able to
citrate dehydrogenase (4, 5). Perplexingly, most, that the accumulation of excessive amounts of field an experienced team at this point, it is en-
if not all, of these lesions mutate a single arginine fumarate might inactivate the prolyl-hydroxylase couraging to see favorable trends that may enable
residue in either of the two isoforms of the en- enzymes that normally keep the HIF transcription negotiation of discovery routes that were, until
zyme (IDH1 or IDH2). The precise selectivity of factors in an inactive state (and, perhaps, likewise now, largely obscure.
the mutational events, coupled with the observa- impose alterations in the epigenetic state of can-
tion that only one allele of either enzyme appears cer cells). Finally, the same reasoning might apply
to be mutated in human cancer, pointed to the to rare mutations in the human genes encoding References and Notes
1. O. Warburg, K. Posener, E. Negelein, Biochem. Z. 152,
possibility that the lesions might be causing the mitochondrial succinate dehydrogenase and its 319 (1924).
enzymes to adopt a new catalytic function. Indeed, assembly factors that are believed to contribute to 2. O. Warburg, Science 123, 309 (1956).
the mutated forms of the IDH1 and IDH2 en- the formation of paragangliomas and pheochro- 3. N. V. Iyer et al., Genes Dev. 12, 149 (1998).
zymes exhibit a reduced affinity for isocitrate and mocytomas (15, 16). The exciting angle on these 4. D. W. Parsons et al., Science 321, 1807 (2008).
5. H. Yan et al., N. Engl. J. Med. 360, 765 (2009).
are endowed with a new catalytic function wherein studies of cancer-causing mutations in the genes 6. L. Dang et al., Nature 462, 739 (2009).
a-ketoglutarate is converted in an NADPH (nicotin- encoding isocitrate dehydrogenase, fumarate hy- 7. A. C. R. Epstein et al., Cell 107, 43 (2001).
amide adenine dinucleotide phosphate, reduced)– dratase, and succinate dehydrogenase is that they 8. R. K. Bruick, S. L. McKnight, Science 294, 1337
dependent manner to 2-hydroxyglutarate (6). formally predict the concept that the metabolic (2001).
9. P. H. Maxwell et al., Nature 399, 271 (1999).
What pathways might be expected to be al- state of a cell can indeed exert control over its 10. M. Ohh et al., Nat. Cell Biol. 2, 423 (2000).
tered in a cell impeded for the production of a- regulatory state, thereby confirming the recipro- 11. Y. Tsukada et al., Nature 439, 811 (2006).
ketoglutarate and concomitantly endowed with cal relationship between the two. 12. J. R. Whetstine et al., Cell 125, 467 (2006).
increased intracellular production of 2-hydroxy- One enduring complication that shows little 13. P. A. Cloos et al., Nature 442, 307 (2006).
14. J. S. Isaacs et al., Cancer Cell 8, 143 (2005).
glutarate? A logical guess would be the family of sign of resolution concerns the manner in which
15. M. A. Selak et al., Cancer Cell 7, 77 (2005).
dioxygenase enzymes that use a-ketoglutarate as cancer cells are grown and studied in tissue cul- 16. H. X. Hao et al., Science 325, 1139 (2009).
an essential cofactor and, simultaneously, are in- ture plates. We routinely grow cancer cells under 17. I thank M. Brown, R. Bruick, B. Tu, and J. Rutter for
hibited in the presence of 2-hydroxyglutarate. In- conditions of unlimited access to glucose—not to helpful editorial comments.
cluded among this family of dioxygenase enzymes mention oxygen, vitamins, known and unknown
are the prolyl-hydroxylase enzymes that modify growth factors present in serum, and every nutri- 10.1126/science.1199908

Metabolism
dria. There are several times, however, when
REVIEW regulated rapid cell division is required, such as
during embryonic development, in wound healing
The Control of the Metabolic Switch (liver regeneration), or in the immune responses
to specific antigens, where clonal selection pro-
in Cancers by Oncogenes and Tumor

vides increased cell numbers with increased im-
mune specificity. Cancer cells share many of these
same requirements for energy, substrates to grow
Suppressor Genes and divide, and control of the redox potential and
ROS in the cell.
What these processes have in common is a
Arnold J. Levine1,2* and Anna M. Puzio-Kuter2
need to synthesize substrates for membranes, nu-
Cells from some tumors use an altered metabolic pattern compared with that of normal cleic acids, and proteins (increase mass), which
differentiated adult cells in the body. Tumor cells take up much more glucose and mainly process means not metabolizing all of the glucose to CO2
it through aerobic glycolysis, producing large quantities of secreted lactate with a lower use of and H2O but instead providing the proper in-
oxidative phosphorylation that would generate more adenosine triphosphate (ATP), water, and termediates for cell growth. This is accomplished,

carbon dioxide. This is the Warburg effect, which provides substrates for cell growth and division in part, by slowing the entry of pyruvate into
and free energy (ATP) from enhanced glucose use. This metabolic switch places the emphasis on mitochondria, decreasing the conversion to acetyl-
producing intermediates for cell growth and division, and it is regulated by both oncogenes and CoA, and slowing the rate of the TCA cycle.
tumor suppressor genes in a number of key cancer-producing pathways. Blocking these metabolic The pyruvate that builds up in aerobic glycolysis
pathways or restoring these altered pathways could lead to a new approach in cancer treatments. is, in part, converted into lactate that is secreted,
eliminating it from the pool and keeping glycol-
n 1926, Otto Warburg demonstrated that can- begun to answer these questions. Although our ysis active. The secreted lactate lowers the pH
I cer cells did not metabolize glucose in the

same way that glucose was catabolized in
normal, adult differentiated cells (1, 2). The can-
understanding of each question is still imperfect,
it is becoming clear that both oncogenes and
tumor suppressor gene products can influence the
of the cellular environment and the extracellular
matrix. This may influence remodeling of the
matrix, permitting blood vessel invasion in re-
cer cells relied on glycolysis, even in the presence switch between aerobic glycolysis and a more sponse to angiogenic factors produced by the
of abundant oxygen (aerobic glycolysis) with a extensive use of the TCA cycle to generate ATP. tumor (10). Furthermore, as a consequence of
reduced use of the tricarboxylic acid (TCA) cy- Furthermore, the altered metabolic processing of glycolysis, tumor lesions can become acidotic,
cle, so that the pyruvate made in glycolysis was glucose observed by Warburg may well contrib- which allows for the selection of motile cells that
commonly converted to lactate, which was se- ute to some of the causal changes in the cancer can break through the basement membrane and
creted from the cell (Fig. 1). Warburg suggested phenotype. There have been a number of reviews metastasize. The last step in glycolysis is cata-
that this observation explained the cancer pheno- that emphasize different aspects of this question lyzed by pyruvate kinase, which receives input
type and was possibly a causal event in cancer and provide a diverse set of answers (3–9). about both anabolic precursors and the energy
formation (1, 2). There were several reasons why Normal cells and cancer cells use both glu- status of the cell. Cancer cells make the fetal iso-
these observations were not understood and did cose and glutamine as substrates to generate form of pyruvate kinase (the M2 isoform), which
not promote additional research. First, metaboliz- energy for the cell (ATP); to produce substrates to is a spliced variant of the gene that adds several
ing glucose by glycolysis to produce pyruvate synthesize amino acids, nucleosides, and fatty amino acids, one of which is a tyrosine. This
and secreted lactate is energetically inefficient. acids; and to regulate the redox potential (number tyrosine is phosphorylated in cells with activated
Most of the adenosine triphosphate (ATP) gen- of oxidized molecules in a compartment divided tyrosine kinase signaling, a hallmark of actively
erated by glucose catabolism (34 out of 36 ATP by number of reduced molecules) so as to mini- growing cells. Pyruvate kinase M2 is stimulated
molecules per molecule of glucose) occurs mize the effects of reactive oxygen species (ROS) in a feedforward loop by fructose 1,6-bisphosphate,
during the TCA cycle, which is used less often that damage membranes and proteins and cause but the phosphotyrosine inhibits this positive
in cancer cells (Fig. 1). Second, it was unclear mutations in a cell. Glucose contributes carbon, regulation. Thus, in cancer cells the last step of
how this observation could contribute to the can- oxygen, and hydrogen for both anabolic processes glycolysis is slowed, resulting in a buildup of
cer phenotype. Third, possible mechanisms to and energy, whereas glutamine contributes nitro- phosphorylated intermediates that can be used in
mediate a switch to glucose utilization in glycolysis gen for synthesis of purines, pyrimidines, and anabolic synthesis and cell growth (11).
from the more efficient movement of pyruvate nonessential amino acids. Metabolism of gluta- Rapidly dividing cells require favorable en-
into the mitochondria to produce acetyl–coenzyme mine also produces the reduced form of nicotin- ergetics [that is, higher ATP/adenosine diphosphate
A (CoA) and enter the TCA cycle were only amide adenine dinucleotide phosphate (NADPH) (ADP) and ATP/adenosine monophosphate (AMP)
poorly understood. Fourth, with the discovery of for the synthesis of fatty acids and the modulation ratios]. Many cancer cells satisfy this problem by
the mutational activation of oncogenes and of the redox potential in a cell. Glucose passing taking up much larger amounts of glucose than do
inactivation of tumor suppressor genes as causal through the pentose phosphate pathway (PPP) normal cells. This results from facilitated glucose
steps in cancer, the relationship between these also generates NADPH and ribose-5-phosphate transport by one or more of several isozymes of
mutant genes and metabolic regulation was for the synthesis of nucleotides (Fig. 1). Normal membrane glucose transporters (GLUT 1 to 9).
unclear. Remarkably, after a long absence of in- adult differentiated cells have a low cell division Once inside the cell, glucose is phosphorylated
terest, research done in the past 10 years has rate (low turnover) and predominantly metabo- by one of several hexokinase enzymes (the first
lize glucose to CO2 and H2O through glycolysis step in glycolysis) to keep it in the cell because of
1 and the TCA cycle. This satisfies the needs of the charge added to glucose (Fig. 1). The high con-
Institute for Advanced Study, Princeton, NJ 08540, USA.
2
Cancer Institute of New Jersey, New Brunswick, NJ 08903, these cells for free energy supplied by efficient centrations of glucose in the cells of a cancer may
USA. ATP generation during oxidative phosphorylation be observed by positron emission tomography
*To whom correspondence should be addressed. E-mail: (complexes 1 to 4 in the oxidative phosphoryl- (PET) scans of radioactive F-19-2-deoxyglucose
alevine@ias.edu ation chain) linked to the TCA cycle in mitochon- (FDG is not metabolized but is located in the

SPECIALSECTION
Lactate Glucose Glu Trans
(Glut 1-4)
p53 Glucose p53

NADP NADPH
Glucose-6-P Ribose-5-phosphate Nucleotide

TIGAR synthesis
Pentose
phosphate
Fructose-6-P pathway
Fructose
2,6-bisphosphate PFK1
Fructose-1,6-bis-P
Glycolysis

α-Ketoglutarate
p53 PGM NADPH
Lipid Amino acid
NADP
synthesis synthesis
LDH Isocitrate
Lactate Pyruvate
Acetyl-CoA OAA
Glutamine Glutamine
Citrate Glut1
NADPH Glutamate
Pyruvate
NADP
Malate
Acetyl-CoA
Glutaminolysis OAA Citrate
TCA Glutamate Glutamine Glutamine
cycle Glut2
Mal α-KG
p53
Mal
OAA
NADH Cytochrome Cytochrome c
dehydrogenase oxidoreductase oxidase SCO2
Aspartate
OXPHOS
Nucleotide
synthesis
Fig. 1. Signaling networks and their regulation of metabolism in pro- is deaminated to form glutamate, which is processed to generate a-
liferating cells. The figure shows aspects of metabolism in proliferating ketoglutarate and maintain the TCA cycle. p53 induction of key players is
cells including glycolysis; lactate production; TCA cycle; oxidative phos- boxed, and p53 inhibition is circled. p53 induces TIGAR, inhibits phos-
phorylation; PPP; glutaminolysis; and the biosynthesis of nucleotides, lip- phoglycerate mutase (PGM), and represses GLUT1 and GLUT 4, resulting
ids, and amino acids. Glucose can be processed through glycolysis for in inhibition of glycolysis and opposing the Warburg effect that is seem
production of ATP and pyruvate, pass through the PPP to generate ribose in many cancers, whereas p53 induction of SCO2 and GLS2 enhances
5-phosphate and NADPH, and also enter into the mitochondrion-localized mitochondrial respiration. Glut Trans indicates glucose transporters; Glut
TCA cycle. Glucose-derived citrate is exported to the cytosol and processed 1, glutaminase 1; Glut 2, glutaminase 2; LDH, lactate dehydrogenase;
to acetyl-CoA, oxaloacetate (OAA), or a-ketoglutarate (a-KG). Glutamine Mal, malate; and OXPHOS, oxidative phosphorylation.
cell), which is indicative of enhanced glucose of the pyruvate in mitochondria that is not con- to reduce GSSG to GSH. Thus, NADPH is a
uptake by cells. Many, but not all, cancers have verted to lactate). NADPH also contributes to a major source of cellular “coolant” when oxidative
this property (3–9) of increasing glucose uptake, proper redox control and protects the cell from reactions run too “hot” (high ROS levels) by using
and this is a confirmation of the Warburg effect. ROS. There are several ways the cell responds to large amounts of glucose to produce both sub-
With large amounts of glucose available in a lower ROS levels, but by far the major molecule strates and energy. However, high levels of ROS
cell, glucose is metabolized through the PPP, involved is glutathione (GSH), which eliminates can be advantageous for cancer cells when they
producing nucleosides and generating NADPH. ROS by accepting an electron and is converted to allow for the stimulation of cell proliferation, in-
The NADPH is essential for fatty acid synthesis, its oxidized form, GSSG (glutathione disulfide). duction of genetic instability, and evasion from
along with acetyl-CoA (which is made from some The enzyme glutathione reductase uses NADPH senescence. Although if levels are too high, then

Metabolism
cancer cells undergo oxidative damage–induced as well as regulators of the response to hypoxia. protein synthesis and mass of a cell (18). Myc
cell death. Thus, ROS levels can be exploited to All of these metabolic pathways (TCA, PPP, and also regulates glutaminolysis at the microRNA
selectively kill cancer cells and therefore be used glycolysis) contain complex regulatory circuits at (miRNA) level by transcriptionally repressing
as a potential therapeutic. the levels of transcription, mRNA splicing, trans- miR-23a and miR-23b, which results in greater
Glutamine contributes both to substrate needs lation, and small molecule feedforward and feed- expression of their target protein, mitochondrial
of a dividing cell and to control of redox poten- back loops. A deeper understanding of these glutaminase-1, and thus up-regulation of gluta-
tials through the synthesis of NADPH. As with regulatory pathways that connect the genetics of mine catabolism.
glucose, excessive amounts of glutamine are taken cancer to the biochemical metabolic pathways Similarly, the loss of p53 functions lead to the
up (by a glutamine transporter) and used by can- may reveal selected metabolic processes that might Warburg effect (Fig. 2). The p53 protein represses
cer cells. After glutamine is taken into the cell, a be good drug targets for slowing or reversing the transcription of the GLUT 1 and 4 transporters
mitochrondrial-associated enzyme, glutaminase-1, cancers. (18). The p53 protein induces the transcription of
converts it to glutamate (a transaminase can Over the past 10 years, evidence has accumu- the TIGAR gene, which lowers the intracellular
use the amino group and capture the nitrogen for lated that the oncogenes myc, nuclear factor kB concentrations of fructose 2,6 bisphosphatase
synthesis of nucleosides, and amino acids or am- (NF-kB), AKT, and the tyrosine kinase receptors (FBPase) and thus decreases glycolysis by di-
monium is produced). Glutamate is converted to (epidermal growth factor, EGF; insulin-like growth verting glucose through the PPP (19) (Fig. 1).
a-ketoglutarate and enters the TCA cycle in the factor 1, IGF-1; Her-2; etc.), which turn on Ras, TIGAR also has functional similarities to the

mitochondria. The malate and citrate produced RAF–mitogen-activated protein kinase (MAP bisphosphate domain of PFK-2/FBPase-2 in re-
in the TCA cycle leave the mitochondria, where kinase), and the phosphatidylinositol 3-kinases gulating glycolysis, ROS levels, and apoptosis
malate is converted to pyruvate plus NADPH and (PI3Ks) and mammalian target of rapamycin and is structurally similar to FBPase2. The acti-
citrate is converted to isocitrate and then to a- (mTOR) pathways (Fig. 2) along with hypoxia- vation of p53 also increases the ubiqutination of
ketoglutarate, generating another molecule of induced factor (HIF), can stimulate the transcrip- phosphoglycerol mutatase, which decreases the
NADPH. Citrate also is converted to acetyl-CoA tion of a number of genes that encode the proteins activity of this glycolytic enzyme. p53 increases
for fatty acid synthesis and oxaloacetate for the that mediate the glycolysis and glutaminolysis the use of the TCA cycle and oxidative phos-
synthesis of nonessential amino acids (Fig. 1). pathways (Fig. 1). The rate of glycolysis can vary phorylation. The p53 protein enhances the tran-
The pyruvate generated in these reactions can be over 100-fold. High AKT and mTOR activities scription of the gene for synthesis of cytochrome
used to produce glucose (reverse glycolysis), which result in high HIF activity. Both the myc and c oxidase 2 (SCO2), which, along with synthesis
enters the PPP, maximizing the production of HIF-1 transcription factors increase the rate of of cytochrome c oxidase 1 (SCO1), assembles into
NADPH. The glutamate can be converted to as- transcription of some of the GLUT transporters oxidative phosphorylation complexes (20). Cells
partate, which contributes to nucleoside synthe- and hexokinase 2, enhancing both glucose uptake with mutant p53 have compromised oxidative
sis. The excessive quantities of glutamine taken and its retention in the cell (11). HIF increases the phosphorylation chains. p53 also promotes syn-
into and used by the cell results in the secretion of rate of transcription of over 100 genes, resulting thesis of a number of proteins that reduce the
alanine and ammonium, which together with lac- in angiogenesis, cell migration, cell survival, and high ROS load in cells. Sestrins 1 to 4 are p53-
tate bathe the extracellular matrix. energy metabolism. regulated genes and produce proteins that react
Glutamine is also a major cancer cell energy Among the HIF-regulated genes are 9 of the with and neutralize ROS (21). p53 also regulates
and anabolic substrate that requires functional 10 enzymes that function in glycolysis (12, 13). the p21 gene, and the p21 protein binds to and
mitochondria. However, Warburg’s hyphothesis HIF is regulated by the cellular hypoxia response. stabilizes the Nrf2 transcription factor, which reg-
had its basis in the theory that glycolysis is pre- Acute hypoxia stabilizes the HIF-1a and HIF-2a ulates a set of complex responses to altered re-
dominately used in cancer cells because of a proteins, which form dimers with HIF-1b and dox potentials and high ROS. P53 transcribes the
dysregulation of mitochondrial oxidative phos- HIF-2b. The stability of HIFa subunits is controlled glutaminase 2 gene, a nuclear gene that produces
phorylation. Research has shown that most can- by HIF prolyl-hydroxylases (PHDs), which use O2 a glutaminase localized in the internal compart-
cer cells do not have defects in mitochondrial and a-ketoglutarate to convert a prolyl residue to ment of mitochondria (22, 23). Unlike gluta-
metabolism except for rare mutations in succinate hydroxproline plus succinate and CO2 (14, 15). minase 1, glutaminase 2 converts glutamine to
dehydrogenase (SDH) or fumarate hydratase (FH), The hydroxyl-proline residues are bound by the glutamate, which can be used to enhance the
both enzymes of the TCA cycle and both initiating von Hippel–Lindau protein complex (VHL tumor rate of the TCA cycle and oxidative phosphoryl-
events of familial paraganalioma or leiomyoma suppressor lost in some types of cancers) con- ation (22, 23). Thus, these two glutaminases,
and of papillary renal cell cancer. taining an E3 ubiquitin ligase, which results in regulated by myc (glutaminase 1) and p53 (glu-
Thus, cancer cells maximize their ability to the HIFa subunit being polyubiquitinated and de- taminase 2), have opposite effects on the cell.
synthesize substrates for membranes, nucleic acids, graded (16). The absence of oxygen stabilizes the Just why this is the case remains to be elucidated.
and proteins. This results in increased cell mass HIF transcription factor. In addition, lactate de- An activated p53 protein also inhibits the activ-
and allows cell division when needed. This can- hydrogenase A and pyruvate dehydrogenase ki- ities of the phosphatidylinositol-3 kinase (PI3K)–
not be accomplished without large amounts of nase are transcriptionally regulated by HIF, both of AKT and mTOR pathways (Fig. 2). P53 regulates
energy (ATP), which are obtained by increasing which keep pyruvate away from the mitochondria. the transcription of four genes, PTEN, IGF-
the use of glucose and glutamine many fold. Pen- The loss of PTEN (phosphatase and tensin homo- binding protein-3 (IGF-1BP-3), tuberous sclero-
alties for this increased flux are an increase in log, a tumor suppressor gene) and concurrent in- sis protein 2 (TSC-2), and the beta subunit of
oxidative intermediates, an altered redox poten- crease of AKT-1 and mTOR lead to HIF activation AMP-activated protein kinase (AMPK), which
tial, and excessive ROS. The metabolic response and the Warburg effect (Fig. 2). The myc tran- all negatively regulate AKT kinase and mTOR
is to focus reactions on producing NADPH, a scription factor activates the transcription of more (24, 25). In addition, sestrins 1 and 2, which are
coolant that feeds into many chemical systems than 1000 genes involved in all phases of cell p53-regulated genes, stimulate AMPK activity
that reduce the ROS activity. This high rate of growth and metabolism. Myc enhances the tran- (26) All of these activities shut down cell growth,
glucose and glutamine flux must be handled by scription of glutaminase-1, the first enzyme in decrease the Warburg effect, lower HIF levels,
increased metabolic enzyme levels or increased glutaminolysis producing glutamate (17), and it and thus reverse the cancer phenotype. In some
enzyme activities. Remarkably, this is accom- transcribes the ribosomal RNA genes and the ri- cases, this results in a p53-directed apoptosis and
plished by oncogenes and tumor suppressor genes bosomal protein genes, increasing the rate of the activation of autophagy (27).

SPECIALSECTION
IGF-1 that contain p53 mutations, demon-
strating the close interactions between
these two pathways (35). The inhi-
bition of lactate dehydrogenase in
IGF-1 receptor Glucose
cancer cells slows their growth, sug-
IGF-BP3 gesting the importance of making and
secreting lactate from cancer cells.
Most hepatocellular carcinomas have
PI3K lost the expression of glutaminase-2
AMP
in mitochondria, even though most of
these tumors do not contain p53 mu-
tations (p53 regulates this gene). Re-
p53 PTEN PIP3 LKB1 β-AMPK turning a cDNA for glutaminase-2 to
these cells in culture and expressing
this protein, which enhances the use
of the TCA cycle, inhibits cell divi-

mTORC2 PDK1
p53 sion (22, 23). Thus, glutaminase-2 is
AMPK
acting like a tumor suppressor gene in
these situations. Indeed, a wild-type
p53 gene and protein are required for
MDM2 Akt TSC2-TSC1 Rheb efficient mitochondrial DNA replica-
tion and mitochondrial maintenance
in cells (36). These types of observa-
p21 mTOR-Raptor tions suggest that the extensive al-
Forkhead
terations of metabolic processes in
cancer can contribute to the pheno-
types of the tumor cells and as such
4EBP1 S6 kinase are themselves causal (necessary but
not sufficient) for these cancers.
Fig. 2. p53 regulation of PI3K, Akt, and mTOR pathways. p53 functions in a complex network to mediate a cell’s Conclusions
adaptation to stress. To do this, p53 regulates the transcription of four genes, PTEN, IGF-BP3, TSC2, and AMPKb, which The observations and ideas re-
then all negatively regulate Akt kinase and mTOR, leading to a decrease in cell growth and a reversal of the cancer
viewed here suggest a unity in the
phenotype. Coordinately, there is an inhibition of proliferation that is promoted through an activation of p53 and
genes and pathways involved in sev-
LKB1. In addition to slowing cell growth, p53-dependent inhibition of the mTOR pathway promotes autophagy, a way
of helping cells survive. 4EBP1, 4E-binding protein 1; IGF-1 receptor, insulin-like growth factor–1 receptor; LKB1, liver eral diseases. The interrelationships
kinase B1; MDM2, murine double minute 2; PDK1, phosphoinositide-dependent kinase-1; PIP3, phosphatidylinositol of the p53, AKT, and mTOR path-
3,4,5-trisphosphate; Raptor, regulatory associated protein of mTOR; Rheb, Ras homolog enriched in brain; S6 kinase, ways (Fig. 2) bring together stress
ribosomal protein S6 kinase; TSC, tuberosclerosis complex. responses and diabetes. Indeed, p53
in adipose tissue can regulate insulin
resistance (37). There is a similar
A number of mutations in genes that encode NADH-dependent reduction of a-ketoglutarate overlap among several genes whose mutations
enzymes in the TCA cycle have been shown to to 2-hydoxyglutarate, an oncometabolite that can predispose an individual to Parkinson’s disease
lead to some types of cancers. Mutations in suc- be a correlative marker for mutations occurring in and the functions of those genes in the p53, AKT,
cinate dehydrogenase and fumarate hydratase isocitrate dehydrogenase enzymes (32). Just why and mTOR pathways (38). The connections among
alter the complex 2 oxidative phosphorylation this is so strongly selected for in these tumors chronic inflammatory responses of the immune
chain, which generates reduced flavine adenine may well be more complex than simply generat- system, with the activation of NF-kB and its as-
dinucleotide (FADH2). These mutations force a ing more NADPH. sociated metabolic changes (Warburg effect) and
switch to the Warburg effect and contribute to se- The large number of genetic alterations ob- PET scan–positive cells, and the formation of
lected inherited and sporadic cancers (27). There served in human cancers in the oncogenes and cancers of those cells are well established (39). It
is some evidence that these mutations result in the tumor suppressor genes involved in the IGF-1/ should not be surprising to observe such a central
inactivation of the PHDs, leading to increases in mTOR pathways (Fig. 2) suggest that drugs may role of metabolic processes in many disorders
HIF-1 and an enhanced glycolytic pathway. In be developed that alter the Warburg effect and and the integration of metabolic pathways with
glioblastoma multiforme, up to 12% of these tu- some of its consequences. Inhibitors of TOR many diverse signal transduction pathways. Meta-
mors have spontaneous point mutations in the gene complex1 (TORC1), which controls protein syn- bolic pathways comprise an evolutionarily con-
for cytosolic isocitrate dehydrogenase 1 (IDH1) (28). thesis and cell cycle progression, are already ap- served underlying feature for most functions of a
This enzyme converts isocitrate to a-ketoglutarate, proved for use in selected cancers. TORC1 is cell and an organism.
generating NADPH. Likewise, mutations have regulated by AMPK, which measures ATP/AMP
been observed in IDH2 at residue Arg172, in the ratios and nutrient availability. Metformin, which References
active site, in patients of low-grade gliomas (29) is used as a treatment for type 2 diabetes, stimulates 1. O. Warburg, K. Posener, E. Negelein, Biochem. Z. 152,
and acute myeloid leukemia (AML) (30), as well AMPK. Diabetic patients treated with metformin 309 (1924).
2. O. Warburg, Science 123, 309 (1956).
as other diseases (31). The somatic mutations in have lower incidences of cancer than diabetics 3. P. P. Hsu, D. M. Sabatini, Cell 134, 703 (2008).
IDH1 and IDH2 identified in gliomas and AML not treated with this drug (33, 34). Indeed, metformin 4. G. Kroemer, J. Pouyssegur, Cancer Cell 13, 472
result in a new ability of the enzyme to catalyze the acts as a synthetic lethal drug on cells in culture (2008).

Metabolism
5. M. G. Vander Heiden, L. C. Cantley, C. B. Thompson, 15. K. S. Hewitson, L. A. McNeill, J. M. Elkins, C. J. Schofield, 27. Z. Feng, H. Zhang, A. J. Levine, S. Jin, Proc. Natl. Acad.
Science 324, 1029 (2009). Biochem. Soc. Trans. 31, 510 (2003). Sci. U.S.A. 102, 8204 (2005).
6. R. J. Deberardinis, N. Sayed, D. Ditsworth, C. B. Thompson, 16. P. H. Maxwell et al., Nature 399, 271 (1999). 28. D. W. Parsons et al., Science 321, 1807 (2008);
Curr. Opin. Genet. Dev. 18, 54 (2008). 17. R. J. DeBerardinis et al., Proc. Natl. Acad. Sci. U.S.A. 10.1126/science.1164382.
7. J. W. Locasale, L. C. Cantley, M. G. Vander Heiden, Nat. 104, 19345 (2007). 29. H. Yan et al., N. Engl. J. Med. 19, 765 (2009).
Biotechnol. 27, 916 (2009). 18. C. V. Dang, Mol. Cell. Biol. 19, 1 (1999). 30. P. S. Ward et al., Cancer Cell 16, 225 (2010).
8. D. A. Tennant, R. V. Durán, H. Boulahbel, E. Gottlieb, 19. K. Bensaad et al., Cell 126, 107 (2006). 31. M. Kranendijk et al., Science 330, 336 (2010); 10.1126/
Carcinogenesis 30, 1269 (2009). 20. S. Matoba et al., Science 312, 1650 (2006); 10.1126/ science.1192632.
9. E. Gottlieb, K. H. Vousden, in The p53 Family, A. J. Levine, science.1126863. 32. L. Dang et al., Nature 17, 966 (2010).
D. Lane, Eds. (Cold Spring Harbor Laboratory Press, 21. A. V. Budanov, A. A. Sablina, E. Feinstein, E. V. Koonin, 33. J. M. Evans, L. A. Donnelly, A. M. Emslie-Smith,
Cold Spring Harbor, NY, 2010), pp. 187–198. P. M. Chumakov, Science 304, 596 (2004). D. R. Alessi, A. D. Morris, BMJ 330, 1304 (2005).
10. T. K. Hunt et al., Antioxid. Redox Signal. 9, 1115 22. W. Hu et al., Proc. Natl. Acad. Sci. U.S.A. 107, 7455 34. S. Jiralerspong et al., J. Clin. Oncol. 27, 3297
(2007). (2010). (2009).
11. H. R. Christofk, M. G. Vander Heiden, N. Wu, J. M. Asara, 23. S. Suzuki et al., Proc. Natl. Acad. Sci. U.S.A. 107, 35. M. Buzzai et al., Cancer Res. 67, 6745 (2007).
L. C. Cantley, Nature 452, 181 (2008). 7461 (2010). 36. A. Bourdon et al., Nat. Genet. 39, 776 (2007).
12. G. L. Semenza, Nat. Rev. Cancer 3, 721 (2003). 24. Z. Feng et al., Cancer Res. 67, 3043 (2007). 37. T. Minamino et al., Nat. Med. 15, 1082 (2009).
13. M. L. Macheda, S. Rogers, J. D. Best, J. Cell. Physiol. 202, 25. Z. Feng, in The p53 Family, A. J. Levine, D. Lane, Eds. 38. R. H. Kim et al., Cancer Cell 7, 263 (2005).
654 (2005). (Cold Spring Harbor Laboratory Press, Cold Spring 39. P. Ak, A. J. Levine, FASEB J. 24, 3643 (2010).
14. R. K. Bruick, S. L. McKnight, Science 294, 1337 (2001); Harbor, NY, 2010), pp. 199–298.

10.1126/science.1066373. 26. A. V. Budanov, M. Karin, Cell 134, 451 (2008). 10.1126/science.1193494
phagosomes. The Atg1/ULK1 complex (Atg1

REVIEW
in yeast and ULK1 in mammals) is an essential
Autophagy and Metabolism

positive regulator of autophagosome formation
(1). When nutrients are abundant, binding of the
ULK1 complex by the mammalian target of ra-
Joshua D. Rabinowitz1,2 and Eileen White2,3,4 pamycin (mTOR) complex 1 (mTORC1) inhibits
autophagy. mTORC1 is an important regulator of
Autophagy is a process of self-cannibalization. Cells capture their own cytoplasm and organelles cell growth and metabolism. It is composed of
and consume them in lysosomes. The resulting breakdown products are inputs to cellular five subunits that include Raptor, which binds
metabolism, through which they are used to generate energy and to build new proteins and ULK1, and mTOR, a serine-threonine kinase. By
membranes. Autophagy preserves the health of cells and tissues by replacing outdated and phosphorylating ULK1 and another complex mem-
damaged cellular components with fresh ones. In starvation, it provides an internal source of ber (the mammalian homolog of yeast Atg13),
nutrients for energy generation and, thus, survival. A powerful promoter of metabolic homeostasis mTOR inhibits autophagy initiation. In starvation,
at both the cellular and whole-animal level, autophagy prevents degenerative diseases. It does mTORC1 dissociates from the ULK1 complex,
have a downside, however—cancer cells exploit it to survive in nutrient-poor tumors. freeing it to trigger autophagosome nucleation and
elongation.
Autophagosome nucleation requires a com-
iving organisms from yeast to humans are conserved from yeast to mammals and regulate plex containing Atg6 or its mammalian homolog,
L capable of eating parts of themselves in

order to survive. This involves the degra-
dation of cellular components, either because they
the cannibalism of intracellular cytoplasm, pro-
teins, and organelles.
Autophagy is the only mechanism to degrade
Beclin 1, that recruits the class III phosphatidylino-
sitol 3-kinase VPS34 to generate phosphatidylino-
sitol 3-phosphate (2). Expansion of autophagosome
are deleterious (e.g., damaged organelles and mi- large structures such as organelles and protein membranes involves two ubiquitin-like molecules,
crobial invaders) or because the resulting break- aggregates. In the absence of stress, basal autoph- Atg12 and Atg8 (called LC3 in mammals), and
down products are needed to support metabolism. agy serves a housekeeping function. It provides a two associated conjugation systems. The E1-like
This process was aptly termed autophagy from routine “garbage disposal” service to cells, elimi- Atg7 and E2-like Atg10 covalently link Atg12
the Greek “auto” or oneself and “phagy” or to eat. nating damaged components that could otherwise with Atg5, which together bind Atg16L1 to form
It has gained attention recently as an essential become toxic. Such cellular refreshing is partic- pre-autophagosomal structures. In the second
contributor to human health and disease. ularly important in quiescent and terminally dif- ubiquitin-like reaction, LC3 is cleaved by the pro-
There are several forms of autophagy, each of ferentiated cells, where damaged components are tease Atg4. Phosphatidylethanolamine is conju-
which involves delivering intracellular cargo to not diluted by cell replication. In starvation, au- gated to cleaved LC3 by Atg7 and a second E2-like
lysosomes for degradation. The predominant form, tophagy provides a nutrient source, promoting enzyme, Atg3, and this lipidated LC3-II asso-
macroautophagy (autophagy hereafter), produces survival. Autophagy is induced by a broad range ciates with newly forming autophagosome mem-
vesicles called autophagosomes that capture and of other stressors and can degrade protein ag- branes. LC3-II remains on mature autophagosomes
deliver cytoplasmic material to lysosomes (1). gregates, oxidized lipids, damaged organelles, until after fusion with lysosomes and is common-
The autophagy-related genes (the atg genes) are and even intracellular pathogens. Although it is ly used to monitor autophagy.
not always possible to resolve the metabolic and The process beginning with the Beclin 1 com-
1
garbage disposal roles for autophagy, it is clear plex gives rise to nascent autophagosome mem-
Department of Chemistry and Lewis-Sigler Institute for that autophagy prevents disease. Defects in auto- branes. These membranes assemble around cargo,
Integrative Genomics, 241 Carl Icahn Laboratory, Washington
Road, Princeton University, Princeton, NJ 08544, USA. E-mail: phagy are linked to liver disease, neurodegenera- encapsulating the cargo in a vesicle that subse-
joshr@genomics.princeton.edu 2Cancer Institute of New tion, Crohn’s disease, aging, cancer, and metabolic quently fuses with a lysosome, generating an auto-
Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, syndrome. lysosome. The contents are then degraded by
USA. 3Department of Molecular Biology and Biochemistry, proteases, lipases, nucleases, and glycosidases.
Rutgers University, 604 Allison Road, Piscataway, NJ 08854, Process of Autophagy
USA. 4Robert Wood Johnson Medical School, University of
Lysosomal permeases release the breakdown
Medicine and Dentistry of New Jersey, 675 Hoes Lane, A series of protein complexes composed of atg products—amino acids, lipids, nucleosides, and
Piscataway, NJ 08854, USA. E-mail: whiteei@umdnj.edu gene products coordinate the formation of auto- carbohydrates—into the cytosol, where they are

SPECIALSECTION
Pentose phosphate pathway (PPP)

Glycogen Glucose-6-phospate Ribulose-5-phosphate
Glycogen
granule
Glycolysis Lipid
droplet
triglycerides
Glycerol
Fatty
Pyruvate acids
New lipid
Citrate synthesis
Acetyl-CoA
OAA Citrate

Malate Aconitate Energy
TCA cycle homeostasis
Fumarate Isocitrate
Ammonia
Succinate
α-KG
Succinyl-CoA
New protein
New
synthesis Amino acids nucleic acid
synthesis
Fatty
acids
Proteins Starvation, ammonia

Sugars stress, damage,
Lipids developmental cues
Carbohydrates Nucleosides
Nucleic acids
Autophagy
Autolysosome
Mitochondria, ribosomes, ER,
peroxisomes, protein aggregates,
cytoplasm, lipid droplets,
glycogen granules,
proteins
Fig. 1. Use of the products of autophagy. Multiple forms of stress activate lines) are used as building blocks for new protein synthesis, for ATP production by
autophagy (bottom right). Degradation of proteins, lipids, carbohydrates, and central carbon metabolism, and (in liver) as substrates for gluconeogenesis (Fig.
nucleic acids liberates amino acids, fatty acids, sugars, and nucleosides that are 3). They also can be combined to yield citrate, which drives lipid synthesis and
released into the cytoplasm for reutilization. Sugars (blue lines), including glucose membrane biogenesis. Catabolism of amino acids yields ammonia, an activator of
released from glycogen granules by glycogenolysis or autophagy, are catabolized autophagy (dotted line). Fatty acids (yellow lines) from lipolysis or from autophagy
by glycolysis and the PPP to generate ATP, and pyruvate for subsequent TCA cycle of membranes or lipid droplets yield acetyl-CoA, which feeds the TCA cycle,
metabolism. Nucleosides (green lines) are used for new nucleic acid synthesis and supporting ATP production and citrate generation. OAA indicates oxaloacetate;
catabolized by the combined action of the PPP and glycolysis. Amino acids (purple a-KG, a-ketoglutarate; and ER, endoplasmic reticulum.
available for synthetic and metabolic pathways prevent bulk autophagy from consuming essen- degradation by either the autophagy or protea-
(Fig. 1). tial components, such as a cell’s final mitochon- some pathways is ubiquitination. Many proteins
drion, remains unclear, and in some cases such accumulate in autophagy-defective mammali-
Substrates of Autophagy consumption may lead to cell death. an cells, indicating that autophagy has a major
Autophagy can be nonselective or selective. Non- Selective autophagy of proteins and of or- role in controlling the cellular proteome and that
selective, bulk degradation of cytoplasm and organelles such as mitochondria (mitophagy), ri- proteasome-mediated degradation cannot com-
ganelles by autophagy provides material to support bosomes (ribophagy), endoplasmic reticulum pensate for defective autophagy (4). To target
metabolism during starvation. It also contributes (reticulophagy), peroxisomes (pexophagy), and proteins for autophagic degradation, ubiquitin
to extensive tissue remodeling, as in Drosophila lipids (lipophagy) occurs in specific situations. on modified proteins is recognized and bound by
morphogenesis (3). Whether mechanisms exist to In mammals, the signal for targeting proteins for autophagy receptors, such as p62 or Nbr1, which

Metabolism
interact with LC3 to deliver cargo to autophago- perfused tissues and tumors (13). Glucagon, a trogen is removed, yeast defective in autophagy
somes (1). predominant hormone of the fasted state, also become severely depleted of internal amino acids.
Mechanisms regulating selective autophagy triggers autophagy in the liver. Adrenergic re- This precludes the synthesis of proteins im-
of organelles are more elaborate. In yeast, Atg32 ceptor activation, which like glucagon activates portant for surviving nitrogen starvation and
localized in mitochondria is a receptor that in- adenylate cyclase and cyclic adenosine mono- accelerates cell death (15). Thus, autophagy
teracts with Atg8 and Atg11 to produce selective phosphate (cAMP) production, also stimulates provides the primary route to nitrogen during
mitochondrial autophagy (5, 6). In mammals, liver autophagy. starvation.
autophagy of depolarized mitochondria, which Unlike microbes, mammalian cells benefit
protects cells from toxic reactive oxygen spe- Autophagy and Starvation from a relatively constant nutrient environment.
cies, is initiated by Pink1-dependent mitochon- All cells have internal nutrient stores for use dur- Nevertheless, autophagy can support mammalian
drial translocation of Parkin. This is followed ing starvation. Glycogen and lipid droplets are cells through nutrient deprivation. For example,
by ubiquitination of mitochondrial proteins and overtly designed for this purpose. Their contents in lymphocytes, the ability to consume environ-
recruitment of p62 to direct mitochondria to auto- are accessed primarily through the actions of ded- mental nutrients is growth factor–dependent. In
phagosomes (7, 8). The pruning of damaged icated enzymes, such as glycogen phosphorylase the absence of growth factor stimulation, energy
mitochondria by autophagy has two homeostatic and hormone-sensitive lipase. Many other cellular charge is maintained through autophagy, with
functions. The first is limiting oxidative damage. components have a dual function as nutrient stores. cells shrinking ~50% in size over 3 months of

The second is in maintaining a functional mito- For example, ribosomes occupy ~50% of the dry self-cannibalization (16).
chondrial pool. weight of rapidly growing microbes. In addition At the organismal level, autophagy is required
to enabling rapid protein synthesis when nutrient at multiple stages of mammalian development.
Regulation of Autophagy conditions are favorable, this provides a store of The first directly follows oocyte fertilization, with
Cells integrate information regarding nutrient amino acids for proteome remodeling when autophagy essential to feed the developing em-
availability, growth factor and hormonal receptor conditions turn for the worse. Autophagy has a bryo before it gains access to the maternal blood
activation, stress, and internal energy through an key role in providing access to such undedicated supply. Autophagy-defective embryos fail to reach
elaborate array of signaling pathways (Fig. 2). In nutrient stores. the blastocyst stage (17). Maternally supplied auto-
mammals, insulin—the master hormone of the Limitation for any of the major elemental nu- phagy proteins enable autophagy-deficient off-
fed state—blocks autophagy. trients triggers autophagy in yeast, with nitrogen spring to complete embryogenesis, revealing a
A major intracellular hub for integrating limitation the strongest stimulus (14). When ni- second requirement for autophagy: when access
autophagy-related signals is mTORC1
(9). In the presence of abundant nu-
trients and growth factors includ- Energy depletion
ing insulin, mTORC1 promotes cell
Stress Glucagon
growth and metabolic activity while
suppressing the ULK1 complex and Limitation for insulin,
autophagy. In deprivation or stress, nu- Hypoxia IKKβ
growth factors, or hormones
merous signaling pathways inactivate p53 LKB1
mTORC1 kinase activity. This both
suppresses cell growth to reduce ener- Depletion Sestrins RAS PTEN
PI3K
of amino acids
gy demand and induces autophagy to
HIFs
enable stress adaptation and survival. A AMPK
MAPK
second mTOR complex, mTORC2,
positively regulates mTORC1. Up-
stream of mTORC1 is the cellular TSC 1/2
RAGs AKT
energy–sensing pathway controlled by
adenosine monophosphate–activated Rheb
protein kinase (AMPK) (10). High
concentrations of AMP signal energy
depletion, activate AMPK, and in- mTORC1
hibit mTORC1, thus promoting au- Ammonia eEF-2 Ca++ FOXOs
kinase
tophagy (Fig. 2). ULK1 complex
Regulation of autophagy also
occurs through the forkhead box or
FOXO transcription factors, whose
activation leads to transcription of
atg genes (11). Similarly, hypoxia Autophagy
and activation of hypoxia-inducible
factors, or HIFs, induces the transcrip-
tion of mitophagy-specific genes and Fig. 2. Signaling pathways that regulate autophagy. Common nutrient, growth factor, hormone, and stress signals
mitophagy (Fig. 2) (12). Less well- that regulate autophagy. Purple lines depict events that positively regulate autophagy. Yellow lines depict those that
characterized mTOR-independent negatively regulate autophagy. Many pathways converge on the AMPK-mTORC1 axis. Green lines depict pathways that
regulators of autophagy also exist. are mTOR-independent. Note that all input signals are framed as autophagy activators; thus, they include limitation for
One is ammonia, a by-product of ami- growth factors and nutrients. IKKb, inhibitor of nuclear factor kB kinase b; PI3K, phosphatidylinositol-3 kinase; PTEN,
no acid catabolism, which stimu- phosphatase and tensin homolog; MAPK, mitogen-activated protein kinase; TSC1/2, tuberosclerosis complexes 1 and 2;
lates autophagy, likely in poorly and EF, elongation factor.

SPECIALSECTION
to the maternal blood supply is sud-
denly lost due to birth. Autophagy-
defective pups die within 24 hours
O O
of delivery. Both circulating and Cardiac O
OH
tissue amino acid levels are re- autophagy
OH O
ATP
duced, and AMPK is activated in the
heart, which shows electrocardio- OH
Fatty Amino
graphic changes analogous to those Ketone
acids acids OH
bodies
observed with severe myocardial O
HO
infarction (18). HO OH
ATP OH
In adult starvation, autophagy Glucose
also has a central role, increasing
within 24 hours in liver, pancreas,
Ketogenesis
kidney, skeletal muscle, and heart;
ATP, NADH Gluconeogenesis
the brain is spared (19). Pharmaco-
logical blockade of autophagy re-

sults in cardiac dysfunction early in
starvation (20). Although autophagy TCA
O
levels return to normal in liver 2 days
O
into starvation, it remains increased Fatty

HO
acids Amino O O
HO
in both cardiac and skeletal muscle. Fatty acids

H2N OH
Liver mass, however, persistently acids Amino
Liver NH2
falls faster than muscle or total body autophagy O
acids
mass. This decline is consistent with H3C
a failure of biosynthesis to balance Hormone- OH
basal consumption of liver by au- sensitive NH2

Muscle autophagy
tophagy (21). As liver mass falls, lipase and
other and proteosomal
breakdown of muscle and adipose lipases degradation
tissue feeds the liver, which exports
glucose and ketone bodies required
by the brain (Fig. 3).
Fig. 3. Role of autophagy in adult mammalian starvation. Depicted pathways predominate after depletion of
Use of Metabolites Released glycogen stores, typically ~12 hours into starvation. Autophagy in liver and heart (but not brain) generates fatty
by Autophagy acids and amino acids, which are catabolized to yield energy. In the liver, this energy drives gluconeogenesis
The breakdown products derived from and ketogenesis. Amino acids are substrates for both ketogenesis and gluconeogenesis; acetyl-CoA from fatty
autophagy have a dual role, providing acids is only for ketogenesis. As starvation continues, degradation of adipose and muscle play an increasing role
substrates for both biosynthesis and in supplying substrates to the liver, which exports glucose and ketone bodies to feed the brain. The relative
energy generation (Fig. 1). In terms importance of ketone bodies increases in prolonged starvation. NADH, reduced form of nicotinamide adenine
of biosynthesis, the abundance of dinucleotide.
ribosomal (relative to messenger)
RNA makes transcriptome remod-
eling straightforward. In contrast, proteome re- bodies for distribution elsewhere in the body available, the peroxisomes are no longer required
modeling demands copious amino acids, and a (Fig. 3). Ribose-phosphate from nucleosides and are cleared by autophagy (22). The function
major role of autophagy is to provide them. can be converted to glucose through the non- of autophagy in removing unneeded peroxisomes
In addition to providing anabolic substrates, oxidative pentose phosphate pathway (PPP). is conserved in mammals. Peroxisomes are
nucleosides and amino acids can be catabolized Amino acids feed into central metabolism at induced in liver by various hydrophobic chem-
for energy generation. RNA breakdown yields nu- multiple points, including pyruvate, tricarboxylic icals, known collectively as peroxisome prolifer-
cleosides, which are degraded to ribose-phosphate. acid cycle (TCA) cycle intermediates, and acetyl– ators. Removal of peroxisome proliferators leads
Six ribose-phosphate molecules are energetically coenzyme A (CoA) (Fig. 1). Pyruvate and TCA to restoration of normal peroxisome abundance
equivalent to five glucose-phosphates, and, like cycle intermediates are substrates for gluconeo- through autophagy (22).
glucose-phosphate derived by glycogen break- genesis. In contrast, mammals cannot convert Autophagy also regulates the abundance of
down, they can yield adenosine triphosphate (ATP) acetyl-CoA into glucose. Because lipid degrada- liver lipid droplets by their constitutive degradation
either aerobically or anaerobically. In contrast, tion yields mostly acetyl-CoA, ketone bodies are (23). Defective autophagy in mice leads to larger
amino acids, like lipids, yield ATP only through essential for feeding the brain and other vital tis- and more plentiful lipid droplets, increased con-
oxidative phosphorylation (Fig. 1). The catastroph- sues during prolonged starvation. centrations of hepatic triglycerides and cholesterol,
ic effects of ischemia are a consequence of the and increased gross liver size. Lipophagy is se-
relative paucity of nucleic acids and glycogen Autophagy as a Regulator lectively decreased by free fatty acids in vitro and
combined with the inefficiency of anaerobic gly- of Metabolism by a high-fat diet (23). Thus, in addition to pro-
colysis. Oxygen is the one nutrient that autoph- Autophagy is important for regulating cellular moting lipid droplet growth, free fatty acids may
agy cannot provide. metabolic capabilities. A striking example comes impair lipid droplet breakdown. Because lipophagy
In addition to being directly catabolized to from yeast capable of living off of methanol or fatty releases free fatty acids, its suppression by them
yield energy, the liver can convert nucleosides, acids, substrates that are burned in peroxisomes. is a case of one of the most prevalent regulatory
amino acids, and lipids into glucose and ketone When more appealing forms of carbon become motifs in metabolism: feedback inhibition. But this

Metabolism
feedback mechanism may backfire in the case of a may also contribute to the difficulty of losing of autophagy might synergize with existing cancer
chronic high-fat diet or obesity. weight (28). treatments (37).
In contrast to the role of autophagy in clearing
lipid droplets from the liver, autophagy is re- Autophagy and Disease Conclusions
quired for the production of the large lipid drop- Cellular garbage disposal by autophagy prevents Autophagy is a major contributor to cellular
lets characteristic of white adipose tissue (24, 25). the buildup of damaged proteins and organelles metabolism. It provides internal nutrients when
White adipose refers to the canonical fat storage that cause chronic tissue damage and disease. external ones are unavailable. It also provides an
tissue that expands in obesity; this is in contrast to Genetic inactivation of autophagy in mice re- essential means of refreshing and remodeling
brown adipose, a mitochondria-rich tissue that vealed that the type of disease depends on the cells. As such, it is required for normal develop-
catabolizes glucose and lipids to generate heat tissue type. In the brain, autophagy suppresses ment, including that of metabolic tissues such as
rather than ATP. Brown adipose tissue contains the accumulation of ubiquitinated proteins, dis- adipose tissue and pancreatic b cells. In adults,
uncoupling protein 1, which allows protons to poses of aggregation-prone proteins and dam- autophagy promotes metabolic homeostasis and
leak across the inner mitochondrial membrane, aged organelles that cause Huntington’s and prevents degenerative disease and cancer. Once
short-circuiting oxidative phosphorylation. Inhi- Parkinson’s diseases, and prevents neurodegen- cancer occurs, however, autophagy may contrib-
bition of autophagy blocks white adipocyte dif- eration (31, 32). In the liver, autophagy suppresses ute to tumor resiliency. Thus, both activation and
ferentiation, and adipose-specific knockout of atg7 protein aggregate and lipid accumulation, oxida- inhibition of autophagy hold promise for im-

results in mice whose white adipocytes manifest tive stress, chronic cell death, inflammation, and proved treatment of common, devastating diseases.
features typical of brown adipose tissue. Consist- cancer (4, 33). In intestinal Paneth cells, it pre-
ent with the rapid energy burning of brown serves cellular function, prevents expression of References
1. A. Kuma, N. Mizushima, Semin. Cell Dev. Biol. 21,
adipocytes, these mice are lean; however, they damage and inflammatory markers, and prevents 683 (2010).
are not healthy—when fed either a regular or the development of Crohn’s disease (34). Although 2. S. F. Funderburk, Q. J. Wang, Z. Yue, Trends Cell Biol.
high-fat diet, they are at increased risk of early the cell-refreshing role of autophagy functions in 20, 355 (2010).
death (24). preventing the above diseases, autophagy’s meta- 3. D. L. Berry, E. H. Baehrecke, Cell 131, 1137 (2007).
4. R. Mathew et al., Cell 137, 1062 (2009).
Autophagy also contributes to both insulin bolic role may also contribute by ensuring con-
5. T. Kanki, K. Wang, Y. Cao, M. Baba, D. J. Klionsky,
secretion and physiological sensitivity to the sistent availability of internal nutrients and enabling Dev. Cell 17, 98 (2009).
hormone. It is essential for the health of pancre- cells to survive periods of poor external nutri- 6. K. Okamoto, N. Kondo-Okamoto, Y. Ohsumi, Dev. Cell
atic b cells and for the expansion of b-cell mass tion in good health. Regardless of the underlying 17, 87 (2009).
that occurs in response to a high-fat diet (26, 27). mechanism, autophagy stimulation is under con- 7. S. Geisler et al., Nat. Cell Biol. 12, 119 (2010).
8. D. Narendra, A. Tanaka, D. F. Suen, R. J. Youle,
In liver, defective autophagy leads to insulin re- sideration for disease prevention. In support of
J. Cell Biol. 183, 795 (2008).
sistance (28). For reasons that are not yet fully this concept, autophagy mediates the protective 9. A. Efeyan, D. M. Sabatini, Curr. Opin. Cell Biol.
clear, hepatic autophagy is decreased in obese effects of dietary restriction on aging-related 22, 169 (2010).
mice, and its restoration through retroviral expres- diseases in model systems (35), and autophagy 10. D. B. Shackelford, R. J. Shaw, Nat. Rev. Cancer 9,
sion of atg7 ameliorates their insulin resistance. suppression contributes to the deleterious con- 563 (2009).
11. J. Zhao et al., Cell Metab. 6, 472 (2007).
sequences of obesity (28). Induction of autoph-
Overall Energetic Impact of Autophagy 12. H. Zhang et al., J. Biol. Chem. 283, 10892 (2008).
agy may result in the fasting rituals common in 13. C. H. Eng, K. Yu, J. Lucas, E. White, R. T. Abraham,
To maintain homeostasis, tissue degradation by many religions as well modern cleansing rituals, Sci. Signal. 3, ra31 (2010).
autophagy must be balanced by new macro- producing health benefits. 14. K. Takeshige, M. Baba, S. Tsuboi, T. Noda, Y. Ohsumi,
molecule synthesis, which is energetically ex- In contrast to normal cells in tissues, tumors J. Cell Biol. 119, 301 (1992).
15. J. Onodera, Y. Ohsumi, J. Biol. Chem. 280, 31582
pensive. Each peptide bond in protein costs often reside in an environment deprived of nu- (2005).
four high-energy phosphate bonds, two for tRNA trients, growth factors, and oxygen as a result of 16. J. J. Lum et al., Cell 120, 237 (2005).
charging and two for ribosome peptide bond insufficient or abnormal vascularization. Thus, 17. S. Tsukamoto et al., Science 321, 117 (2008).
formation. Assuming a free energy of ATP hy- the effects of autophagy in cancer are paradox- 18. A. Kuma et al., Nature 432, 1032 (2004).
19. N. Mizushima, A. Yamamoto, M. Matsui, T. Yoshimori,
drolysis of –50 kJ/mol under physiological condi- ical: Although autophagy can prevent initiation Y. Ohsumi, Mol. Biol. Cell 15, 1101 (2004).
tions, synthesizing 1 g of protein consumes 1.8 kJ of some cancers, it also may support tumor growth. 20. H. Kanamori et al., Am. J. Pathol. 174, 1705 (2009).
of energy. For a typical human, this means that Autophagy localizes to hypoxic tumor regions 21. M. Komatsu et al., J. Cell Biol. 169, 425 (2005).
rebuilding 10% of the body’s protein content most distant from nutrient-supplying blood ves- 22. M. Oku, Y. Sakai, FEBS J. 277, 3289 (2010).
23. R. Singh et al., Nature 458, 1131 (2009).
would consume at least 2000 kJ, or 20% of daily sels, where it sustains tumor cell survival (36).
24. R. Singh et al., J. Clin. Invest. 119, 3329 (2009).
dietary energy intake. In vitro estimates of au- Whether the primary role of autophagy in tumors 25. Y. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 106,
tophagic rates have generally been at or above the is to provide metabolic substrates or prevent 19860 (2009).
10% per day level, for example, 1% of protein buildup of damaged components is not yet known. 26. C. Ebato et al., Cell Metab. 8, 325 (2008).
per hour for cultured hepatocytes (21). Accord- But either way, inhibition of autophagy may sup- 27. H. S. Jung et al., Cell Metab. 8, 318 (2008).
28. L. Yang, P. Li, S. Fu, E. S. Calay, G. S. Hotamisligil,
ingly, although in vivo rates of autophagy are pre- press the growth of established tumors (37). Cell Metab. 11, 467 (2010).
sumably lower, rebuilding the structures degraded Many of the pathways that control autophagy 29. A. Rodriguez et al., Cell Metab. 3, 211 (2006).
by autophagy may be a major contributor to mam- are deregulated in cancer (Fig. 2), and cancer 30. A. M. Cuervo, J. F. Dice, J. Biol. Chem. 275, 31505
malian caloric requirements. Consistent with this therapeutics targeting these pathways activate (2000).
31. T. Hara et al., Nature 441, 885 (2006).
possibility, knockout of p62, which brings cargo autophagy. Some do so directly by inhibiting 32. M. Komatsu et al., Nature 441, 880 (2006).
to autophagosomes, results in decreased calorie mTOR, whereas others inhibit upstream nutrient 33. M. Komatsu et al., Cell 131, 1149 (2007).
burning and eventually obesity (29). During aging, or signaling pathways. Cytotoxic cancer thera- 34. K. Cadwell et al., Nature 456, 259 (2008).
both autophagy and total caloric expenditures pies activate autophagy, presumably by inflicting 35. P. Kapahi et al., Cell Metab. 11, 453 (2010).
36. K. Degenhardt et al., Cancer Cell 10, 51 (2006).
decrease in tandem (30). The possibility of a caus- damage. The functional role of autophagy in these 37. E. White, R. S. DiPaola, Clin. Cancer Res. 15, 5308
ative link, in which decreased autophagy leads to settings needs to be established. A particularly in- (2009).
reduced energy burned for self-regeneration, teresting possibility is that autophagy favors tumor
is intriguing. Decreased autophagy in obesity cell survival. If this proves correct, then inhibition 10.1126/science.1193497

SPECIALSECTION
of posttranslational signaling as a mechanism
REVIEW controlling of circadian cycles (4), and recent
work has shown that the SCN neuronal coupling
Circadian Integration of network itself has intrinsic oscillatory function

that can emerge in the absence of cell-autonomous
Metabolism and Energetics

oscillators (5). RNA interference screening of
mammalian cells also indicates coupling of the
peripheral clock to phosphatidylinositol 3-kinase
Joseph Bass1,2,3,4* and Joseph S. Takahashi5,6 signaling (6). Further research is warranted to
elucidate the impact of posttranslational signal-
Circadian clocks align behavioral and biochemical processes with the day/night cycle. Nearly all ing pathways on the core clock and its physio-
vertebrate cells possess self-sustained clocks that couple endogenous rhythms with changes in logical outputs.
cellular environment. Genetic disruption of clock genes in mice perturbs metabolic functions of Fibroblast cell lines display ~24 hours oscil-
specific tissues at distinct phases of the sleep/wake cycle. Circadian desynchrony, a characteristic of lation of core clock genes, demonstrating that
shift work and sleep disruption in humans, also leads to metabolic pathologies. Here, we review the clock is expressed not only in neurons but
advances in understanding the interrelationship among circadian disruption, sleep deprivation, also in peripheral tissues (7). Intrinsic oscillation

obesity, and diabetes and implications for rational therapeutics for these conditions. of clocks in liver cells can be entrained by food,
whereas oscillation of the brain clock is resilient
he rising and setting of the sun has cap- level, it has been suggested that increased activ- and entrained primarily by light (8). A recurring
T tivated naturalists interested in the cause

of daily rhythmic phenomenon, prompt-
ing de Marian to demonstrate the existence of
ity during what was “rest” time in the premod-
ern world, together with sleep disruption, have
been associated with an increased prevalence of
theme in understanding the coupling between
circadian and metabolic systems is the recogni-
tion that the two systems are reciprocally regulated;
an internal clock in the 18th century by placing obesity, diabetes, and cardiovascular disease, in food entrains the liver clock, whereas light acts
the Mimosa plant in a dark box and showing addition to certain cancers and inflammatory through the brain clock to control feeding time.
that its leaves continued to open and close every disorders. This review highlights advances in
24 hours. More than two centuries later, genetic understanding the molecular coupling between Crosstalk Between the Clock and Metabolic
studies in fruit flies paved the way for discovery metabolic and clock networks and its relevance Transcription Networks
that the circadian clock is encoded by a set of to gene-environment and brain-behavioral sys- Nuclear hormone receptors and the phase
transcriptional activators and repressors that com- tems important in energy balance and metabolic alignment of metabolic gene expression cycles.
prise an autoregulatory transcription-translation disease. Direct evidence for metabolic input into the core
feedback loop. The circadian clock in mammals clock includes the finding that the orphan nu-
is expressed within pacemaker neurons of the Core Transcriptional Components of the Clock clear hormone receptor (NHR) reverse-erb alpha
suprachiasmatic nucleus (SCN) that in turn main- and Posttranslational Regulation (REV-ERBa) (a repressor) and the opposing ret-
tain proper phase alignment of peripheral tissue Features of the circadian clock in all organisms inoic acid orphan receptors (RORa and b) (acti-
clocks present in nearly all cells. Thus, the brain include its persistence under constant condi- vators) constitute a short feedback loop controlling
SCN clock provides “standard time” for all pe- tions, a periodicity that is temperature compen- Bmal1 transcription (9, 10) (Fig. 2). Peroxisome
ripheral tissue clocks. In experimental models, sated, and its entrainment to light from the sun proliferator–activated receptor a (PPARa) and
clock disruption leads to disorders in glucose (Fig. 1). In mammals, cell-autonomous circadian the coactivator peroxisome proliferators–activated
metabolism, confirming a role for these genes as clocks are generated by a transcriptional auto- receptor gamma coactivator 1-a (PGC1a) also
key regulators of metabolism and supporting the regulatory feedback loop composed of the tran- modulate Bmal1 transcription through this feed-
hypothesis proposed by McKnight and colleagues scriptional activators CLOCK and BMAL1 and back loop (11), indicating that REV-ERBa is a
that circadian cycles are intimately interconnected their target genes Period (Per) and Cryptochrome nodal point for metabolic input into the clock.
with metabolic cycles (1). Accumulating evidence (Cry), which rhythmically accumulate and form NHR profiling has revealed rhythmic clustering
has revealed that multiple clock genes participate a repressor complex that interacts with CLOCK- of these factors in metabolic tissues across the
in metabolic homeostasis, suggesting that these BMAL1 to inhibit their own transcription (2). day/night cycle, suggesting extensive coupling
proteins have evolved overlapping (or conver- This autoregulatory loop is posttranscriptionally between circadian and nuclear receptor signaling
gent) functions both as intrinsic “hands” of the regulated by casein kinases (CK1e and CK1d), networks (12). These findings raise the possibil-
clock and as regulators of metabolism. Although which target the PER proteins for degradation ity that disruption of NHR cycles may perturb
still at an early stage, emerging studies in humans via the Skp1, Cullin1, F-box protein (SCF)/b-TrCP the clock and, conversely, that delay, advance,
suggest parallels in the role of circadian genes and ubiquitin ligase complex, and by adenosine mono- or reduced amplitude of circadian oscillations
metabolic homeostasis. At the epidemiological phosphate (AMP) kinase, which targets the CRY may impair NHR function. Knock-in mice of
proteins for degradation via the SCF/FBXL3 the NHR co-repressor NCor display increased
1
Department of Medicine, Northwestern University, Feinberg ubiquitin ligase complex by the 26S proteosome energy expenditure and a shift in the oscillation in
School of Medicine, Chicago, IL 60611, USA. 2Department of [reviewed in (2)] (Fig. 2). the abundance of RNAs encoding oxidative,
Neurobiology and Physiology, Northwestern University, Evanston,
The prevailing model of the circadian clock glycolytic, and respiratory genes, indicating that
IL 60208–3520, USA. 3Weinberg College of Arts and Sciences,
Northwestern University, Evanston, IL 60208–3520, USA. 4Center involves the transcription-translation feedback disruption of the phase of expression of NHRs
for Sleep and Circadian Biology, Northwestern University, loop, but less is known about nontranscriptional contributes to metabolic dysregulation (13). Mis-
Evanston, IL 60208–3520, USA. 5Department of Neuroscience, mechanisms that may generate circadian oscil- timing of gene expression rhythms as a cause of
University of Texas Southwestern Medical Center, Dallas, TX lations. In cyanobacteria, cycles of protein phos- metabolic dysregulation has also been suggested
75390–9111, USA. 6Howard Hughes Medical Institute, University
of Texas Southwestern Medical Center, Dallas, TX 75390–9111, phorylation are sufficient to generate biological by studies in Rev-erba mutant animals, in which
USA. rhythms in the absence of transcription (3). In a phase shift in oscillating rhythms of metabolic
*To whom correspondence should be addressed. E-mail: the mammalian SCN, changes in cyclic AMP gene transcription, rather than changes in total
j-bass@northwestern.edu levels alter period length, an additional example abundance of RNA, correspond with altered

Metabolism
energy balance (14). Misalignment between gene genes (16). Alternatively, the clock activator loop glucagon and inhibition of cyclic AMP signaling
transcription cycles within metabolic tissues and drives D-element–binding protein expression, pro- (18). Conceptually, the question of timing versus
the behavioral cycle (of fasting and feeding) may viding indirect regulation of gluconeogenic genes expression as a cause of metabolic disorders after
be sufficient to alter energy homeostasis. For (17). This raises the question as to whether the disruption of clock genes is akin to the difference
instance, high-fat feeding provided at the in- effects of clock-gene disruption relate to direct between a musical performer playing the wrong
correct circadian time leads to greater weight gain alterations in “timing” per se or to indirect effects notes or playing the right notes at the wrong time.
in mice than isocaloric feeding at the normal arising from independent activity of the clock One experimental approach to tease apart the role
circadian time (15). factors on metabolic networks. The dichotomy of circadian timing per se in physiology would be
Direct versus indirect role of clock tran- between circadian versus noncircadian actions to investigate whether physiological defects
scription factors in metabolic gene regulation. of clock proteins may not be fully valid, because could be corrected by alignment of the internal
It is likely that disruption within the core clock the rhythmic abundance in the expression level period with the external light cycle (i.e., a test of
may be transmitted to metabolic outputs through of these proteins in turn may produce rhythmic “resonance”). In plants, various period-length mu-
alterations in NHRs or directly by actions of clock changes in metabolism. For example, CRY, a tants have improved photosynthesis and growth
activators or repressors. For example, PER2 di- rhythmically expressed clock repressor, modu- when exposed to external light cycles that matched
rectly occupies promoters of certain metabolic lates gluconeogenesis through interference with the endogenous circadian period (19).

Environmental Inputs and Behavioral and Circadian
Internal Circadian Organization Physiologic Outputs Disruptors
∆ Period
SCN
(high fat)
original phase new phase
Sleep
Feeding
Brain Clock
SCN environmental
Wakefulness light cycle
Environmental
light/dark internal
cycle circadian time
Extra-SCN ∆ Phase
period
(shift work)
PVN LHA amplitude
ARC
∆ Amplitude
PIT
(night eating, insulin resistance)

Non-Autonomous
Circadian Control phase
Liver
Phase-Resetting Curves
Autonomous Circadian Control
Peripheral Clocks
Muscle
1
pe
Environmental
ty
nutrient cycle Glucose homeostasis type 0

fasting/feeding Fat Lipogenesis
Sterol turnover old CT
Oxidative metabolism
Respiration
Pancreas
new CT
Fig. 1. Central and peripheral clocks coordinate external cues with behavior right are the three possible ways to disrupt the clock by changing period,
and metabolic outputs. Light entrains the master pacemaker in the SCN, which phase, or amplitude, each of which can trigger disorders of metabolism. Phase
in turn synchronizes extra-SCN and peripheral clocks. Brain clock outputs resetting can be broadly classified into two groups based on phase response
include behavioral rhythms (i.e., sleep and feeding), whereas peripheral after delivery of the agent at sequential time points across the 24-hour cycle.
clock outputs include metabolic rhythms (e.g., glucose and lipid homeosta- Type 1 or weak resetting indicates that the slope of the plot relating the new to
sis). The hierarchical organization of the mammalian clock is highlighted, the old circadian phase is 1 (interventions that cause different phase shifts at
with “nonautonomous” regulation of peripheral tissue clocks denoting the different circadian times). Type 0 or strong resetting indicates that the slope of
regulation of peripheral tissue oscillators through direct neural and humoral the new to the old circadian phase is 0 (i.e., interventions that cause the same
signals, and “autonomous” regulation indicating the intrinsic regulation of phase at all circadian times). PVN, paraventricular nucleus; PIT, pituitary; ARC,
local cellular oscillators independently of the brain clock. Highlighted to the arcuate nucleus; LHA, lateral hypothalamic area.

SPECIALSECTION
CORE CLOCK PGC1α

PPARα
ROR
REV-ERB
Indirect Outputs
CLOCK BMAL1
E-box Gene
P NAMPT NAD SIRT1
CRY
P PER +
CKIε/δ PER Histone modification /
CRY Other Transcriptional Regulation
NAD-dependent (FOXO, STAT3, PGC1α)
sirtuins?
? LKB

AMPK ATP/AMP Post-Transcriptional
P Regulation
CRY
FBXL3 Proteasome
Direct Outputs
Metabolic Flux
Oscillating ∆ glucose
Transcription Factors Oscillating ∆ ATP/AMP
RNAs ∆ O2
DBP within ∆ glucocorticoids
NHRs Metabolic ∆ catecholamines
TEF Networks
REV-ERB
Gluconeogenesis
HLF Mitochondrial biogenesis
Oxidative phosphorylation
E4BP4
Amino acid turnover
Lipogenesis
Bile acid synthesis
Fig. 2. Direct and indirect outputs of the core clock mechanism. The core clock gluconeogenesis and oxidative metabolism. The clock also receives reciprocal input
consists of a series of transcription/translation feedback loops that synchronize from nutrient signaling pathways (including SIRT1 and AMPK), which function as
diverse metabolic processes through both direct and indirect outputs, including rheostats to couple circadian cycles to metabolic flux, especially in peripheral tissues.
Reciprocal control of clock by metabolic sig- of circadian and metabolic cycles, although this tissues. An unresolved question is whether pe-
naling. An intriguing question remains the extent remains to be tested. For example, PER2 forms ripheral organ clocks are principally entrained
to which NHRs modulate circadian systems ac- physical interactions with PPARa and REV- through direct neural wiring or through circulating
cording to changing environmental conditions, ERBa (16), in turn modulating transcription of hormones, such as glucocorticoids (24). The im-
such as humoral or nutritional factors. For ex- the gluconeogenic factor G6pase. Conversely, pact of glucocorticoids on hepatic entrainment has
ample, variation in the concentration of gluco- oscillation in NHR ligands may affect not only important implications for health under conditions
corticoid hormone, retinoic acid, heme, and fatty the phase and amplitude of circadian rhythms but of circadian misalignment, such as phase resetting
acids affect glucocorticoid receptors (GRs), ret- also physiological outputs of the circadian sys- during jet lag or shift work (25). The finding that
inoic acid receptor (RAR), REV-ERBa, neuronal tem. For example, glucocorticoid receptor bind- liver and brain respond to different entraining sig-
Per-Arnt-Sim (PAS) domain protein 2 (NPAS2), ing to the promoter of PER2 modulates leptin nals points toward a possible weak point in the
and PPARs. Therefore, variation in cellular con- production and glucose tolerance (22). Non- system; conditions such as insulin resistance,
centrations of any one of these ligands might autonomous signals, such as glucocorticoids or where signaling through glucocorticoid, catecho-
influence Bmal1 transcription and thereby mod- other systemic cues, may have an especially im- liminergic, or peptideric hormones may be at-
ulate local cellular circadian rhythms. Within portant role in sustaining oscillations of PER2 tenuated, may cause misalignment between the
brain, heme and carbon monoxide may modu- even in the absence of rhythmic oscillation of phase of central and peripheral oscillators. A
late NPAS2 activity (20), whereas within vascular CLOCK-BMAL1 (23). It may be possible to ex- related phenomenon is food anticipatory activity
cells, retinoic acid influences circadian oscilla- ploit tissue-specific differences in NHR-clock in- (FAA) caused by food presentation at the in-
tions through activation of RARa and RXRa teractions for therapeutic purposes. correct circadian time. Although clock-gene func-
(21). Likewise, rhythmic variation in NHR lig- NHRs may also participate in entrainment of tion in FAA behavior has been debated, abrogation
ands may exert distinct effects on local tissue central and peripheral clocks. In vertebrates, a hi- of melanocortinergic signaling influences this
clock function at different times in the day/night erarchy of signals within SCN pacemaker neurons behavior, consistent with a noncircadian timing
cycle. in the brain and downstream extra-SCN neurons mechanism (26). Finally, body temperature has
Local differences in NHR expression may generates entraining cues to maintain phase align- been shown to be a powerful entraining agent for
give rise to tissue-specific differences in coupling ment between oscillators in multiple peripheral peripheral oscillators. Indeed, most signals that

Metabolism
synchronize peripheral oscillators affect either The clock also functions in ultradian variation minogen activation inhibitor-1 (PAI-1) has been
body temperature or the heat shock pathway, so (cycles occurring multiple times in 24 hours) of shown to exhibit circadian regulation (44, 45).
this may be a final common pathway for resetting endoplasmic reticulum (ER) stress signaling, which Thus, inflammation, thrombosis, cardiomyocyte
clocks in mammals (27). in turn modulates SREBP activation through a metabolism, vascular tone, and response to vas-
posttranscriptional pathway (35). Rhythmic os- cular injury each represent phenotypes affected
How Circadian Disruption Causes Metabolic cillation of phosphorylation of inositol-requiring by circadian clock function.
Pathologies: Experimental Genetic Models and enzyme 1a (IRE1a), a transducer of the ER stress Circadian systems in glucose homeostasis and
Human Clinical Studies response, triggers rhythmic cleavage and trans- diabetes. Glucose concentrations in the blood are
REV-ERB and bile acid synthesis. Further studies location of SREBP into the nucleus. The ER stress highly rhythmic because of changes in insulin
on the repressor REV-ERBa have uncovered a response detects unfolded or improperly folded sensitivity and insulin secretory capacity of endo-
connection between the clock and the master path- proteins; thus, rhythmic activation of IRE1a in- crine pancreas (46). Individuals with type 2 dia-
way of hepatic lipid metabolism involving the tegrates circadian, stress-signaling, and lipogenic betes, and even their first-degree relatives not yet
sterol regulatory element–binding protein (SREBP). pathways. Indeed, ultradian rhythms within liver affected with the disease, display altered rhyth-
SREBPs control both fatty acid and sterol bio- appear with a 12-hour periodicity in the expres- micity in glucose tolerance (47). Although early
synthesis through modulation of rate-limiting en- sion of many clock-controlled RNAs (36) and morning insulin resistance has been ascribed to
zymes in these pathways. Using a combination even in rhythmic oscillation of the metabolite the surge in growth hormone during slow-wave

of genetic loss and gain of function approaches, nicotinamide adenine dinucleotide (NAD+). These sleep, rhythmic variation in insulin sensitivity is
Le Martelot et al. observed that the nuclear recep- shorter cycles are harmonics of the 24-hour cycle in part due to autonomic rhythms generated by
tor REV-ERBa controls oscillation in the abun- and may in turn produce rhythmic patterns in phys- afferent input from hypothalamus to liver, down-
dance and activation of the SREBPs through iologic pathways such as lipogenesis. In Cry1/Cry2 stream of the circadian clock (48). Ever since the
modulation of the enzyme INSIG2 (insulin-induced double-knockout mice, which harbor disruption inception of insulin use in clinical practice, re-
gene 2), an insulin-responsive factor that regu- within the repressor limb of the core clock, loss capitulating the endogenous rhythm of insulin
lates SREBP release from the endoplasmic reticu- of circadian oscillation corresponded with con- production, and achieving a proper match in the
lum. REV-ERBa knockout mice develop increased stitutive IRE1a activation and accumulation of variation in insulin requirement throughout the
lipogenesis through up-regulation of SREBP1c hepatic lipids. In contrast, in Clock ∆19 mutant mice, day/night cycle, has been a pragmatic clinical
and SREBP2 target genes independently of nu- which are deficient in the clock activation limb, goal.
trient state. In contrast, REV-ERBa–overexpressing there were opposite effects on SREBP activation. Rhythmic production of insulin regulated by
mice have decreased SREBP target gene transcrip- The finding that ablation of activators and repres- peripheral b-cell clocks was revealed by contin-
tion and correspondingly reduced circulating sors each produce physiologic effects builds evi- uous perifusion of isolated islets from the rat,
lipid concentrations. The effects of REV-ERBa dence that lipogenesis is driven by the circadian which has a 24-hour rhythm (49). Live-cell imag-
on bile acid metabolism are mediated through al- clock rather than an epiphenomenon of clock- ing in islets isolated from Per2-Luciferase mice
teration of oxysterol synthesis and liver X receptor gene disruption. shows that they express a self-sustained oscillator
(LXR) activity (14), although effects of the tran- The aforementioned studies in mice also have with period length matching that of the liver and
scription factor, the small heterodimeric protein implications for metabolic functions of the clock pituitary (50). Clock ∆19 mutant mice develop age-
(SHP), and E4BP4 (adenoviral E4 protein–binding in humans, because clock genes oscillate within dependent hyperglycemia in both the light and
protein) have also been implicated in this process human adipocytes (37) and alterations in clock- dark phases of the cycle, corresponding with pe-
(28). Because REV-ERBa expression is controlled gene expression are correlated with obesity (38). riods of fasting and feeding (29). These animals
by CLOCK-BMAL1, the rhythmic regulation of These findings increase the need to delineate the also develop susceptibility to diet-induced obesi-
bile acid production may represent one of the first relationship between chronotype (e.g., whether ty; however, rather than displaying the anticipated
direct molecular outputs of circadian clock on one is a “lark” or a “night owl”), clock genotype, hyperinsulinemia, they instead have inappro-
metabolic physiology. and metabolic physiology in humans. priately low concentrations of insulin. Clock∆19
Clock network and lipogenesis. In addition to Circadian regulation of cardiovascular func- mutant mice display a steeper drop in blood sugar
clock control of bile acid synthesis, mounting tion, inflammation, and thrombosis. It is axiomatic in response to treatment with insulin, a sign that
evidence has implicated both a direct and indirect in clinical medicine that certain cardiovascular these animals have enhanced insulin sensitivity,
effect of clock transcription factors on other as- catastrophes, including myocardial infarction and thereby masking their b-cell deficiency (50).
pects of lipogenesis. These observations stem from thrombosis, cluster early in the morning. Yet, the Bmal1 mutant mice also have impaired glucose
the finding that Clock ∆19 mutant mice develop mechanistic underpinnings of timing in cardio- tolerance (51), increased insulin sensitivity, and a
hypertriglyceridemia (29) due to effects within vascular disease are not understood. Many as- progressive myopathy with aging that causes ca-
both enterocytes and liver (30). At the level of the pects of fatty acid metabolism, a key fuel for chexia, which limits interpretation of glucose
intestine, the clock regulates triglyceride packag- cardiac muscle, exhibit strong circadian varia- turnover studies. Studies in isolated islets re-
ing into chylomicrons (globules that transport tion, and clock disruption affects chronotropic vealed first that the clock oscillator is expressed
dietary lipids), whereas in the liver, clock disruption function (39). Ablation of Bmal1 also increases and self-sustained in this tissue and, second, that
triggers lipid accumulation (30). The clock- the extent of arterial wall lesions after endothelial glucose responsiveness in islets is diminished
controlled gene Nocturnin also affects the inter- injury (40), suggesting multiple ways through when the clock is disrupted. After middle age, the
related processes of lipogenesis, osteogenesis (bone which clock genes may influence susceptibility mutants also have smaller islet size, reduced pro-
formation), and energy homeostasis (31–33). Ef- to myocardial damage. Similarly, autonomic and liferation, and transcriptome-wide decreases in
fects of circadian gene mutations on lipid absorp- mineralocorticoid control of vascular tone, fac- proliferative gene expression. Studies in tissue-
tion are strain-dependent in mice, with severe tors in cardiovascular disease risk, have been tied specific knockout mice have supported the hy-
steatorrhea (excess fecal fat) and malabsorption to the clock (41, 42). In individuals with meta- pothesis that function of the clock activators in
occurring in the ICR (Institute for Cancer Re- bolic syndrome, one predictor of cardiovascular the liver opposes their function in the pancreas
search) strain, thereby masking the effects of the risk is the absence of normal nocturnal variation (51). Whereas ablation of Bmal1 exclusively with-
Clock ∆19 mutation on hepatic triglyceride pro- in blood pressure, so-called “nondippers” (43). in the islet does not affect activity behavior, feed-
duction, and diet-induced obesity (34). Production of the prothrombotic molecule plas- ing, or body weight, these mice display a much

SPECIALSECTION
greater impairment of glucose tolerance than the indicates that lack of orexin signaling increases pulation that is intended to simulate deleterious
global knockout, as predicted. Islets from both susceptibility to obesity (rather than the original effects of jet lag or shift work, caused impaired
global and pancreas-specific knockouts have expectation that orexin, a potent wakefulness- glucose tolerance and hypoleptinemia. Whether
normal insulin content, and influx of calcium in inducing peptide, would induce adiposity) (57). circadian disruption might also affect endocrine
response to glucose is intact. However, exocytosis Orexin receptor 2 mutations also account for pancreas insulin secretion, hepatic gluconeogenesis,
is impaired, suggesting that the clock controls the canine narcolepsy, and orexin deficiency is a and glucose disposal in skeletal muscle in humans
latest stage in stimulus-secretion coupling. hallmark of the disease in humans (58). Activity awaits further study; however, these results empha-
Findings in experimental genetic models of of the orexin neuron is modulated by glucose and size the clinical linkages between circadian function
clock-gene ablation may also have implications integrates signals downstream of leptin-responsive and metabolic homeostasis.
for understanding emerging evidence that the neurons within the arcuate nucleus. Leptin also
circadian system participates in human glucose affects sleep, possibly independently of effects Coupling and Outputs: How Do Clocks Sense
metabolism. For instance, in genome-wide associa- on body weight, raising the need to further define and Respond to Nutrient Signals?
tion studies, variation in the Melatonin 1b receptor leptin actions in this process (59). Manipulation Under homeostatic conditions, the clock acts as a
(MTNR1B) and in Cry2 are both associated with of orexin signaling, an integrator of energetic and driver of metabolic physiology (Fig. 3). However,
blood glucose concentrations
[(52) and reviewed in (53)].

MTNR1B, the cognate receptor
Sleep deprivation
of the circadian-regulated hor- Prolonged wakefulness
Muscle
mone melatonin, is expressed Fatty acid uptake High-fat diet
in many metabolic tissues, Glycolytic metabolism
whereas Cry2 encodes a clock
repressor. These findings un- Fat
derscore the need to incorpo- Lipogenesis Insulin re
sista
Adiponectin production nce
rate temporal considerations Ins
uli
at the planning stages in future ion ns
Liver et
studies to account for circadian r
ec
ec
re
Glycogen synthesis
s
tio
variation. Similarly, temporal
n
n
uli
Cholesterol synthesis
Ins
considerations may aid in anal- WAKE

Bile acid synthesis
Me
ysis of experimental genetic
ids
laton
FEEDING
rtico
models because testing at dif- Pancreas Muscle
in secretion
ne Glucoco
ferent times and under different Insulin secretion Oxidative metabolism

environmental light cycles may
uncover unanticipated effects. Fat
Sleep and forced circadian Lipid catabolism
etic to
misalignment: genetic models Leptin secretion

and human studies. Ties be-
path
SLEEP
tween circadian disruption and
Sym
FASTING Liver
metabolic disturbance have
Gluconeogenesis
sis
garnered attention, including

ne
large cross-sectional sampling ge Glycogenolysis

eo
c on Mitochondrial biogenesis
of populations subjected to Glu
shift work. Extensive studies io n
Grow
th hormone Leptin se
cret Pancreas
also indicate a correlation be-
Glucagon secretion
tween sleep time and body
mass index (BMI). Disruption
in specific phases of sleep may Fig. 3. The clock partitions behavioral and metabolic processes according to time of day. The clock coordinates ap-
be connected to metabolic func- propriate metabolic responses within peripheral tissues with the light/dark cycle. For example, the liver clock promotes
tion. Subtle tones sufficient to gluconeogenesis and glycogenolysis during the sleep/fasting period, whereas it promotes glycogen and cholesterol
selectively deprive subjects of synthesis during the wake/feeding period. Proper functioning of peripheral clocks keeps metabolic processes in synchrony
slow-wave sleep without produc- with the environment, which is critical for maintaining health of the organism. Different tissues exhibit distinct clock-
ing conscious wakefulness were controlled properties; thus, ablation of the clock in certain tissues will cause opposing effects on metabolic function as
sufficient to impair glucose tol- uncovered through dynamic challenges at different times in the cycle under different nutrient conditions. Aging, diet, and
environmental disruption such as shift work may also affect the integration of circadian and metabolic systems.
erance (54). Neuroanatomic
studies also indicate intercon-
nections between regions of hypothalamus im- circadian signals, may thus provide opportunities with perturbations in either circadian or metabolic
portant in circadian signaling, energetics, and to intervene not only in disorders of sleep but also systems, such as forced behavioral misalignment
sleep (55, 56). At the molecular level, orexin in related metabolic complications. with shift work or, conversely, high-fat feeding, a
(also termed hypocretin), originally discovered In humans exposed to a light/dark cycle vicious cycle ensues in which disruption of meta-
as a neuropeptide produced in the feeding- lengthened to 28 hours, out of synchrony with the bolic pathways damp and lengthen circadian os-
stimulatory neurons of lateral hypothalamus, is endogenous clock, the sleep/wake cycle is driven cillations (61). The identity of metabolic sensors
positioned at the intersection of neuronal systems at 28 hours, whereas the melatonin and body- that may act as intermediates in coupling circa-
controlling sleep, circadian output, and metabolism temperature rhythm free-runs with a ~24-hour dian cycles with physiologic systems remains
(56). Analysis of orexin receptor 2 knockout mice period (60). Such “forced desynchrony,” a mani- to be identified. For instance, do changes in cell-

Metabolism
nutrient signaling in turn produce changes in that couple nutrient availability, metabolism, and 32. C. B. Green et al., Proc. Natl. Acad. Sci. U.S.A. 104,
circadian clock function? Does metabolic dis- the clock. 9888 (2007).
33. M. Kawai, A. M. Delany, C. B. Green, M. L. Adamo,
ease lead to altered amplitude or phase of cir- C. J. Rosen, Endocrinology 151, 4861 (2010).
cadian cycles within brain or peripheral organs? Conclusion 34. T. Kudo, T. Tamagawa, M. Kawashima, N. Mito,
Two lines of research have begun to address In just the past 20 years, the mystery of biological S. Shibata, J. Biol. Rhythms 22, 312 (2007).
these questions: first, involving the cellular path- timing has been transformed through genetic 35. G. Cretenet, M. Le Clech, F. Gachon, Cell Metab. 11,
47 (2010).
way of AMP concentrations, and second, involv- discovery. As a consequence, the availability of 36. M. E. Hughes et al., PLoS Genet. 5, e1000442
ing NAD+ metabolism. Using phosphopeptide molecular clock genes has now provided tools to (2009).
mapping, Lamia et al. identified a consensus mo- understand the physiological functions of the 37. X. Wu et al., Obesity (Silver Spring) 15, 2560 (2007).
tif for phosphorylation by AMP-activated protein circadian system in unprecedented detail. As we 38. X. Wu et al., Int. J. Obesity (London) 33, 971 (2009).
39. D. J. Durgan, M. E. Young, Circ. Res. 106, 647
kinase (AMPK), a sensor of AMP/ATP (adeno- experimentally dismantle the clock, the interde- (2010).
sine triphosphate) ratio, within the CRY protein pendence of timing and energetics seems in- 40. C. B. Anea et al., Circulation 119, 1510 (2009).
(62). AMP kinase activator 5-aminoimidazole-4- extricable. Major gaps in our understanding 41. A. M. Curtis et al., Proc. Natl. Acad. Sci. U.S.A. 104,
carboxamide-1-b-D-ribofuranoside (AICAR) include (i) the connection between brain and 3450 (2007).
42. N. Allaman-Pillet et al., Mol. Cell. Endocrinol. 226,
promoted degradation of CRY, which was abro- peripheral tissue clocks in metabolic homeosta-
59 (2004).
gated by mutation of the AMPK consensus mo- sis, (ii) the interplay between circadian and sleep

43. D. E. Ayala et al., Chronobiol. Int. 26, 1189 (2009).
tif. AMPK knockout mouse embryonic fibroblasts disruption in energetics, (iii) the relationship be- 44. E. J. Westgate et al., Circulation 117, 2087 (2008).
(MEFs) also displayed altered rhythmicity, lead- tween nutrient state and circadian homeostasis, 45. J. A. Schoenhard et al., J. Mol. Cell. Cardiol. 35,
ing to the proposal that AMP concentration di- and (iv) the impact of circadian clock systems on 473 (2003).
46. K. S. Polonsky et al., N. Engl. J. Med. 318, 1231
rectly couples circadian rhythms to nutrient state human physiology. Ultimately, such studies will (1988).
in peripheral cells. yield deeper insight into the interconnections 47. G. Boden, X. Chen, M. Polansky, Diabetes 48,
A second example in metabolic coupling between genes, behavior, and metabolic disease. 2182 (1999).
with the core clock originated with the finding 48. A. Kalsbeek et al., PLoS ONE 3, e3194 (2008).
49. E. Peschke, D. Peschke, Diabetologia 41, 1085
that the mammalian ortholog of the yeast sirtuin References and Notes (1998).
deacetylases (for silent information regulator)— 1. J. Rutter, M. Reick, S. L. McKnight, Annu. Rev. Biochem. 50. B. Marcheva et al., Nature 466, 627 (2010).
proteins that activate or silence chromatin ac- 71, 307 (2002). 51. K. A. Lamia, K. F. Storch, C. J. Weitz, Proc. Natl. Acad.
2. J. S. Takahashi, H. K. Hong, C. H. Ko, E. L. McDearmon, Sci. U.S.A. 105, 15172 (2008).
cording to availability of fuel—comprises part Nat. Rev. Genet. 9, 764 (2008). 52. J. Dupuis et al., Nat. Genet. 42, 105 (2010).
of an additional feedback loop with the core 3. M. Nakajima et al., Science 308, 414 (2005). 53. H. Mulder, C. L. Nagorny, V. Lyssenko, L. Groop,
clock (63, 64). Sirtuins are present in transcrip- 4. J. S. O’Neill, E. S. Maywood, J. E. Chesham, Diabetologia 52, 1240 (2009).
tion complexes with the clock and in turn mod- J. S. Takahashi, M. H. Hastings, Science 320, 949 54. E. Tasali, R. Leproult, D. A. Ehrmann, E. Van Cauter,
(2008). Proc. Natl. Acad. Sci. U.S.A. 105, 1044 (2008).
ulate activity of clock transcription factors.
5. C. H. Ko et al., PLoS Biol. 8, e1000513 (2010). 55. C. B. Saper, T. E. Scammell, J. Lu, Nature 437,
CLOCK-BMAL1 activates the major pathway 6. E. E. Zhang et al., Cell 139, 199 (2009). 1257 (2005).
for mammalian NAD+ synthesis, involving its 7. A. Balsalobre, F. Damiola, U. Schibler, Cell 93, 929 56. A. Adamantidis, L. de Lecea, Trends Endocrinol. Metab.
regeneration from nicotinamide mediated by nic- (1998). 19, 362 (2008).
otinamide phosphoribosyltransferase (NAMPT) 8. K. A. Stokkan, S. Yamazaki, H. Tei, Y. Sakaki, M. Menaker, 57. H. Funato et al., Cell Metab. 9, 64 (2009).
Science 291, 490 (2001). 58. S. Taheri, J. M. Zeitzer, E. Mignot, Annu. Rev. Neurosci.
(65) (66). NAD+ concentration in cells varies 9. N. Preitner et al., Cell 110, 251 (2002). 25, 283 (2002).
across the light/dark cycle, consistent with a role 10. H. Duez, B. Staels, Diab. Vasc. Dis. Res. 5, 82 (2008). 59. A. D. Laposky, M. A. Bradley, D. L. Williams, J. Bass,
for NAD+ as an oscillating metabolite linking 11. C. Liu, S. Li, T. Liu, J. Borjigin, J. D. Lin, Nature 447, F. W. Turek, Am. J. Physiol. Regul. Integr. Comp. Physiol.
metabolic cycles with the clock. Both NAMPT 477 (2007). 295, R2059 (2008).
12. X. Yang et al., Cell 126, 801 (2006). 60. F. A. Scheer, M. F. Hilton, C. S. Mantzoros, S. A. Shea,
and SIRT1, similar to the NHRs and AMPK, 13. T. Alenghat et al., Nature 456, 997 (2008). Proc. Natl. Acad. Sci. U.S.A. 106, 4453 (2009).
are regulated not only by the clock but also by 14. G. Le Martelot et al., PLoS Biol. 7, e1000181 61. A. Kohsaka et al., Cell Metab. 6, 414 (2007).
the nutritional status of the organism. For ex- (2009). 62. K. A. Lamia et al., Science 326, 437 (2009).
ample, fasting increases NAMPT expression in 15. D. M. Arble, J. Bass, A. D. Laposky, M. H. Vitaterna, 63. G. Asher et al., Cell 134, 317 (2008).
F. W. Turek, Obesity (Silver Spring) 17, 2100 (2009). 64. Y. Nakahata et al., Cell 134, 329 (2008).
an AMPK-dependent manner in skeletal muscle, 16. I. Schmutz, J. A. Ripperger, S. Baeriswyl-Aebischer, 65. K. M. Ramsey et al., Science 324, 651 (2009).
whereas fasting and caloric restriction increase U. Albrecht, Genes Dev. 24, 345 (2010). 66. Y. Nakahata, S. Sahar, G. Astarita, M. Kaluzova,
SIRT1 activity across multiple tissues. Thus, reg- 17. W. J. Roesler, P. J. McFie, C. Dauvin, J. Biol. Chem. 267, P. Sassone-Corsi, Science 324, 654 (2009).
ulation of the clock by NAD+ and SIRT1 allows 21235 (1992). 67. J. T. Rodgers, P. Puigserver, Proc. Natl. Acad. Sci. U.S.A.
18. E. E. Zhang et al., Nat. Med. 16, 1152 (2010). 104, 12861 (2007).
for fine-tuning and synchronization of the core 19. A. N. Dodd et al., Science 309, 630 (2005). 68. G. Asher et al., Cell 142, 943 (2010).
molecular clock with the environment. Because 20. E. M. Dioum et al., Science 298, 2385 (2002). 69. We thank R. Allada, F. Turek, B. Chung and K.-M. Ramsey
NAD+-dependent deacetylases regulate gluco- 21. P. McNamara et al., Cell 105, 877 (2001). for helpful suggestions and B. Marcheva for figures.
neogenesis and many other pathways (67), it 22. A. Y. So, T. U. Bernal, M. L. Pillsbury, K. R. Yamamoto, This work was supported by NIH (PO1 AG011412 and
will be important to further delineate the role of B. J. Feldman, Proc. Natl. Acad. Sci. U.S.A. 106, R01HL097817), Chicago Biomedical Consortium Searle
17582 (2009). Funds, Islet Biology Core of the University of Chicago
the clock in NAD+-driven metabolism (Fig. 2). 23. B. Kornmann, O. Schaad, H. Bujard, J. S. Takahashi, Diabetes Research and Training Center, American
A second NAD+-regulated pathway has recently U. Schibler, PLoS Biol. 5, e34 (2007). Diabetes Association, and Juvenile Diabetes Research
been linked to circadian feeding cycles: PARP-1 24. A. Balsalobre et al., Science 289, 2344 (2000). Foundation to J.B., and NIH P50 MH074924 and R01
activity is circadian in the liver, causing the 25. S. Kiessling, G. Eichele, H. Oster, J. Clin. Invest. 120, MH078024 to J.S.T. J.S.T. is an investigator in the Howard
2600 (2010). Hughes Medical Institute. J.S.T. is a cofounder of ReSet
rhythmic addition of poly-adenosine-diphosphate- 26. G. M. Sutton et al., J. Neurosci. 28, 12946 (2008). Therapeutics, Inc., and J.S.T. and J.B. are members of its
ribose residues to the CLOCK protein. Because 27. E. D. Buhr, S. H. Yoo, J. S. Takahashi, Science 330, scientific advisory board. J.B. is also an advisor and
PARP-1 is regulated by NAD+, this provides yet 379 (2010). receives support from Amylin Pharmaceuticals. Patents
another pathway for metabolic signals to reg- 28. H. Duez et al., Gastroenterology 135, 689 (2008). have been applied for related to J.B.’s work on
29. F. W. Turek et al., Science 308, 1043 (2005). therapeutic applications of NAD and melatonin.
ulate the core clock pathway (68). Collectively, 30. J. E. Baggs et al., PLoS Biol. 7, e52 (2009).
these findings identify incoming (AMPK and 31. M. Kawai et al., Proc. Natl. Acad. Sci. U.S.A. 107,
PARP-1) and outgoing (NAD+/Sirtuin) sensors 10508 (2010). 10.1126/science.1195027

SPECIALSECTION
particularly active pharmaceutical ingredients
REVIEW (APIs), some of which are too complex to be chem-
ically synthesized and yet have a value that justi-
Manufacturing Molecules fies the cost of developing a genetically engineered

microorganism. The cost of starting materials is
Through Metabolic Engineering

generally a small fraction of their cost, and rel-
atively little starting material is necessary so avail-
ability is not an issue. Most APIs fall into three
Jay D. Keasling1,2,3 classes of natural products, and many of the bio-
synthetic pathways for their precursors have been
Metabolic engineering has the potential to produce from simple, readily available, inexpensive reconstituted in heterologous hosts.
starting materials a large number of chemicals that are currently derived from nonrenewable Alkaloids are nitrogen-containing, low mo-
resources or limited natural resources. Microbial production of natural products has been achieved lecular weight compounds found primarily in and
by transferring product-specific enzymes or entire metabolic pathways from rare or genetically derived from plants and widely used as drugs.
intractable organisms to those that can be readily engineered, and production of unnatural Two recent studies conclude that the large group
specialty chemicals, bulk chemicals, and fuels has been enabled by combining enzymes or of benzyl isoquinoline alkaloids (BIAs) will

pathways from different hosts into a single microorganism and by engineering enzymes to have one day be producible in Escherichia coli and
new function. Whereas existing production routes use well-known, safe, industrial microorganisms, Saccharomyces cerevisiae (2). Unfortunately, the
future production schemes may include designer cells that are tailor-made for the desired BIAs are only one of four major alkaloid groups,
chemical and production process. In any future, metabolic engineering will soon rival and all of which are produced through different path-
potentially eclipse synthetic organic chemistry. ways. As the metabolic pathways for other
alkaloids are discovered in their
he term “metabolic engineer- natural producers, many more of
T ing” was coined in the late

1980s–early 1990s (1). Since
that time, the range of chemicals that
these valuable molecules could be
produced microbially.
Polyketides and nonriboso-
can be produced has expanded sub- mal peptides (NRPs) have found
stantially, in part due to notable ad- Sucrose broad use as APIs, veterinary
vances in fields adjacent to metabolic agents, and agrochemicals. Natu-
Starch 6-C sugars
engineering: DNA sequencing efforts rally occurring polyketides and
have revealed new metabolic reac- Cellulosic Bulk NRPs are produced by a number
biomass 5-C sugars chemicals
tions and variants of enzymes from of bacteria and fungi using large,
many different organisms; extensive Fine
modular enzymes. Their titers and
databases of gene expression, meta- chemicals yields in the native producers have
bolic reactions, and enzyme struc- been improved through traditional
tures allow one to query for desired Drugs strain engineering and advanced
reactions and design or evolve novel Fuels metabolic engineering. More re-
enzymes for reactions that do not cently, some of the most valuable
exist; new genetic tools enable more molecules have been produced with
precise control over metabolic path- engineered industrial hosts (3).
ways; new analytical tools enable the Recombination of various synthase
metabolic engineer to track RNA, Fig. 1. Conversion of sugars to chemicals by means of microbial catalysts. modules allows one to produce a
protein, and metabolites in a cell to nearly infinite range of chemicals
identify pathway bottlenecks; and detailed models pathway; and (vi) ways to maximize yields, titers, (4, 5), opening up the possibility that they may
of biology aid in the design of enzymes and meta- and productivities (Fig. 1). Unfortunately, these one day be used to produce fine and bulk chemicals.
bolic pathways. Yet even with these substantial de- design decisions cannot be made independently of Isoprenoids have found use as fragrances and
velopments, microbial catalysts are not as malleable each other: Genes cannot be expressed, nor will the essential oils, nutraceuticals, and pharmaceuticals.
as those in synthetic organic chemistry, and meta- resulting enzymes function, in every host; products Many isoprenoids have been produced microbially,
bolic engineers must weigh many trade-offs in the or metabolic intermediates may be toxic to one host including carotenoids and various plant-derived
development of microbial catalysts: (i) cost and avail- but not another host; different hosts have different terpenes (6–8), taking advantage of terpene syn-
ability of starting materials (e.g., carbon substrates); levels of sophistication of genetic tools available; thases to form the most complicated part of the
(ii) metabolic route and corresponding genes encod- and processing conditions (e.g., growth, produc- molecules and hydroxylases to introduce hydrox-
ing the enzymes in the pathway to produce the tion, product separation and purification) are not yl group that can be subsequently functionalized
desired product; (iii) most appropriate microbial host; compatible with all hosts. Even with these many chemically or biologically (7, 9). Isoprenoids are
(iv) robust and responsive genetic control system for challenges, metabolic engineering has been suc- one of the few classes of natural products where
the desired pathways and chosen host; (v) methods cessful for many applications, and with continued there are alternative precursor production path-
for debugging and debottlenecking the constructed developments more applications will be possible. ways. An example of using metabolic engineer-
ing and synthetic chemistry together to produce
1
Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA Starting Materials, Products, an API is the semisynthesis of the antimalarial
94608, USA. 2Physical Biosciences Division, Lawrence Berkeley and Metabolic Routes drug artemisinin with S. cerevisiae engineered to
3
National Laboratory, Berkeley, CA 94720, USA. Synthetic Biol-
ogy Engineering Research Center, Department of Chemical and
One area where metabolic engineering has a produce artemisinic acid, the most complex part
Biomolecular Engineering, University of California, Berkeley, CA sizable advantage over synthetic organic chem- of the molecule, and synthetic chemistry to pro-
94720, USA. E-mail: keasling@berkeley.edu istry is in the production of natural products, duce artemisinin from the microbially sourced

Metabolism
artemisinic acid (6, 7, 9). Beyond producing is the production of transportation fuels. For are many other molecules that one might want to
natural products, laboratory evolution or rational many of the same reasons that it is desirable to produce, such as short, highly branched hydro-
engineering of terpene cyclases, terpene hydrox- produce petroleum-derived chemicals using bio- carbons (e.g., 2,2,4-trimethyl pentane or isooctane)
ylases, and a host of other terpene-functionalizing logical systems, it is desirable to produce trans- that would be excellent substitutes for petroleum-
enzymes (8, 10–12) and combinatorial expres- portation fuels from readily available, inexpensive, derived gasoline. Additionally, most petroleum
sion of these evolved enzymes in a heterologous renewable sources of carbon. There is a long his- fuels are mixtures of large numbers of components
host will enable the production of unnatural ter- tory of using microorganisms to produce alcohols, that together create the many important properties
penes, some of which might be more effective primarily ethanol and butanol. Although much of of the fuels. It should be possible to engineer
than the natural product for the treatment of hu- the work on these alcohols was done by traditional single microbes or microbial consortia to produce
man disease. strain mutagenesis and selection, more recent work a mixture of fuels from one of the biosynthetic
Although individual metabolic pathways have focused on engineering yeasts and bacteria to pathways or from multiple biosynthetic path-
been developed to produce natural products de- produce ethanol or butanol from a variety of sugars ways. Indeed, some enzymes produce mixtures
rived from a single pathway, there is an oppor- while eliminating routes to side products and of products from a single precursor—maybe these
tunity to synthesize multisubstituent APIs (e.g., improving the tolerance of the host to the alcohol enzymes could be tuned to produce a fuel mixture
Taxol) or other molecules from the products of (14). Larger, branched-chain alcohols can be ideal for a particular engine type or climate.
multiple biosynthetic pathways. This will require produced by way of the Ehrlich pathway. By To make these new fuels economically viable,

simultaneous expression of multiple precursor incorporating broad substrate-range 2-keto acid we must tap into inexpensive carbon sources (name-
pathways in a single microorganism, as well as decarboxylases and alcohol dehydrogenases, ly, sugars from cellulosic biomass). Given the va-
“ligases” that can assemble multiple substituents several microbes have now been engineered to riety of sugars in cellulosic biomass, the fuel producer
together into a single molecule. The benefit would produce these fuels (15, 16). These alcohols are must be able to consume both five- and six-carbon
be the synthesis of complicated molecules that generally considered better fuels than ethanol and sugars. Because many yeasts do not consume five-
might not otherwise be produced. butanol and can also be used to produce a variety carbon sugars, recent developments in engineering
Although not as valuable as pharmaceuticals, of commodity chemicals. yeast to catabolize these sugars will make pro-
many fine chemicals have been produced eco- Recent advances in metabolic pathway and duction of these fuels more economically viable
nomically from natural and engineered micro- protein engineering have made it possible to (22). Engineering fuel-producing microorgan-
organisms, including amino acids, organic acids, engineer microorganisms to produce hydrocar- isms to secrete cellulases and hemicellulases to
vitamins, flavors, fragrances, and nutraceuticals. bons with properties similar or identical to those depolymerize these sugar polymers into sugars
For fine chemicals, profit margins are generally of petroleum-derived fuels and thus compatible before uptake and conversion into fuels has the
much lower than for APIs and may be affected by with our existing transportation infrastructure. potential to substantially reduce the cost of pro-
substrate availability and cost. Some of these mol- Linear hydrocarbons (alkanes, alkenes, and esters) ducing the fuel.
ecules are sufficiently complicated that they can- typical of diesel and jet fuel can be produced by
not be produced economically by any route other way of the fatty acid biosynthetic pathway (17–19). Hosts and Expression Systems
than biological production, whereas others have For diesel in cold weather and jet fuel at high From the applications cited above, it should be
chemical routes. For some important products altitudes, branches in the chain are beneficial— evident that the product, starting materials, and
(fragrances, flavors, amino acids), heterologous regularly branched and cyclic hydrocarbons of production process all affect host choice. Some
hosts have been engineered to enhance their pro- different sizes with diverse structural and chemical of the most important qualities one must consider
duction. Yet we have barely begun to investigate properties can be produced via the isoprenoid bio- when choosing a host are whether the desired
what will be possible to produce. synthetic pathway (20, 21). Both the fatty acid– metabolic pathway exists or can be reconstituted
In contrast, bulk chemicals such as solvents derived and the isoprenoid-derived fuels diffuse (or in that host; if the host can survive (and thrive)
and polymer precursors are rarely produced from are pumped) out of the engineered cells and phase under the desired process conditions (e.g., ambient
microorganisms, because they can be produced separate in the fermentation, making purification versus extremes of temperature, pH, ionic strength,
inexpensively from petroleum by chemical catal- simple and reducing fuel cost. etc.); if the host is genetically stable (both with the
ysis. Due to fluctuations in petroleum prices and Although the pathways described above pro- introduced pathway and not susceptible to phage
recognition of dwindling reserves, trade imbal- duce a wide range of fuel-like molecules, there attack); and if good genetic tools are available to
ances, and political considerations, it is now pos-
sible to consider production of these inexpensive
chemicals from low-cost starting materials such
as starch, sucrose, or cellulosic biomass (e.g., ag- Met Met
ricultural and forest waste, dedicated energy
crops, etc.) with a microbial catalyst. For example,
1,3-propanediol (1,3-PDE), a useful intermediate
in the synthesis of polyurethanes and polyesters, (-) (+)
is now being produced from glucose by E. coli
engineered with genes from Klebsiella pneumo-
niae and S. cerevisiae (13). There is an opportunity Gene R Gene A Gene B Gene C Gene 1 Gene 2 Gene 3 Gene P
to produce many other bulk chemicals (e.g., poly-
mer precursors) by using metabolically engineered
cells, but the key will be to produce the exact Substrate Metabolite Product
molecule needed for existing products rather than
something “similar but green” that will require
extensive product testing before it can be used. Fig. 2. Use of synthetic regulators to modulate metabolic pathways that have a toxic intermediate.
By far the highest-volume (and lowest- Regulatory proteins or RNAs bind the toxic metabolite and down-regulate the biosynthetic pathway and up-
margin) application for engineered metabolism regulate the consumption pathway.

SPECIALSECTION
DNA repository Metabolic pathway Ghost envelope

and registry and cell design
Constructed
chromosome
Engineered
microbial catalyst
Enzyme, Commercial Cell

pathway, and DNA synthesis production
cell CAD FAB facility

software
Fermentation
Desire
Desired
products
Fig. 3. The future of engineered biocatalysts. Pathways, enzymes, and genetic those elements are synthesized at a FAB and incorporated into a ghost
controls are designed from characteristics of parts (enzymes, promoters, etc.) envelope to obtain the new catalyst. The design of the engineered catalyst is
by means of pathway and enzyme CAD software. The chromosomes encoding influenced by the desired product and the production process.
manipulate the host. Widely used, heterologous that might otherwise be used to produce the de- of a culture (29). Constitutive promoters (26) and
hosts include E. coli, S. cerevisiae, Bacillus sired molecule of interest, particularly important promoters that respond to a change in growth
subtilis, and Streptomyces coelicolor, to name a for production of low-margin chemicals, while condition or to an important intermediary metab-
few. Although E. coli and S. cerevisiae excel in underexpressed genes will create pathway bottle- olite (30) allow for inexpensive, inducer-free
the genetic tools available, E. coli has the dis- necks. Furthermore, because intermediates of a gene expression, which is particularly important
advantage of being susceptible to phage attack. foreign metabolic pathway can be toxic to the het- where cost is an issue (Fig. 2). Although there are
And while B. subtilis and S. coelicolor have the erologous host (6), which results in decreased pro- many inducible promoters for bacteria, the small
ability to easily express polyketide synthases, duction of the desired final compound, it is number of inducible promoters for yeast and
they have fewer genetic tools available than either essential that the relative levels of the enzymes other potential industrial hosts makes regulation
S. cerevisiae or E. coli. Although minimal, bacte- be coordinated. of metabolic pathways in those organisms more
rial hosts may have scientific interest (23), mini- Central to any genetic manipulation is the challenging than in bacteria.
mal hosts that require addition of many nutrients or vector used to carry and/or harbor the transform- Because production of complicated mole-
that cannot cope with stresses in processing will ing DNA in the host. Important features of the cules often requires several enzymes, it is desira-
probably not find a niche in industrial chemical or cloning vector include segregational stability, ble to coordinate expression of the genes encoding
fuel production where cost is critical. Thus, it is minimal and consistent copy number in all cells these enzymes to prevent accumulation of toxic
essential to have genetic tools for existing industrial of a culture, and the ability to replicate and express intermediates and bottlenecks in biosynthetic
hosts that can grow on simple, inexpensive carbon large sequences of DNA. There is growing rec- pathways. There are many ways to coordinate ex-
sources and salts or on an inexpensive, undefined ognition that one or only a few copies of a gene are pression of multiple genes, such as using a non-
medium with minimal additions (24, 25). needed, particularly for metabolic engineering native RNA polymerase or transcription factor to
The key issue necessitating good genetic tools applications. With the ability to vary promoter induce multiple promoters (31); grouping multi-
is the introduction of foreign genes encoding the (26) and ribosome binding strength (27), as well as ple, related genes into operons; varying the ribo-
metabolic pathway and control over their expres- the stabilities of the mRNA (28) and the resulting some binding strength for the enzymes encoded in
sion to maximize yields and titers. The genes protein produced from it, there are many factors the operon (27); or controlling segmental mRNA
encoding the transformational enzymes in meta- other than copy number that can be manipulated to stability of each coding region to regulate the
bolically engineered cells do not need to be highly alter enzyme production. amount of each enzyme produced (32). One of
expressed, but must be produced in catalytic Promoters play an essential role in controlling the limitations to expressing multiple genes in
amounts sufficient to adequately transform the biosynthetic pathways. Inducible promoters are yeast is the lack of internal ribosomal entry se-
metabolic intermediates into the desired products one of the easiest and most effective ways to quences (IRESs) that are available for higher
at a sufficient rate. Expression of the desired genes regulate gene expression, but it is essential that eukaryotes. The development of yeast IRESs would
at too high a level will rob the cell of metabolites the promoter be induced consistently in all cells allow one to express genes encoding metabolic

Metabolism
pathways without the need for a promoter for trial processing conditions. Specific, engineered 3. B. A. Pfeifer, S. J. Admiraal, H. Gramajo, D. E. Cane, C. Khosla,
each gene. transporters would be incorporated into the mem- Science 291, 1790 (2001).
4. H. G. Menzella et al., Nat. Biotechnol. 23, 1171
brane to pump the desired product out of the cell (2005).
Debottlenecking, Debugging, and keep it out and to import the desired starting 5. V. Siewers, R. San-Bento, J. Nielsen, Biotechnol. Bioeng.
and Process Optimizing material. The biosynthetic pathway would be 106, 841 (2010).
Even with a kit full of tools, building a biosyn- constructed from a parts registry containing all 6. V. J. J. Martin, D. J. Pitera, S. T. Withers, J. D. Newman,
J. D. Keasling, Nat. Biotechnol. 21, 796 (2003).
thetic pathway is made difficult without accurate known enzymes by means of retrosynthesis soft- 7. D. K. Ro et al., Nature 440, 940 (2006).
blueprints. In almost all areas of engineering, ware (38), and done so to maximize yield and 8. E. Leonard et al., Proc. Natl. Acad. Sci. U.S.A. 107,
there are models and simulation tools that allow minimize the time required to grow the organism 13654 (2010).
one to predict which components to assemble to and produce the desired chemical from the desired 9. M. C. Chang, R. A. Eachus, W. Trieu, D. K. Ro, J. D. Keasling,
Nat. Chem. Biol. 3, 274 (2007).
obtain a larger system with a desired function or starting material. In the event that an enzyme does 10. Y. Yoshikuni, T. E. Ferrin, J. D. Keasling, Nature 440,
characteristic. Similar biological design tools are not exist for a particular reaction or set of reactions, 1078 (2006).
in their infancy. However, metabolic models that one would use computer-aided design (CAD) 11. C. Schmidt-Dannert, D. Umeno, F. H. Arnold,
incorporate cell composition and gene regulation software to design the desired enzyme (39). Nat. Biotechnol. 18, 750 (2000).
12. J. A. Dietrich et al., ACS Chem. Biol. 4, 261 (2009).
have become relatively predictive and may one day Once the cell has been designed in the com-
13. C. E. Nakamura, G. M. Whited, Curr. Opin. Biotechnol.
be used to design metabolic pathways and predict puter, the genetic control system would be

14, 454 (2003).
the level of gene expression needed to achieve a designed to control expression of all the genes 14. E. J. Steen et al., Microb. Cell Fact. 7, 36 (2008).
particular flux through a reaction or pathway (33). at the correct time and at the appropriate levels. 15. G. K. Donaldson, A. C. Eliot, D. Flint, L. A. Maggio-Hall,
Regardless of how sophisticated the design Redundancies in the genetic control system V. Nagarajan, U.S. Patent 20070092957 (2007).
16. S. Atsumi, T. Hanai, J. C. Liao, Nature 451, 86 (2008).
tools and how good the blueprint, there will always would be engineered to ensure that design param- 17. E. J. Steen et al., Nature 463, 559 (2010).
be “bugs” in the engineered system. Analogous to eters are maintained regardless of the transient 18. H. R. Beller, E.-B. Goh, J. D. Keasling, Appl. Environ.
software debuggers that allow one to find and fix changes the cell encounters during the production Microbiol. 76, 1212 (2010).
errors in computer code, the development of sim- process. Simulations and scenario planning would 19. A. Schirmer, M. A. Rude, X. Li, E. Popova, S. B. del Cardayre,
Science 329, 559 (2010).
ilar tools for biological debugging would reduce test various designs, including genetic control sys- 20. S. T. Withers, S. S. Gottlieb, B. Lieu, J. D. Newman,
development times for optimizing engineered cells. tem failure. Safety for the plant operators and the J. D. Keasling, Appl. Environ. Microbiol. 73, 6277
For the development of microbial chemical facto- environment would be an essential design criterion. (2007).
ries, functional genomics can serve in the role of When the genetic controls were fully designed and 21. N. S. Renninger, D. J. McPhee, World Patent 200804555
(2008).
debugging routines (34), because imbalances in a tested, the chromosome(s) would be designed and 22. H. W. Wisselink, M. J. Toirkens, Q. Wu, J. T. Pronk,
metabolic pathway often elicit a stress response constructed. Gene location, modularity, and ease of A. J. van Maris, Appl. Environ. Microbiol. 75, 907
in central metabolism (due to protein overpro- construction are but a few of the important (2009).
duction or accumulation of toxic intermediates or considerations in designing the chromosome. The 23. D. G. Gibson et al., Science 329, 52 (2010).
24. H. H. Wang et al., Nature 460, 894 (2009).
end products) (6, 35). Information from one or chromosome would be ordered from a commer-
25. G. Pósfai et al., Science 312, 1044 (2006).
more of these techniques can be used to diagnose cial DNA manufacturer. Depending on the state 26. P. R. Jensen, K. Hammer, Appl. Environ. Microbiol. 64,
the problem and modify expression of genes in of the technology at the time, the chromosome 82 (1998).
the metabolic pathway or in the host to improve would arrive in pieces and be assembled in the 27. H. M. Salis, E. A. Mirsky, C. A. Voigt, Nat. Biotechnol. 27,
titer and/or productivity. constructed envelope or would be completely as- 946 (2009).
28. C. D. Smolke, T. A. Carrier, J. D. Keasling, Appl. Environ.
Many desirable chemicals will be toxic to the sembled at the factory and sent to another loca- Microbiol. 66, 5399 (2000).
producer, particularly at the high titers needed for tion to be introduced into the ghost cell. One can 29. A. Khlebnikov, K. A. Datsenko, T. Skaug, B. L. Wanner,
industrial-scale production. Taking advantage of even envision a day when cell manufacturing is J. D. Keasling, Microbiology 147, 3241 (2001).
the cell’s native stress response pathways can be done by different companies, each specializing in 30. W. R. Farmer, J. C. Liao, Nat. Biotechnol. 18, 533
(2000).
an effective way to alleviate part or all of the certain aspects of the synthesis—one company 31. H. Alper, G. Stephanopoulos, Metab. Eng. 9, 258
toxicity (36). Even better, transporters could be constructs the chromosome, one company builds (2007).
used to pump the desired product outside the cell, the membrane and cell wall (the “bag”), one com- 32. B. F. Pfleger, D. J. Pitera, C. D. Smolke, J. D. Keasling,
reducing intracellular toxicity and purifying the pany fills the bag with the basic molecules needed Nat. Biotechnol. 24, 1027 (2006).
33. J. S. Edwards, R. U. Ibarra, B. O. Palsson, Nat. Biotechnol.
product from the thousands of contaminating to boot up the cell. 19, 125 (2001).
intracellular metabolites (37). Until this future arrives, manufacturing of mo- 34. J. H. Park, K. H. Lee, T. Y. Kim, S. Y. Lee, Proc. Natl. Acad.
lecules will be done with well-known, safe, in- Sci. U.S.A. 104, 7797 (2007).
Designer Cells for Designer Chemicals dustrial microorganisms that have tractable genetic 35. L. Kizer, D. J. Pitera, B. F. Pfleger, J. D. Keasling,
Appl. Environ. Microbiol. 74, 3229 (2008).
One can envision a future when a microorganism systems. Continued development of tools for ex-
36. H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, G. Stephanopoulos,
is tailor-made for production of a specific chem- isting, safe, industrial hosts, cloning and expressing Science 314, 1565 (2006).
ical from a specific starting material, much like genes encoding precursor production pathways, 37. M. J. Dunlop, J. D. Keasling, A. Mukhopadhyay,
chemical engineers build refineries and other and the creation of novel enzymes that catalyze Syst. Synth. Biol. 4, 95 (2010).
chemical factories from unit operations (Fig. 3). unnatural reactions will be necessary to expand 38. K. L. Prather, C. H. Martin, Curr. Opin. Biotechnol. 19,
468 (2008).
The chemical and physical characteristics of the the range of products that can be produced from 39. J. B. Siegel et al., Science 329, 309 (2010).
product and starting materials would be con- biological systems. When more of these tools are 40. This work was supported in part by the Synthetic Biology
sidered in the design of the organism to minimize available, metabolic engineering should be just as Engineering Research Center, which is funded by
both production and purification costs (e.g., powerful as synthetic chemistry, and together the National Science Foundation Award No. 0540879 and by
the Joint BioEnergy Institute, which is funded by the U.S.
operating the engineered cell at the boiling point two disciplines can greatly expand the number of Department of Energy, Office of Science, Office of
of volatile, toxic products to drive production and products available from renewable resources. Biological and Environmental Research, through contract
reduce product toxicity). The cell envelope DE-AC02-05CH11231. Competing financial interests:
would be designed to be resistant to the specific The author is a founder of and owns equity in Amyris
References and Notes and LS9.
desired chemical, and the cell wall would be de- 1. J. E. Bailey, Science 252, 1668 (1991).
signed to make the organism tolerant to indus- 2. K. M. Hawkins, C. D. Smolke, Nat. Chem. Biol. 4, 564 (2008). 10.1126/science.1193990

RESEARCH ARTICLE
houses, and objects. We thus sought to under-
stand which of these stimuli are processed in the
How Learning to Read Changes

VWFA area before reading and how their cortical
representation, which gets refined during the
school years (28), is affected by literacy.
the Cortical Networks for Vision A second issue is that, at present, most func-
tional imaging studies of illiteracy only contrasted
and Language schooled versus unschooled adults. Because these

studies did not include “ex-illiterate” adults who
did not attend school but learned to read during
Stanislas Dehaene,1,2,3,4* Felipe Pegado,1,2,3 Lucia W. Braga,5 Paulo Ventura,6 adulthood, they confounded the effects of school-
Gilberto Nunes Filho,5 Antoinette Jobert,1,2,3 Ghislaine Dehaene-Lambertz,1,2,3 ing and literacy. The only important exception (4)
Régine Kolinsky,7,8 José Morais,7 Laurent Cohen9,10,11 focused solely on how brain anatomy is changed
by literacy. In this study, we separated the func-
Does literacy improve brain function? Does it also entail losses? Using functional magnetic tional effects of schooling and literacy by compar-
resonance imaging, we measured brain responses to spoken and written language, visual faces, houses, ing illiterates, ex-illiterates, and adults schooled
tools, and checkers in adults of variable literacy (10 were illiterate, 22 became literate as adults, and in childhood.

31 were literate in childhood). As literacy enhanced the left fusiform activation evoked by writing, Populations studied and verification of literacy
it induced a small competition with faces at this location, but also broadly enhanced visual responses level. We scanned a total of 63 Portuguese and
in fusiform and occipital cortex, extending to area V1. Literacy also enhanced phonological activation Brazilian participants. Our sample included 32 un-
to speech in the planum temporale and afforded a top-down activation of orthography from schooled adults (10 illiterates and 22 ex-illiterates
spoken inputs. Most changes occurred even when literacy was acquired in adulthood, emphasizing with variable reading skills), and 31 schooled and
that both childhood and adult education can profoundly refine cortical organization. literate adults. The latter group included 11 lit-
erate subjects matched to the illiterates in socio-
ractically all adult neuroimaging experi- on cortical processing? Two theoretical positions economic status (SES) (29). Reading skills were
P ments are performed in highly educated

college students. The observed brain archi-
tecture therefore reflects the influence of culture
can be contrasted. The first view, derived from
animal studies of environmental enrichment and
sensory plasticity, emphasizes that perceptual
verified through behavioral tasks of letter identi-
fication, word and pseudo-word reading (with or
without speed pressure), and sentence reading
and education over and above spontaneous brain learning entails beneficial modifications of cor- (Fig. 1, fig. S1, and table S1). All tests revealed
development (1, 2). Indeed, the acquisition of tical maps, including sharpened receptive fields the same ordering of literacy, from Brazilian il-
reading, a major event in children’s lives, is now and neuronal tuning curves correlated with be- literates (ILB) to Portuguese ex-illiterates (EXP),
recognized as capable of changing both brain havioral improvements (20–22). Without denying Brazilian ex-illiterates (EXB), low-SES Brazilian
anatomy (3, 4) and brain activation (5–9). In the these positive effects, the second view empha- literates (LB2), Portuguese literates (LP), and Bra-
auditory modality, literacy leads to phonemic sizes that reading is a cultural invention too re- zilian literates (LB1). We therefore relied on whole-
awareness, the ability to manipulate the smallest cent to involve dedicated genetic or developmental brain linear regressions with reading performance
units of spoken language [i.e., phonemes (10)], mechanisms. Thus, during education, reading pro- (number of stimuli read per minute) across all
and alters online speech processing (11–14). At cesses must invade and “recycle” cortical space groups to identify the brain regions influenced by
the visual level, developmental neuroimaging devoted to evolutionary older functions, opening literacy. Once identified, each brain site was sub-
studies in normal and dyslexic children show the possibility that these functions suffer as read- mitted to restricted comparisons of subgroups to
that, with reading acquisition, a specific brain site ing expertise sets in (2, 23). Much like expertise evaluate the effects of schooling and literacy with
in left occipito-temporal cortex, which has been for nonface stimuli induces a reduction in face re- maximal sensitivity (29).
termed “visual word form area” (VWFA), starts sponses (24–26), reading, which recruits an iden- We used three types of whole-brain functional
to respond to orthographic stimuli in the learned tical cortical site in all cultures (27), might entail magnetic resonance (fMRI) runs: a “localizer” with
script (15–19). a reorganization of nearby responses to faces, horizontal and vertical checkerboards, written and
These observations leave many important
questions unanswered. First, does literacy primar-
ily lead to cooperative or to competitive effects A Reading Speed (pseudowords) B Accuracy (pseudowords)
1
INSERM, Cognitive Neuroimaging Unit, Gif sur Yvette 91191,
pseudowords/ min
France. 2Commissariat à l’Energie Atomique, Direction des

Sciences du Vivant, I2BM, Neurospin center, Gif sur Yvette
% correct
91191, France. 3University Paris-Sud 11, 91405 Orsay, France.

4
Collège de France, 11 Place Marcelin Berthelot, 75005 Paris,
France. 5SARAH Network–International Center for Neurosciences
and Rehabilitation, QL 13, Lago Norte, 71.535-005 Brasilia, DF
Brazil. 6Faculty of Psychology, University of Lisbon, 1600-214 Lisbon,
Portugal. 7Faculty of Psychology, Université Libre de Bruxelles
(ULB), 1050 Brussels, Belgium. 8Fonds de la Recherche Scientifique
(FNRS), 1000 Brussels, Belgium. 9Université Pierre et Marie Curie-
Paris 6, Faculté de Médecine Pitié-Salpêtrière, 75013 Paris, France.
10
Assistance Publique–Hôpitaux de Paris, Groupe hospitalier Pitié-
Salpêtrière,DepartmentofNeurology,75651Paris,France. 11INSERM,
Centre de Recherches de l’Institut du cerveau et de la moelle Fig. 1. The six groups of participants and their reading skills. Box plots show (A) the speed and
épinière, UMRS 975, Paris, France. (B) accuracy in reading a list of pseudowords (central horizontal line, median; box, 25th and 75th per-
*To whom correspondence should be addressed. E-mail: centiles; whiskers, minimum and maximum). Additional data on word and sentence reading are provided
stanislas.dehaene@gmail.com in fig. S1.

RESEARCH ARTICLE
spoken sentences, motor commands, and calcu- The visual word form area: A major correlate versus rest: −48, −56, −6, Z = 3.53). This finding
lation problems (fig. S2); three visual runs eval- of literacy. Our next analyses focused on visual indicates that adult literacy suffices to establish an
uating cortical responses to faces, houses, tools, responses in the VWFA. The effect of reading increased VWFA response to orthographic patterns.
letter strings, false fonts, and moving checker- performance on occipito-temporal cortex during Literacy not only amplified letter string re-
boards, while the participant focused on detecting sentence reading was replicated when passive sponses, but also increased the cortical selectivity
a target star (fig. S3); and four auditory lexical- viewing of letter strings was contrasted to rest for this category relative to others. When we tested
decision runs with spoken stimuli. To verify com- [main peak = −46, −80, 4, Z = 5.75; subpeaks at the impact of reading performance on the differ-
pliance, during the localizer, participants either −40, −70, −12, Z = 4.50; and the VWFA proper, ence between letter strings and other visual cate-
heard or saw short verbal instructions to perform −46, −58, −10, Z = 4.11; right occipital region, gories, only the VWFA appeared (peak at −44,
simple calculations or to click the left or right 24, −86, −10, Z = 5.25, corrected P < 0.05 by −56, −14, Z = 5.00; subpeak −44, −68, −12, Z =
button. With spoken instructions, we observed false detection rate (FDR) analysis (29)]. In this 3.85). This effect showed a highly significant hemi-
classical regions for calculation and hand move- part of the experiment, which involved viewing spheric asymmetry, peaking at the classical VWFA
ments, without modulation by literacy, indicat- meaningless pseudowords during an easy target- coordinates (−44, −56, −12, Z = 5.07). Thus,
ing similar comprehension and compliance in all detection task, only these visual regions were literacy results in the emergence of a cortical site
groups. With written instructions, however, acti- modulated by literacy, confirming their role in increasingly more responsive to writing than to
vation at the same locations was strongly modu- automatic orthographic coding (16). Notably, the other visual categories (17, 30).
lated by reading performance, varying from zero impact of schooling on the VWFA was replicated Group analyses left open the possibility of a
activation in illiterates to a level equivalent to when the illiterates were compared with the selective but spatially variable response to written

spoken instructions in literates (see fig. S4). While matched low-SES literates (ILB < LB2), both for strings in every subject. Literacy would then merely
unsurprising, these results validate our group def- written sentences versus rest (−40, −50, −14, Z = displace this response to a reproducible site, with-
initions and literacy measure and establish that, 6.77) and strings versus rest (−48, −60, −10, Z = out changing its amplitude. This possibility was
with spoken materials, all groups followed instruc- 3.54). The VWFA was also identified when we refuted, however, through an individual analysis
tions quite well. Thus, any subsequent differences searched for activation positively correlated with in which the voxel most responsive to written
cannot be attributed to lower attention or compre- reading performance within the unschooled par- sentences versus checkerboards was first identi-
hension in illiterates. ticipants only (illiterates and ex-illiterates; sen- fied in each participant, within 10 mm of the
Responses to written sentences enhanced by tences versus rest: −42, −54, −6, Z = 6.25; strings group peak (similar results were obtained with 20
literacy. We first examined, in the localizer run,
which regions were modulated by reading per- VWFA Left frontal
Response at [-44, -50, -14]
formance during the viewing of simple sentences Response at [-46, -2, 52]
consisting of serially presented written words (Fig. LB1 6 LB1
4
2 and fig. S7). A massive effect was seen in the LP LP
left ventral occipito-temporal cortex, at classical 3 LB2 4 LB2
VWFA coordinates (−40, −50, −14, Z score = 6.86); EXB EXB
2
with posterior subpeaks (−46, −70, −18, Z = 5.39; EXP 2 EXP
−32, −80, −8, Z = 3.96); and at a right occipital 1 ILB ILB
site (22, −86, −10, Z = 5.17). These regions were 0
strictly visual, as attested by their lack of Checkers Spoken Written
Checkers Spoken Written
activation to spoken sentences. Modulation by Occipital
reading performance was also seen in a vast left- Response at [22, -86, -10] Response at [-54, 26, -6]
hemisphere language network, which was also 6
activated by spoken language in all groups: left 1
posterior, middle, and anterior superior temporal 0.5
4
sulcus (STS; −50, −44, 6, Z = 7.10; −54, −12, −12,
0
Z = 5.42); left temporal pole (−50, 12, −24, Z =
2 -0
0.5
5
4.13); left and right premotor cortex (−46, −2, 52,
Z = 8.50; 46, 4, 40, Z = 5.48); left inferior frontal -1
gyrus (−54, 26, −6, Z = 5.76); and left supple- 0
Checkers Spoken Written Checkers Spoken Written
mentary motor area (−4, 2, 62, Z = 6.33). A sig-
nificant left-hemispheric asymmetry of this effect Left superior temporal sulcus
Response at [-50, -44, 6] Response at [-50, 12, -24]
was observed in all areas except temporal pole
and occipital cortex. Direct comparison of spoken 1.5
6
versus written stimuli showed that, in the literate 1
participants, frontal regions became equally 4
0.5
activated by spoken versus written language,
whereas temporal areas overlapped but still showed 2 0
a significant difference favoring spoken language -0.5
(fig. S8). 0
This analysis thus uncovered three simple ef- Checkers Spoken Written Checkers Spoken Written
fects: with the acquisition of literacy, written ma- Fig. 2. Effects of literacy on brain responses to written sentences during the localizer. Axial and
terials (i) activate right occipital cortex at the sagittal slices show voxels where activation was modulated by literacy during exposure to written
same level as checkerboards; (ii) induce a strong sentences relative to rest (voxel P < 0.001, cluster P < 0.05 corrected). Colored labels refer to
activation in left ventral visual cortex, at the clas- participant groups and are ordered according to reading performance (see Fig. 1). Plots report
sical site of the visual word form area (VWFA); activation to visual checkers, spoken and written language relative to rest in the localizer run, in
(iii) gain access to left perisylvian temporal and arbitrary units (mean T 1 SE). To avoid circularity, we generated plots solely from the spoken and
frontal language areas. written commands data, which were independent from the voxel-selection criterion.

RESEARCH ARTICLE
A z =-14 or 40 mm), and then analyzed for its responses to sibility that frequency of exposure to faces might
VWFA: -44, -50, -14 strings and false fonts in the independent visual increase with socioeconomic status and influence
2 LB1 runs. We observed a significant lateral-to-mesial fusiform responses (32). When studied at the whole-
L R LP shift of the word-responsive peak (from x = −48 brain level, the ILB > LB2 contrast indicated a
1.5
LB2 in illiterates to −46 in ex-illiterates and −44 in lit- highly significant reduction of face response with
1 EXB erates, P = 0.006), but its activation to strings also literacy (P < 0.001, cluster P < 0.05 corrected) in
0.5 EXP
increased strongly with reading performance (linear two bilateral posterior fusiform clusters (right: 40,
ILB
0 regression, r2 = 46.5%, P < 0.0001), as did the −80, 0, Z = 5.93, with an anterior subpeak, 38,
selectivity index for strings relative to false fonts −50, −12, Z = 4.70; left, −44, −70, −12, Z =
(P = 0.001). Thus, literacy genuinely increases both 4.58, with a subpeak precisely at the VWFA, −42,
B the strength and specificity of cortical responses −54, −14, Z = 3.91). The same contrast did not reach
z =-20
Face increase : 38, -42, -20 to the learned script in the VWFA. corrected-level significance for houses or tools.
4 We also searched for nonmonotonic effects In summary, at the VWFA site, learning to
3 across our six groups, which might arise if the ex- read competes primarily with the cortical repre-
L R
illiterates had to mobilize a broader network than sentation of checkers and faces. Further analyses
2
either literates or illiterates in order to read. Indeed, showed that this competition was spatially re-
1 during sentence reading, Brazilian ex-illiterates stricted. We implemented analyses inspired by
showed greater activity than Brazilian literates in Golarai et al. (28), who showed that cortical peaks

bilateral mesial fusiform areas (−34, −60, 0, Z = with adult-like selectivity to faces and places al-
C z =-8
5.34; 38, −50, −2, Z = 4.54) and right posterior ready exist in 7 to 11 year olds and, with increas-
House increase : -28, -46, -8
parietal cortex (24, −62, 38, Z = 4.70; 12, −58, ing age, progressively expand into the surrounding
2
60, Z = 4.16) (fig. S5). Thus, to achieve their cortex. For each subject, we first searched for
L R
1 modest reading performance, ex-illiterates enga- the peak response to faces versus houses within
ge a broader and more bilateral ventral network 10 mm of the group coordinates of the VWFA.
0 than literates and recruit additional posterior We then examined an orthogonal regression test-
-1
parietal regions associated with serial effortful ing how the activation to faces varied with liter-
reading (31). This observation is similar to the acy, at the peak and in increasingly larger annuli
D z =-10 developmental finding that reading in young of 2, 4, 6, or 8 voxels surrounding it (fig. S6).
Global increase : 24, -84, -10 children initially involves a broad bilateral visual There was no change in peak face responses with
LB1 network (18) that progressively restricts to the reading performance (P = 0.47), nor in annuli of
6 L R LP VWFA as greater expertise sets in (19). radius 2 or 4, but in the more distant annuli of
LB2 Competition with other visual categories in radius 6 or 8, face activation decreased with read-
4 EXB occipito-temporal cortex. At the peak coordi- ing performance (regression across all groups, re-
EXP
2 ILB
nates of the VWFA (−44, −50, −14, identified by spectively P = 0.037 and P = 0.015). Similar
the localizer), analysis of the independent visual findings were obtained for the individual peak of
runs showed a strong response to strings but also responsitivity to houses versus faces: no change
E Increased response to horizontal checkers to other visual categories, particularly faces and in peak activation (P = 0.20), but a decrease in
-12, -88, 2 16, -88, 2 tools (Fig. 3). This finding confirms that this area house-driven activation in the larger annulus of
z=2
4 6 plays a broad role in visual shape analysis. Read- radius 8 (P = 0.045). For tools, a category for
2
4
2 ing being a recent invention, we expected that which no selective region exists in ventral visual
2
0 L R 0 written words would not activate a fully dedi- cortex (33), we did not find an annular reduction,
Horiz Verti Horiz Verti cated cortical site but would only partially “re- but a more diffuse reduction in activation with
Fig. 3. Effects of literacy on visual responses to cycle” existing cortical mechanisms for visual reading performance, significant over both a large
different categories of stimuli. (A) Visual responses in the recognition (2, 23), inducing a cortical competi- sphere of 16 mm (P = 0.039) and in the 50 voxels
VWFA. (Inset) Statistical parameter map (SPM) map of tion that would increase with reading expertise. best responsive to tools versus houses (P = 0.019),
modulation by literacy of activation to written sentences We tested the predicted cortical competition by but again not at the peak itself (P = 0.27).
in the localizer (P < 0.001, cluster corrected P < 0.05). searching for a decreasing response to visual stimu- Overall, our results indicate that the develop-
Plot showing activation in the independent visual runs at li with increasing reading performance. At the in- mental competition induced by the expansion of
the isolated peak (mean T 1 SE). Activation increased in dependently defined VWFA peak, the responses orthographic representations in the ventral visual
response to letter strings (P = 0.005) and decreased for to checkerboards slightly diminished with read- system is modest, does not directly affect the
checkerboards (P = 0.013) and, to a lesser extent, for ing performance, both across all groups (linear peak responses to faces and houses, but interferes
faces (overall P = 0.09, ILL > LB2, P = 0.003). (B and C) regression, P = 0.013), for illiterates compared to with their expansion into the surrounding cortex.
SPM maps (voxel P < 0.001 uncorrected) and peak plots all other groups (P = 0.025), and for illiterates These conclusions fit with previous studies of visual
showing increases with reading performance of face compared to SES-matched literates (ILB > LB2 development (28), expertise (24, 25), and plasticity
responses in right anterior fusiform, and of house re-
comparison, P = 0.039). For houses and tools, of sensory maps (20), which reveal a displacement
sponses in bilateral parahippocampal regions. (D) SPM
only marginal decreasing trends were found (same of map boundaries due to cortical competition.
map of enhancement by literacy of the response to
written sentences in lateral occipital cortex (inset, P < three tests, P = 0.08, 0.025, 0.09 for houses and Positive effects of literacy on visual organization.
0.001, cluster corrected P < 0.05), and plot showing its 0.13, 0.025, 0.06 for tools). For faces, the de- Competition could also have a positive effect on
replication in response to other visual categories. (E) creasing tendency was stronger: The regression cortical responses to non–reading-related visual
Modulation by literacy of the greater response to hor- with reading performance across all participants categories: By reducing the dispersion of their
izontal than to vertical checkerboards in primary visual was marginal (P = 0.09), but the more focused neural responses, it might force them to a more
cortex (inset, voxel P < 0.001 uncorrected). In (B), (C), comparisons were significant (ILB > other groups, consistent cortical site. Indeed, a whole-brain
and (E), plots are provided for illustration, because they P = 0.025; ILB > LB2, P = 0.003). Comparing search revealed positive correlations of reading
do not arise from independent data; plots (A) and (D) illiterates with SES-matched literates indeed argu- performance with face and house responses in
are from independent data. ably provides a purer test, controlling for the pos- ventral occipito-temporal cortex (Fig. 3). Reading

RESEARCH ARTICLE
performance modulated positively the face-versus- sites corresponding to primary visual cortex (16, probably reflect a facilitation of speech compre-
rest contrast in the right anterior fusiform gyrus −88, 2, Z = 5.10; and −12, −88, 2, Z = 4.75, hension in literate participants (8). In the con-
(38, −42, −20, Z = 4.84, FDR) and induced a FDR). These sites exhibited a strong response to verse direction, however, activation to spoken
significant right-hemispheric shift of face responses horizontal, but not vertical checkerboards, and sentences essentially doubled from illiterates to
in occipito-temporal cortex (24, −88, −10, Z = the modulation by reading performance was seen literates in left and right superior temporal regions
8.02; 36, −62, −12, Z = 6.72; and 38, −40, −22, only with horizontal checkerboards (Fig. 3). This just posterior to Heschl’s gyrus (planum tempo-
Z = 4.77, FDR). Similarly, reading performance effect was significantly stronger in left than in rale; −38, −28, 18, Z = 5.52; 42, −14, 16, Z = 5.43),
modulated the house-versus-rest contrast in bi- right V1 (asymmetry effect for horizontal > ver- with bilateral subpeaks near Heschl’s gyrus (−60,
lateral mesial fusiform and parahippocampal regions tical checkerboards, peak at −4, −88, −4, Z = 5.37, −14, 10; Z = 4.28; 66, −2, 24, Z = 4.41) and a
(peaks at 34, −58, −12, Z = 5.19; 24, −36, −16, Z = FDR). Both occipital sites also showed a positive significant left-hemispheric asymmetry. The ef-
4.83), with a right-hemispheric asymmetry (36, correlation with literacy when written sentences fect was replicated in the independent auditory
−60, −12, Z = 5.53). Altogether, these influences were presented (table S2). Overall, these results lexical decision runs, with both words and pseudo-
on word, face, and place responses resulted in a suggest that literacy results in a form of percep- words (correlations with reading performance:
better differentiated mosaic of category-specific tual learning (20–22) that refines the earliest r2 = 0.20, P = 0.0002; and r2 = 0.18, P = 0.0005).
regions in ventral visual cortex in literates (Fig. stage of cortical visual processing. At this stage, The enhanced temporal response was restricted
4). Notably, however, face and house increases learning is generic enough to generalize to checker- to spoken language, with no trace of activation to
were found neither when we compared illiterates board stimuli presented at the trained location. The written sentences at this site.
with their SES-matched literate group (ILB < greater effect in left area V1 fits with the larger The planum temporale is involved in phono-

LB2 comparison), nor when we tested for the letter-identification span in the right visual field logical coding of speech (36) and is sensitive to
effect of literacy in nonschooled participants only in left-to-right readers (35). the congruity between a speech sound and a simul-
(table S2). Plots showed that these effects differ- Enhanced responses to spoken language. taneous visually presented letter (37), an effect that
entiated the participants living primarily in urban Finally, we examined how literacy affected spoken is reduced or absent in dyslexic subjects (38). Our
areas (LB1, LP, and to a lesser extent, EXP) ver- language processing (Fig. 5). Several regions results make this region a prime candidate for the
sus those living in rural areas (ILB, EXB, and showed a decreasing activation to spoken sen- enhanced phonemic processing that accompanies
LB2), regardless of their schooling and reading tences with greater reading performance: left pos- reading acquisition [(10–12); see also (39)]. They
scores (Fig. 3). Although these observations sug- terior STS (−44, −52, 18, Z = 5.45), left and right also suggest that the reduced planum temporale
gest an influence of familiarity rather than literacy middle temporal gyri (−66, −22, −10, Z = 5.25; activation seen in dyslexic children, rather than
or schooling per se, they are nevertheless impor- 48, −30, −8, Z = 5.34), and midline anterior cin- being a cause of dyslexia (38), could be a con-
tant in showing how ventral fusiform organization gulate cortex (4, 42, 42, Z = 4.11). These reductions sequence of abnormal reading acquisition.
can be affected by cultural variables (1).
Literacy led to another effect: a general en-
hancement of occipital responses. We probed the iIliterates z =-12
y =-56
right occipital location identified as being modu-
lated by reading performance during written sen-
tences versus rest in the localizer run (coordinates
22, −86, −10). During the independent visual faces
runs, the activation of this region to every visual
category correlated positively with reading scores, houses
with the lowest correlation achieved with the L R
checkerboards (r2 = 0.08, P = 0.02; all other cat-

egories, r2 ranging from 0.17 to 0.22, P < 0.0007). tools ex-illiterates
Furthermore, these effects were genuinely related
to literacy, not just schooling (table S2). When strings
extended to a whole-brain search, with a main
contrast for increasing activation to all visual cate-
gories, this effect was significant not only in right checkers
occipital (24, −84, −10, Z = 14.6) but also in left
occipital cortex (−48, −80, −4, Z = 9.35) and a
right occipito-parietal cluster (24, −76, 36, Z =
6.75), always with significant right-hemispheric literates
asymmetry. Thus, literacy enhanced occipital re-
sponses to essentially all the contrasted black-
and-white visual stimuli used in our study.
We also examined whether early retinotopic
responses were affected. The localizer comprised
horizontal and vertical checkerboards, designed
to isolate the meridians of early visual maps. In left hemisphere right hemisphere
the Roman alphabet, words appear as horizontally x coordinate
extended strings, and expert readers show en- Fig. 4. Mosaic of preferences for different visual categories in ventral visual cortex. Slices at right show
hanced behavioral processing of letter strings the activation difference between a given category and all the others [for greater comparability between
presented at the familiar foveal and horizontal groups with different numbers of subjects, the figure does not show statistical t maps, but blood oxygen
location (31, 34). We therefore predicted that level–dependent (BOLD) signal maps arbitrarily thresholded at 0.66% of the mean BOLD signal over
literacy might increase the responses to horizon- the whole brain; similar results were seen with t maps]. Graphs at left shows the evolution of the signal
tal relative to vertical checkerboards. Indeed, this relative to rest for the different categories (mean T 1 SE), at successive cortical sites tracing a horizontal
effect was observed at two symmetrical occipital line through the classical coordinates of the VWFA (−42, −57, −12; dotted line).

RESEARCH ARTICLE
During auditory lexical decision, but not sen- an orthographic code rather than a generic picture of early schooling by regressing out the effect of
tence listening, a second site in left inferior code (44). reading performance and testing for a remaining
temporal cortex also increased its activation as a Previous psycholinguistic research has dem- difference between the Brazilian ex-illiterates (EXB)
function of reading performance (−48, −52, −8, onstrated that orthography affects spoken lan- and the low-SES Brazilian literates (LB2 group),
Z = 8.62). This site showed no activation in il- guage processing (11–14), but it remains debated who only differed in early schooling.
literates, but a strong one in all literate groups whether an orthographic code is activated online The results were clear-cut. Essentially all of
(Fig. 5). Its coordinates strongly suggest a top- whenever we hear a spoken word, or whether or- the above effects of literacy were present in ex-
down activation of the VWFA and the neighbor- thography merely changes the nature of phono- illiterates who learned to read during adulthood
ing lateral inferior temporal cortex (40). Indeed, logical representations (12). Our results show that (table S2). Such was the case for the increased
its activation to spoken words and pseudowords both phenomena coexist: The planum temporale VWFA response to letter strings; the capacity to
was positively correlated to its activation by increase suggests enhanced phonological coding, activate the spoken language network through
written strings in the independent visual runs (r = compatible with a recent study using low-resolution reading (except in left temporal pole); the general
+0.46; no such correlation was found with other electroencephalography (12), whereas the VWFA visual increase in right occipital cortex; the greater
visual categories). Overlap with the VWFA was activation indicates an additional and optional or- response of area V1 to horizontal checkerboards
also established by first identifying the peak thographic recruitment. and written sentences; and the enhanced planum
response to written sentences versus checker- Effects of early schooling and late literacy. temporale and top-down VWFA activation to
boards in each participant, and then correlating The above fMRI findings are based on global spoken words and pseudowords. Thus, the neural
its activation during spoken lexical decision with correlations of brain activation with reading per- modifications induced by adult literacy education

the participant’s reading performance (P = 0.005). formance, and therefore reflect the joint influences were considerable. Furthermore, the vast majority
Altogether, those results confirm that the VWFA of schooling and literacy. To separate these var- were unaffected by early schooling (table S2).
can be activated in a top-down manner during iables, we performed additional regression analy- There were only two interesting exceptions. The
speech processing (41–43), even in a lexical ses on all previously identified peaks (29). One first was the reduced activation to faces in the
decision task that does not require orthographic analysis evaluated the impact of literacy acquired VWFA, which was indeed particularly prominent
processing. Because this activation is present during adulthood by testing the effect of read- in the LB2 group, who benefited from early
only inasmuch as the participants can read, our ing performance within unschooled subjects only. schooling, relative to the EXB group. This finding
findings suggest that it reflects the recruitment of Another analysis, conversely, probed the impact suggests that competitive interactions between
written words and faces in ventral visual cortex
A primarily occur when reading is acquired in
Spoken sentences in localizer Auditory lexical decision
childhood, a time when visual maps are known
to be highly malleable (28). The other effect of
Response at [-38, -28, 18] Response at [-38, -28, 18] early schooling concerned a marginal left pre-
5 motor increase in activation to written sentences.
6
LB1 This region overlaps with Exner’s writing center
4 and is thought to code handwriting gestures (45).
LP
4
LB2 3 Whereas early-schooled participants were fluent
EXB in handwriting, it is possible that the ex-illiterates
2 2
EXP did not receive enough training to automatically
ILB 1 activate a gesture code from the mere vision of
0
written sentences.
Checkers Spoken Written Words Pseudowords We caution that these conclusions may only be
commands valid for the moderate level of reading fluency
achieved by our ex-illiterate participants. Whether
B early-schooling effects truly reflect a limit on adult
Spoken sentences in localizer Auditory lexical decision sensory plasticity or would vanish with more in-
tense reading practice remains an open question.
Response at [-44
[-44, -50
-50, -14] Response at [-44,
[ 44, -50,
50, -14]
14] However, our results also indicate that, in liter-
ates, most of the observed effects do not change
4 LB1 2 further as reading expertise increases (table S2;
LP 1.5 the only exceptions were increases in planum tem-
3 LB2 porale and left pSTS, and reduced VWFA acti-
EXB 1
2 vation to checkerboards). In particular, the VWFA
EXP 0.5 activation to words and strings increases briskly
1 ILB from illiterates to ex-illiterates and then reaches
0
a plateau uncorrelated with ultimate proficiency,
Checkers Spoken Written Words Pseudowords
in agreement with developmental evidence that
commands
minimal literacy training suffices to establish it
Fig. 5. Effects of literacy on brain responses to spoken language. (A) SPM maps of the effect of literacy on in 6 year olds (18).
the activation to spoken sentences (left), words and pseudowords (right; voxel P < 0.001, cluster-size Conclusion. Literacy, whether acquired in
corrected P < 0.05). Plots show the effect observed in the left planum temporale: Activation to spoken childhood or through adult classes, enhances brain
language, words, and pseudowords was doubled or more after the acquisition of literacy (peak selected responses in at least three distinct ways. First, it
using the localizer run, left panel, and replicated in the independent lexical decision runs, right panel). (B) boosts the organization of visual cortices, partic-
SPM maps of the effect of literacy on activations in ventral occipito-temporal cortex to spoken language. ularly by inducing an enhanced response to the
Literacy modulated activation to words and pseudowords during auditory lexical decision (right), but not known script at the VWFA site in left occipito-
during mere listening to spoken commands. Plots show the response at the a priori location of the VWFA, temporal cortex and by augmenting early visual
as defined by the effect of literacy on written sentences (same location as in Figs. 2 and 3A). responses in occipital cortex, in a partially retino-

topic manner. Second, literacy allows practically 13. P. Ventura, J. Morais, R. Kolinsky, Cognition 105, 547 39. P. E. Turkeltaub, L. Gareau, D. L. Flowers, T. A. Zeffiro,
the entire left-hemispheric spoken language net- (2007). G. F. Eden, Nat. Neurosci. 6, 767 (2003).
14. J. C. Ziegler, A. Petrova, L. Ferrand, J. Exp. Psychol. Learn. 40. L. Cohen, A. Jobert, D. Le Bihan, S. Dehaene, Neuroimage
work to be activated by written sentences. Thus Mem. Cogn. 34, 643 (2008). 23, 1256 (2004).
reading, a late cultural invention, approaches the 15. B. A. Shaywitz et al., Biol. Psychiatry 52, 101 (2002). 41. Y. N. Yoncheva, J. D. Zevin, U. Maurer, B. D. McCandliss,
efficiency of the human species’ most evolved 16. L. Cohen, S. Dehaene, Neuroimage 22, 466 (2004). Cereb. Cortex 20, 622 (2010).
communication channel, namely speech. Third, 17. C. I. Baker et al., Proc. Natl. Acad. Sci. U.S.A. 104, 42. A. S. Desroches et al., Brain Res. 1356, 73 (2010).
9087 (2007). 43. J. R. Booth et al., Hum. Brain Mapp. 19, 155 (2003).
literacy refines spoken language processing by 18. S. Brem et al., Proc. Natl. Acad. Sci. U.S.A. 107, 7939 44. F. Kherif, G. Josse, C. J. Price, Cereb. Cortex
enhancing a phonological region, the planum (2010). 10.1093/cercor/bhq063 (2010).
temporale, and by making an orthographic code 19. U. Maurer et al., Neuroimage 33, 749 (2006). 45. M. Longcamp, J. L. Anton, M. Roth, J. L. Velay,
available in a top-down manner. These largely 20. D. B. Polley, E. E. Steinberg, M. M. Merzenich, J. Neurosci. Neuroimage 19, 1492 (2003).
26, 4970 (2006). 46. This work was supported by Institut National de la
positive changes should not hide that literacy, like 21. W. Li, V. Piëch, C. D. Gilbert, Nat. Neurosci. 7, 651 (2004). Santé et de la Recherche Médicale (INSERM),
other forms of expertise, also leads to cortical 22. M. Sigman et al., Neuron 46, 823 (2005). Commissariat à l’Energie Atomique (CEA), Collège de
competition effects (23–26). At the VWFA site, a 23. S. Dehaene, Reading in the Brain (Penguin Viking, France, Agence Nationale de la Recherche (project
significantly reduced activation was found for New York, 2009). CORELEX), Fundação para a Ciência e a Tecnologia,
24. I. Gauthier, T. Curran, K. M. Curby, D. Collins, the European Community FEDER funding (Project
checkerboards and faces. The intriguing possibil-
Nat. Neurosci. 6, 428 (2003). PTDC/PSI/66077/2006, “Cognitive consequences of
ity that our face perception abilities suffer in pro- 25. A. Harel, S. Gilaie-Dotan, R. Malach, S. Bentin, Cereb. literacy”), SARAH Network of Rehabilitation Hospitals,
portion to our reading skills will be explored in Cortex 20, 2304 (2010). and by two grants from the Belgian French community
future research. 26. B. Rossion, C. C. Kung, M. J. Tarr, Proc. Natl. Acad. (Fonds de la Recherche Fondamentale Collective

Sci. U.S.A. 101, 14521 (2004). 2.4586.07 and Action de Recherche Concertée
References and Notes 27. D. J. Bolger, C. A. Perfetti, W. Schneider, Hum. Brain Mapp. 06/11-342). We gratefully acknowledge A. Amadon,
1. J. Henrich, S. J. Heine, A. Norenzayan, Behav. Brain Sci. 25, 92 (2005). M.-H. Baju, L. Labruna, D. Le Bihan, L. Hertz-Pannier,
33, 61 (2010). 28. G. Golarai et al., Nat. Neurosci. 10, 512 (2007). P. Pinel, and the NeuroSpin infrastructure groups at
2. S. Dehaene, L. Cohen, Neuron 56, 384 (2007). 29. Materials and methods are available as supporting NeuroSpin; C. Carvalho, L. Querido, S. Fernandes, and
3. A. Castro-Caldas et al., Eur. J. Neurol. 6, 23 (1999). material on Science Online. T. Fernandes at the Faculty of Psychology, University of
4. M. Carreiras et al., Nature 461, 983 (2009). 30. R. Gaillard et al., Neuron 50, 191 (2006). Lisbon; and D. Y. Suguieda, R. S. Vera, A. G. Tauil,
5. K. M. Petersson, C. Silva, A. Castro-Caldas, M. Ingvar, 31. L. Cohen, S. Dehaene, F. Vinckier, A. Jobert, A. Montavont, E. Amemiya, L. G. N. Nunes, S. B. de Oliveira, and
A. Reis, Eur. J. Neurosci. 26, 791 (2007). Neuroimage 40, 353 (2008). C. L. M. Gillis at SARAH Brasilia.
6. G. Li et al., Hum. Brain Mapp. 27, 144 (2006). 32. E. Eger, S. R. Schweinberger, R. J. Dolan, R. N. Henson,
7. K. M. Petersson, A. Reis, S. Askelöf, A. Castro-Caldas, Neuroimage 26, 1128 (2005). Supporting Online Material
M. Ingvar, J. Cogn. Neurosci. 12, 364 (2000). 33. P. E. Downing, A. W. Chan, M. V. Peelen, C. M. Dodds, www.sciencemag.org/cgi/content/full/science.1194140/DC1
8. A. Castro-Caldas, K. M. Petersson, A. Reis, N. Kanwisher, Cereb. Cortex 16, 1453 (2006). Materials and Methods
S. Stone-Elander, M. Ingvar, Brain 121, 1053 (1998). 34. T. A. Nazir, N. Ben-Boutayab, N. Decoppet, A. Deutsch, SOM Text
9. K. M. Petersson, A. Reis, A. Castro-Caldas, M. Ingvar, R. Frost, Brain Lang. 88, 294 (2004). Figs. S1 to S8
Neuroimage 10, 45 (1999). 35. K. Rayner, Psychol. Bull. 124, 372 (1998). Tables S1 and S2
10. J. Morais, P. Bertelson, L. Cary, J. Alegria, Cognition 24, 36. C. Jacquemot, C. Pallier, D. LeBihan, S. Dehaene, References
45 (1986). E. Dupoux, J. Neurosci. 23, 9541 (2003).
11. H. Cheung, H. C. Chen, Lang. Cogn. Process. 19, 1 (2004). 37. N. van Atteveldt, E. Formisano, R. Goebel, L. Blomert, 23 June 2010; accepted 20 October 2010
12. L. Perre, C. Pattamadilok, M. Montant, J. C. Ziegler, Neuron 43, 271 (2004). Published online 11 November 2010;
Brain Res. 1275, 73 (2009). 38. V. Blau et al., Brain 133, 868 (2010). 10.1126/science.1194140
REPORTS
Carbon Nanotubes with
(2), could be building blocks for various thermal-
ly stable elastic and viscoelastic materials. Aligned,
sparse CNT arrays (3), films (4), and sponges
Temperature-Invariant Viscoelasticity packed with short, straight CNTs (5) have shown
fatigue resistance, supercompressibility, and com-
from –196° to 1000°C

pressive elasticity, respectively. Reports of creep
and buckling of aligned CNTs (6, 7) demonstrate
that a specific assembly of CNTs can show
Ming Xu,1 Don N. Futaba,1* Takeo Yamada,1 Motoo Yumura,1 Kenji Hata1,2* viscoelasticity.
Our strategy to make a viscoelastic CNT
Viscoelasticity describes the ability of a material to possess both elasticity and viscosity. Viscoelastic material was to assemble traversing long CNTs
materials, such as rubbers, possess a limited operational temperature range (for example, for silicone with a very high density of intermittent physical
rubber it is –55° to 300°C), above which the material breaks down and below which the material interconnections, analogous to an aggregate of
undergoes a glass transition and hardens. We created a viscoelastic material composed from a random hair. We made a CNT network where each CNT
network of long interconnected carbon nanotubes that exhibited an operational temperature range made contacts with numerous other CNTs. A
from –196° to 1000°C. The storage and loss moduli, frequency stability, reversible deformation level, combination of reactive ion etching (RIE) to the
and fatigue resistance were invariant from –140° to 600°C. We interpret that the thermal stability catalyst film, water-assisted chemical vapor dep-
stems from energy dissipation through the zipping and unzipping of carbon nanotubes at contacts. osition (8), and compression was used for the
1
iscoelasticity describes the ability of a terials, represented by rubbers, are inherently tem- Technology Research Association for Single Wall Carbon
V material to both dissipate energy (viscous)

and reversibly deform (elastic) and per-
meates all levels of our lives from human tissues,
perature dependent (1). This is because molecular
motion that is the origin of viscoelasticity is a
thermally activated process.
Nanotubes (TASC) and Nanotube Research Center, National
Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba, 305-8565, Japan. 2Japan Science and Technology
Agency (JST), Kawaguchi, 332-0012, Japan.
shoe soles, ear plugs, and mattresses to vibration Carbon nanotubes (CNTs), with their excep- *To whom correspondence should be addressed. E-mail:
isolators. Viscoelastic properties of existing ma- tional mechanical properties and thermal stability kenji-hata@aist.go.jp (K.H.); d-futaba@aist.go.jp (D.N.F.)

REPORTS
synthesis. RIE exposure reduced the catalyst with a change of the modulus as we approached As far as we know, commercial DMAs are lim-
density needed to create randomly oriented and the failure strain. The larger enclosed area of the ited to 600°C. As exemplified by the vibration
sparse CNTs. Water-assisted chemical vapor hysteresis loop for the CNT material meant more isolator demonstration (fig. S2), the CNT mate-
deposition synthesized very long and clean CNTs energy dissipated in one cycle (Fig. 1C). Quanti- rial showed viscoelasticity beyond the limita-
(~68% double-walled CNTs, ~22% single-walled tatively, the viscoelastic properties (storage mod- tion at –190°C (immersed in liquid nitrogen) and
CNTs, and ~10% tripled-walled CNTs; 0.009 ulus, loss modulus, and damping ratio) by DMA at >900°C (exposed to butane torch). To extend
g/cm3; height = 4.5 mm; carbon purity = 99.9%) showed that the CNT material possessed similar the temperature range studied, we implemented
that are essential to increase interconnection stiffness (storage modulus = 1 MPa) and higher impact tests at –196°C, 25°C, and 1000°C by
among CNTs and thereby increase cohesive- dissipation ability (loss modulus = 0.3 MPa) and using a steel ball and analyzed the ball tracks.
ness and thermal stability. Compression increased damping ratio (0.3) than silicone rubber at room The ball tracks were identical for all cases as
the CNT material density fourfold (0.036 g/cm3) temperature (fig. S6A). The viscoelastic prop- observed by SEM and three-dimensional (3D)
(Fig. 1A). erties of the CNT material strongly depend on mapping (Fig. 1, E and F), which suggested un-
Scanning electron microscope (SEM) obser- the internal structure. For example, an increase in varying viscoelastic properties across this 1200°C
vation of the CNT material revealed an intertube density results in a significant increase in both temperature range. The absence of catalyst ma-
structure where individual CNTs traversed later- moduli. We compressed the as-prepared CNT terials in our CNT material is crucial for our
ally, making interconnections with other CNTs. material to increase the density four times, and high thermal stability because oxidation would
This feature allowed the CNT material to bear this resulted in an increase of the storage (5 times) always limit the practical applications in air above
strain without fracture (Fig. 1B). The slow elastic and loss (10 times) moduli and the damping ratio ~ 400°C, particularly when catalyst material is

recovery after release implied viscoelasticity. (2 times). In this way, our CNT material was spe- present (9).
Stress-strain behavior from shear-mode dynamic cifically tuned to exhibit similar viscoelastic prop- Further DMA characterization from –140° to
mechanical analysis (DMA) showed up to 100% erties as silicone rubber at room temperature. 600°C demonstrated temperature-invariant fre-
strain, high nonlinearity, and a closed hysteresis The viscoelastic properties (storage modulus, quency stability, the same level of reversible
without abrupt changes, which were typical of loss modulus, and damping ratio) measured by deformation, and fatigue resistance. Frequency-
viscoelastic, energy-dissipative, and highly de- DMA in ambient N2 were nearly constant over dependence test (Fig. 2, A to C) showed constant
formable materials, for example, rubber. Silicone an exceptionally wide temperature range (–140° viscoelastic properties (storage modulus, loss mod-
rubber was chosen as our strictest benchmark in to 600°C) in contrast to silicone rubber, which ulus, and damping ratio) of the CNT material in
terms of thermal stability because it is the most showed large variation because of hardening at the range of 0.1 to 100 Hz. Furthermore, the CNT
thermally resistant rubber. The observed dual –55°C and degradation at 300°C (Fig. 1D). Var- material showed identical frequency-stability
slope of the stress-strain curve was associated iation between CNT samples was within ~5%. from –140° to 600°C.
Fig. 1. Invariant energy dissipation of the CNT

material, from –196° to ~1000°C. (A) Diagram
showing the synthesis procedure. (B) Photograph
of the flexible CNT material. (Inset) SEM image. (C)
Stress-strain curves of CNT material and silicone
rubber. (D) Temperature dependence of the stor-
age modulus (black), loss modulus (blue), and damp-
ing ratio (red) of the CNT material (silicone rubber in
gray for comparison). (E) Schematic of the impact
test. (F) Split images of the ball tracks performed
at –196°, 25°, and 1000°C (top, SEM; bottom, 3D
mapping from laser microscopy). Bottom graph, all
track profiles for all three cases.

REPORTS
Strain dependence tests (Fig. 2, D to F) at room temperature was estimated as ~100% results from the instability of gap between pres-
showed that the critical strain, that is, the maxi- (fig. S6B). At the failure strain, the structure broke sure heads at large strain because of thermal ex-
mum strain allowing reversible deformation, was down, indicated by the degradation in the non- pansion and contraction.
~5%. Similarly, the CNT material retained the linearity and hysteresis loops at 100% strain (fig. Cyclic tests at 100 Hz showed identical vis-
same level of reversible deformation from –140° S9). The failure strain ranged from 50 to 100% coelastic moduli and stress-strain behavior even
to 600°C. The failure strain for the CNT material between –140°C and 600°C, which we suspect after 1,000,000 cycles at 1% strain (Fig. 3, B and

Fig. 2. Viscoelastic properties of the CNT material over a wide temperature from –140° to 600°C. (D to F) Storage modulus, loss modulus, and damping
range. (A to C) Storage modulus, loss modulus, and damping ratio of the ratio of the CNT materials as function of strain amplitude (1 to ~1000%) at
CNT materials as function of frequency (0.1 to ~100 Hz) at temperatures temperatures from –140°C to 600°C.
Fig. 3. Fatigue resistance of the CNT material across –140° to 600°C. Cyclic test (1% strain, 100 Hz, 106 cycles) at (A) –140°C, (B) 25°C, and (C) 600°C.
Stress-strain curve of fatigue resistance test (102-th 104-th, 106-th cycle) at (D) –140°C, (E) 25°C, and (F) 600°C.

REPORTS
E) that proved excellent fatigue resistance of the This observation at 100% strain corresponded detached through zipping and unzipping (Fig.
CNT material at room temperature. Identical fa- with the measured failure strain. From these re- 4E). This process would dissipate energy because
tigue resistance was observed at –140° and 600°C, sults, we propose that the strain was absorbed at of the energy consumed to overcome the large van
as evidenced by similar viscoelastic moduli and low level by reversible unfolding of the traversing der Waals (vdW) attraction (12) between CNTs
cyclic behavior (Fig. 3, A, C, D, and F). This CNTs and beyond 100% strain by an irreversible when unzipped, yet no energy is required for
phenomenon means that not only are the visco- process of straightening, slipping, and bundling of zipping. We interpret that sliding among CNTs
elastic properties temperature invariant but also CNTs (Fig. 4C). was not a significant contribution because the
unvarying over many cycles. Transmission electron microscope (TEM) friction coefficient (0.003) is small (13). Because
The porous nature of the CNT material allows observation further revealed the intertube struc- the CNT material possessed a very high density of
for rapid and efficient heat dissipation, which pre- ture of the CNT material, which showed insight these “detachable” nodes, we interpret that they
vents significant heat accumulation, the common into the mechanism of energy dissipation (Fig. were the source of the high energy dissipation
cause for property degradation. We interpret that 4D). We found an intertube structure resembling ability. Within our model, under the critical strain,
our CNT material exhibited this high level of re- a 3D highway network, where each CNT made the nodes perpendicular to the strain direction
versible deformation from the entanglement of contact with numerous other CNTs. This peculiar could reversibly zip and unzip and thereby dis-
the long traversing CNTs throughout the material, intertube structure was characterized by a high sipate energy. Under increased strain, the number
like a network of springs creating elasticity. density of connections (nodes), that is, two to of detachable nodes gradually decreased through
To provide insight into the mechanism of vis- four CNTs intermittently contacting in parallel either unzipping or alignment (Fig. 4C), and even-
coelasticity, we carried out SEM observations for only short spans (~150 nm). These nodes tually the ability to dissipate energy decreased.

(Fig. 4A) and Herman’s orientation factor (HOF) were separated mainly by isolated CNTs (struts). Beyond the critical strain, this zipping/unzipping
calculations (Fig. 4B) under increasing shearing In addition, no CNT ends were observed, imply- process was no longer reversible, and upon cycling
strains up to 1000%. HOF describes the degree of ing long, continuous CNTs, and the long CNTs CNTs zipped at different places and/or became
alignment (0 is random, and 1 is aligned) and was were both randomly oriented and traversing. We bundled and aligned, resulting in degradation. A
calculated from the fast Fourier transform (FFT) interpret that this intertube structure of struts and similar irreversible process caused by the slipping
analysis of SEM images (10). Up to 100% strain, nodes was the key for structural cohesiveness among CNTs under large strain has been observed
the traversing, randomly aligned CNTs structur- that allowed for large deformations. This struc- in CNT yarns (11).
ally deformed into mutually aligned CNTs, and ture differs from typically bundled CNT material We estimated the loss modulus to address the
the HOF steadily increased from 0.07 to ~0.48. (11) where CNTs are straight and contacts the validity of the zipper model. The loss modulus
Beyond 100% strain, the HOF plateaued, showing same CNTs over long spans. (G″) of the nodes was approximated as a summa-
no increase in alignment, and the intermittently Although not directly observed, we believe tion over all nodes multiplied by the energy per
contacting CNTs became increasingly bundled. that the CNTs in a node reversibly attached and node to unzip and by a geometrical factor, <cosq>,
to account for the fraction aligned perpendicular to
the strain direction
EDissipated
G″ ¼
gġ 2p
w

1 N l
≈
2p • ∑ ∫ EvdW dl • 〈cosq〉 ð1Þ
gġ w
with vdWadhesion energy per unit length to unzip

two CNTs, EvdW; node density, N; node length, l =
150 nm (by TEM, fig. S7A); shear strain and rate,
g and ġ, respectively; strain angular frequency, w;
and the angle between the node and the direction
perpendicular to the strain, q. The vdW adhesion
energy, EvdW, was estimated as 0.36 nJ/m as cal-
culated from the binding energy of two parallel
cylinders following the Lennard-Jones potential
(14). The CNT node density (4.5 × 1015 nodes per
cm3) was estimated by multiplying the CNT tube
density (4.24 × 1010 tubes per cm2), as estimated
from the CNT bulk density (0.009 g/cm3) and
individual tube density (1.5 × 10−13 g/cm), and the
node density per tube (2.12 × 104 per tube), as
estimated by the node frequency (1/300 nm) ob-
tained by TEM images (fig. S7). With use of these
values, the calculated G″ was 0.51 MPa and
agreed well with experiment (0.3 MPa), which
demonstrated that the energy dissipation stemmed
Fig. 4. Energy dissipation model. (A) SEM images at varying shear strains. (B) Herman orientation factor from the unzipping vdW interaction at the nodes.
(HOF) as a function of shear strain. (Inset) 2D FFT of the SEM images at 0% and 100% strain. (C) In summary, the temperature invariance of the
Schematic description of the change in intertube structure with strain. (Inset) TEM image of intertube vdW interaction (15) and the thermal stability of
structure at 1000% strain. (D) TEM image of the as-prepared intertube structure. (Inset) Selected section CNT material provided this temperature invariant
indicating nodes. (E) Schematic of the zipping and unzipping of nodes. viscoelasticity.

REPORTS
References and Notes 8. K. Hata et al., Science 306, 1362 (2004). 15. G. Lenaz, G. Milazzo, Bioelectrochemistry of Biomacromolecules
1. R. Lakes, Viscoelastic Materials (Cambridge Univ. Press, 9. S. Osswald, E. Flahaut, H. Ye, Y. Gogotsi, Chem. Phys. (Birkhäuser, Basel, Switzerland, ed. 1, 1997).
New York, ed. 1, 2009). Lett. 402, 422 (2005). 16. We acknowledge partial funding by TASC. M.X.
2. E. T. Thostenson, C. Li, T. W. Chou, Compos. Sci. Technol. 10. Materials and methods are available as supporting acknowledges technical consultations from Y. Hayamizu,
65, 491 (2005). material on Science Online. A. Izadi-Najafabadi, and Y. Seki.
3. J. Suhr et al., Nat. Nanotechnol. 2, 417 (2007). 11. M. Zhang, K. R. Atkinson, R. H. Baughman, Science 306,
4. A. Cao, P. L. Dickrell, W. G. Sawyer, M. N. Ghasemi-Nejhad, 1358 (2004). Supporting Online Material
12. L. Qu, L. Dai, M. Stone, Z. Xia, Z. L. Wang, Science 322, www.sciencemag.org/cgi/content/full/330/6009/1364/DC1
P. M. Ajayan, Science 310, 1307 (2005).
238 (2008). Materials and Methods
5. X. C. Gui et al., Adv. Mater. 22, 617 (2010).
13. B. Bhushan, X. Ling, A. Jungen, C. Hierold, Phys. Rev. B Figs. S1 to S9
6. Q. Zhang et al., J. Phys. D Appl. Phys. 43, 315401 (2010).
7. S. Pathak, Z. G. Cambaz, S. R. Kalidindi, J. G. Swadener, 77, 165428 (2008). 9 July 2010; accepted 12 October 2010
Y. Gogotsi, Carbon 47, 1969 (2009). 14. B. Chen et al., Appl. Phys. Lett. 83, 3570 (2003). 10.1126/science.1194865
Video-Rate Molecular Imaging in Vivo (>1 MHz) offers superior sensitivity (D I/I < 10−8
in 1 s).
with Stimulated Raman Scattering

Despite the advantages of SRS contrast, it has
not been applied in living animals or humans for
two reasons: First, previous SRS images required
Brian G. Saar,1*† Christian W. Freudiger,1,2* Jay Reichman,3 C. Michael Stanley,3 ~1 min per frame, which is much too slow because

Gary R. Holtom,1 X. Sunney Xie1‡ living animals and humans inevitably move on the
microscopic scale (movie S1). To achieve high-
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial speed imaging (Fig. 1C), we modulated the inten-
resolution and sensitivity compared with magnetic resonance imaging. Stimulated Raman scattering sity of the Stokes beam at 20 MHz and used a
(SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without home-built, all-analog lock-in amplifier with a re-
adding toxic or perturbative labels. However, SRS imaging in living animals and humans has not been sponse time of ~100 ns (fig. S2). The colinear laser
feasible because light cannot be collected through thick tissues, and motion-blur arises from slow beams were tuned to match a vibrational frequency
imaging based on backscattered light. In this work, we enable in vivo SRS imaging by substantially of interest (Fig. 1B) and raster-scanned across the
enhancing the collection of the backscattered signal and increasing the imaging speed by three orders sample by a resonant galvanometer mirror with a
of magnitude to video rate. This approach allows label-free in vivo imaging of water, lipid, and protein line rate of 8 kHz (100 ns per pixel at 512 × 512
in skin and mapping of penetration pathways of topically applied drugs in mice and humans. pixels with up to 25 frames/s). The power levels used
here were previously used for in vivo imaging, and
ptical imaging techniques are complemen- Coherent anti-Stokes Raman scattering no photodamage was observed (8). Whereas previous
O tary to magnetic resonance imaging (MRI)

for in vivo applications. Although the pen-
etration depth of MRI is much higher, optical tech-
(CARS) (4, 5) has been used for fast imaging of
biological samples, primarily with lipid contrast,
(6, 7), at speeds up to video rate (8). However,
SRS microscopy was limited by the 100-ms re-
sponse time of a commercial lock-in amplifier
(SR844, Stanford Research Systems), our new
niques offer superior spatiotemporal resolution. CARS suffers from spectral distortion (9), limited system is three orders of magnitude faster.
Label-free optical imaging techniques for in vivo sensitivity arising from an unwanted nonresonant Second, SRS microscopy involves measure-
application to humans are attractive because dyes background (10), nonlinear concentration depen- ment of the intensity loss of the transmitted pump
or fluorescent labels may be toxic or perturbative. dence (11), and coherent image artifacts (12) that beam, which is not feasible in thick, nontransparent
Recent developments in applying two-photon auto- make quantitative interpretation and applications samples (for instance, a human arm). We must rely
fluorescence microscopy (1) or second harmonic gen- beyond lipid imaging difficult. on backscattering of the forward-going signal by tis-
eration microendoscopy (2) in vivo in humans have The recent development of stimulated Raman sue. This signal was previously collected by the ex-
revealed a wealth of structural and functional infor- scattering (SRS) microscopy overcame these citation objective (14), which works for samples
mation that cannot be obtained with other methods, limitations (13–16) and provided better vibration- with extremely high scattering (18). However, in
but those techniques are limited to relatively few al contrast (17, 18). In SRS microscopy, the biological tissues, the epi-directed signal collected
specific molecular signatures. Raman spectros- sample is excited by colinear and tightly focused through the objective is too weak for high-speed
copy offers label-free contrast based on char- pump and Stokes beams at wp and wS, respectively. imaging: Even with >60-s integration time, the
acteristic vibrational frequencies for all major If the difference in frequency (Dw = wp – wS) signal-to-noise ratio obtained from tissue was
chemical species in tissue, such as lipids, water, matches a molecular vibration in the sample at the poor (14).
DNA, and proteins, as well as a variety of small frequency Wvib (Fig. 1, A and B), the Stokes beam To quantitatively understand the light collection
molecules such as drugs or metabolites. Coherent intensity increases and the pump beam intensity in epi-SRS, we performed nonsequential ray-tracing
Raman scattering (3) methods allow stimulated decreases as a result of the coherent excitation of simulations (20) by simulating the propagation of
excitation of coherent motions of vibrational molecular vibrations. When using biocompatible a large number (106) of rays emitted from the focal
oscillators and can offer vibrational imaging with excitation, the intensity changes (DI) of the beams volume into a bulk medium with user-defined scat-
subcellular spatial resolution and image acquisition are small compared with their intensities (DI/I < tering parameters, which is used as a model for tis-
speed that is more than four orders of magnitude 10−3). We used a high-frequency modulation transfer sue (Fig. 1, D and E). Scattering events along a ray’s
higher than that of ordinary Raman microscopy. method to detect the signal (fig. S1) (14, 19). In trajectory are modeled probabilistically (21), and
this approach, we modulated the intensity of the we measured the final distribution of the light after
1
Department of Chemistry and Chemical Biology, Harvard Stokes beam with an electro-optic modulator and it is emitted from the simulated tissue volume. We
University, Cambridge, MA 02138, USA. 2Department of Phys- measured the modulation transfer to the pump beam found that, as expected, 40 to 45% of the light is
ics, Harvard University, Cambridge, MA 02138, USA. 3Chroma with a lock-in amplifier (LIA) after blocking the backscattered in a thick tissue sample because
Technology, 10 Imtec Lane, Bellows Falls, VT 05101, USA. modulated Stokes beam with an optical filter. scattering dominates over absorption in most tissues
*These authors contributed equally to this work. Because laser intensity fluctuations and variations (22). Because of multiple scattering, the diffuse
†Present address: MIT Lincoln Laboratory, 244 Wood Street,
Lexington, MA 02420, USA.
in sample transmission associated with raster cloud of backscattered light has a radius of ~5 mm
‡To whom correspondence should be addressed. E-mail: scanning the focus through a turbid sample oc- at the front aperture of the objective lens (Fig. 1F),
xie@chemistry.harvard.edu cur at low frequencies, high-frequency detection largely independent of the excitation numerical

REPORTS

Fig. 1. SRS microscopy. (A) Energy diagram of SRS. If the difference frequency of traveling light to illuminate the detector active area. The modulated Stokes beam is
the excitation beams (Dw = wp – wS) matches a vibrational frequency in the blocked by an optical filter (FI), and the transmitted pump beam is detected and
sample, Wvib, a molecule is excited from the vibrational ground state (n = 0) to a demodulated by the LIA. Images are constructed by scanning the focal spot in two
vibrational excited state (n = 1), passing through a virtual state (dashed lines). This dimensions with the use of a galvanometer mirror (GM) and resonant galvano-
results in a photon in the pump field being annihilated (stimulated Raman loss) meter mirror (RGM). EOM, electro-optic modulator. (D) Ray-tracing simulations
and a photon in the Stokes field being created (stimulated Raman gain), which can allow quantitative understanding of the distribution of the relative intensity (RI) of
be probed as a contrast for microscopy. (B) Raman spectra of chemical compounds the backscattered light at the tissue surface from a focus at a depth of 100 mm into
imaged in this work. (C) Our experimental setup for in vivo SRS microscopy the tissue (scattering mean free path = 200 mm, anisotropy = 0.9) emitting in the
detected the modulation transfer from the Stokes beam to the backscattered pump forward direction with a 0.4 numerical aperture. (E) Depth profile of tissue
beam. (Inset) The epi-detector assembly. The specimen (SP) is excited by focusing showing sample ray trajectories colored according to one of three final outcomes.
light with an objective lens (OL) through a small hole in the center of the large-area (F and G) Simulations of collection efficiency of backscattered, forward-traveling
epi-detector (PD) with a 10-mm outer diameter and a 2-mm aperture in the center. light versus detector radius for (F) different scattering mean free paths of the
Multiple scattering within the tissue sample redirects a large portion of the forward- sample and (G) different numerical apertures (NA) of the excitation objective.
aperture (Fig. 1G). A typical microscope objective Fig. 2. SRS skin imag- A B C
has a front-aperture radius of ~1 to 2 mm, so more ing in living mice. (A)
than 90% of the backscattered light is not collected SRS image of lipids of the
by the objective, unless it is surrounded by specially- stratum corneum shows
designed parabolic collection mirrors (23). We intercellular spaces be-
solved this problem by placing the photodetector tween hexagonal corneo-
directly in front of the objective lens and exciting cytes; (B) SRS water image
(3250 cm−1) of the same
through an aperture in the center of the detector SRS 2845 cm -1 SRS 3250 cm -1 CARS 3250 cm -1
region shows a homoge-
(Fig. 1C). In this geometry, we found in mouse skin D E F
nous distribution of water.
that we collected ~28% of the laser light impinging (C) CARS water image ac-
onto the sample. Given the angular distribution of quired simultaneously with
the backscattered light (fig. S3), a filter to block (B) shows artifacts from
the modulated Stokes beam while transmitting the the nonresonant back-
pump beam had to be specially designed (fig. S4). ground of lipids. (D) SRS
Using this system, we imaged skin in vivo in lipid and (E) water images
mice. Figure 2 shows single SRS video-rate frames of the viable epidermis SRS 2845 cm -1 SRS 3250 cm -1 SRS 2950 cm -1
obtained by the CH2 stretching (primarily lipids; show sebaceous glands G 50 ms

Fig. 2, A and D), OH stretching (primarily water; with positive and negative
Fig. 2, B and E), and CH3 stretching (primarily contrast, respectively. (F)
protein; Fig. 2, F and G) vibrations. The lipid dis- SRS images of the viable SRS 2950 cm -1
tributions (movie S2) are as expected from previous epidermis at the CH3
work (14), but water can only be measured in vivo stretching vibration (2950 cm−1) mainly highlight proteins, as well as residual lipid-rich structures. A capillary
because the skin hydration changes in excised tissue. with individual red blood cells (arrow) is visible. The cells are imaged without motion blur due to video-rate
Imaging water is of particular interest in studying acquisition speed. (G) SRS in vivo flow cytometry. An x-t plot acquired by line-scanning across a capillary at the
the transport properties of water-soluble drugs and position of the arrow in (F) is shown. Individual red blood cells are captured on the fly. Images in (A) to (E) are
their effect on the hydration of the skin barrier (24). acquired in epi-direction, whereas those in (F) and (G) are acquired in transmission, all with 37-ms-per-frame
Figure 2C highlights that CARS imaging of acquisition speed and 512 × 512 pixel sampling. Scale bars, 25 mm. The Raman spectra are shown in Fig. 1B.
water is distorted by the nonresonant background, This effect is not observed in SRS because it is free the viable epidermis (movie S3), due primarily to
which introduces an image artifact: It shows a from this nonresonant background (14). Signal the contrast of CH3 oscillators in hemoglobin. By
positive contrast “honeycomb” pattern for the lipid- averaging can further improve the signal-to-noise performing line scans versus time, we reconstructed
rich areas of the stratum corneum layer, which do ratio if the sample remains still enough (fig. S5). a plot of the cells as they pass through the scan line
not contain water. Thus, the contrast in CARS is The protein images (Fig. 2, F and G, and fig. (Fig. 2G), allowing for in vivo flow cytometry (25)
inverted compared with the real water distribution. S6) show red blood cells moving in a capillary of based on intrinsic chemical contrast.

REPORTS
Fig. 3. In vivo imaging A Z=0 µm B Z=0 µm C Z=0 µm small animals and humans. The approach presented
of drug penetration of here can also be applied to other modulation trans-
trans-retinol. (A to C) fer imaging techniques, including two-color two-
SRS images of hair and photon absorption, pump-probe (28), and stimulated
stratum corneum in the emission (29) microscopy. High-speed vibrational
ear of a living mouse. (D imaging is likely to play an increasingly important
to F) Depth projection of role in medical diagnostics in humans.
3D image stacks of the SRS 2950 cm-1 -1
SRS 2845 cm SRS 1596 cm-1
viable epidermis. Images

are acquired at the indi- D Z Projection E Z Projection F Z Projection References and Notes
1. K. König et al., Microsc. Res. Tech. 70, 398 (2007).
cated Raman shifts. Con- 2. M. E. Llewellyn, R. P. J. Barretto, S. L. Delp,
trast coming primarily M. J. Schnitzer, Nature 454, 784 (2008).
from protein (2950 cm−1) 3. M. D. Levenson, S. S. Kano, Introduction to Nonlinear
and lipid (2845 cm−1) Laser Spectroscopy (Academic Press, San Diego, 1988).
shows the morphology of 4. A. Zumbusch, G. R. Holtom, X. S. Xie, Phys. Rev. Lett. 82,
4142 (1999).
the skin with all its struc-
SRS 2950 cm-1 -1
SRS 2845 cm SRS 1596 cm-1 5. C. L. Evans, X. S. Xie, Annu. Rev. Anal. Chem. 1,
tural elements and sub- 883 (2008).
cellular resolution [see 6. T. B. Huff, J. X. Cheng, J. Microsc. 225, 175 (2007).
nuclei in (E)]. Nuclei are visible as dark structures in the 2845 cm–1 image (E), because the nuclei have low 7. T. Hellerer et al., Proc. Natl. Acad. Sci. U.S.A. 104,

concentrations of lipids. Hairs are visible as solid, periodic structures in the protein image (D) and are surrounded 14658 (2007).
by oil secreted from the sebaceous glands in the lipid image (E). We are able to visualize with SRS that drug 8. C. L. Evans et al., Proc. Natl. Acad. Sci. U.S.A. 102,
16807 (2005).
penetration of the topically applied trans-retinol (C) occurs along the hair shaft (F). Images are collected in 9. H. A. Rinia, M. Bonn, M. Müller, J. Phys. Chem. B 110,
transmission with 37-ms-per-frame acquisition speed and 512 × 512 pixel sampling. Scale bars, 25 mm. The 4472 (2006).
Raman spectra are shown in Fig. 1B. 10. F. Ganikhanov, C. L. Evans, B. G. Saar, X. S. Xie, Opt. Lett.
31, 1872 (2006).
11. L. Li, H. Wang, J.-X. Cheng, Biophys. J. 89, 3480 (2005).
Fig. 4. SRS skin imaging in living humans. A B 12. J. X. Cheng, A. Volkmer, X. S. Xie, J. Opt. Soc. Am. B 19,
(A to C) SRS images of the stratum cor- 1363 (2002).
neum and viable epidermis tuned into CH3 13. E. Ploetz, S. Laimgruber, S. Berner, W. Zinth, P. Gilch,
stretching vibration of proteins (2950 cm−1) Appl. Phys. B 87, 389 (2007).
showing the stratum corneum (A), the 14. C. W. Freudiger et al., Science 322, 1857 (2008).
15. Y. Ozeki, F. Dake, S. Kajiyama, K. Fukui, K. Itoh, Opt.
viable epidermis (B), and a hair on the
Exp. 17, 3651 (2009).
skin surface (C). (D) SRS image of DMSO 16. P. Nandakumar, A. Kovalev, A. Volkmer, New J. Phys. 11,
penetrating the skin at the same region as 033026 (2009).
shown in (C). We find that DMSO also 17. B. G. Saar et al., Angew. Chem. Int. Ed. 49,
accumulates in the hair shaft. We used SRS 2950 cm-1 SRS 2950 cm-1 5476 (2010).
deuterium labeling to create a unique vi- 18. M. N. Slipchenko et al., Analyst (London) 135,
bration of d6-DMSO at 2120 cm−1 for C D 2613 (2010).
19. Methods, additional results, and movies are available as
specific imaging. Images are acquired in supporting material on Science Online.
epi-direction on the forearm of a volunteer 20. Zemax User's Guide (Zemax Development Corporation,
(X.S.X.). Image acquisition time is 150 ms Bellevue, WA, 2010).
for (A) and (B) and 37 ms for (C) and (D), 21. S. L. Jacques, C. A. Alter, S. A. Prahl, Lasers Life Sci. 1,
all with 512 × 512 pixel sampling. Scale 309 (1987).
22. W. F. Cheong, S. A. Prahl, A. J. Welch, IEEE J. Quant. Elec.
bars, 50 mm. The Raman spectra are shown 26, 2166 (1990).
in Fig. 1B. 23. C. Combs et al., J. Microsc. 228, 330 (2007).
SRS 2950 cm-1 SRS 2120 cm-1 24. A. V. Rawlings, C. R. Harding, Dermatol. Ther. 17
(suppl, 1), 43 (2004).
Figure 3 demonstrates the ability of SRS mi- depth of 50 mm can be seen when tuned in to the 25. D. A. Sipkins et al., Nature 435, 969 (2005).
croscopy to follow the penetration of a small- CH3 stretching band. High contrast is available for 26. M. R. Prausnitz, S. Mitragotri, R. Langer, Nat. Rev. Drug
Discov. 3, 115 (2004).
molecule drug (trans-retinol, which stimulates nuclei (Fig. 4, A and B), and the varying nuclear 27. J. Lademann et al., Skin Pharm. Phys. 21, 150 (2008).
collagen synthesis) into mouse skin. Contrast size can be used to find the boundary between the 28. D. Fu, T. Ye, T. Matthews, G. Yurtsever, W. Warren,
using the CH3 and CH2 stretching highlights viable epidermis and the stratum corneum. Figure J. Biomed. Opt. 12, 054004 (2007).
the morphology of the skin. By tuning in to the 4C shows a hair disappearing down the hair shaft. 29. W. Min et al., Nature 461, 1105 (2009).
30. We thank J. MacArthur for advice on electronics and
polyene stretch of trans-retinol, we see that the In this case, deuterated dimethyl sulfoxide (DMSO), K. Sherwood, W. Faustino, and X. Zhang for helpful
drug has penetrated via the hair shaft into the se- a penetration-enhancing small molecule, was ap- discussions. C.W.F. was supported by a Boehringer
baceous gland, one of three hypothesized pene- plied to the skin. By tuning to the C-D stretching Ingelheim Fonds Ph.D. fellowship. This work was supported
tration routes into skin (26, 27). Averaging the vibration at 2125 cm−1, contrast from DMSO ap- by the Bill and Melinda Gates Foundation and an NIH T-R01
award (1R01EB010244-01) to X.S.X. Patent applications
three-dimensional (3D) data (movie S4), we see peared. DMSO can again be found to accumulate based on this work have been filed by Harvard Univ. Imaging
the localization of trans-retinol surrounding the in the area surrounding the hair, though it does not was performed in accordance with Harvard Univ. Faculty of
hair and in the top of the sebaceous gland (Fig. completely penetrate into the hair itself. Such Arts and Sciences Institutional Animal Care and Use
3F). In our previous work, performed excised tissue, mechanistic insight into the transport of small mol- Committee protocol number 29-01.
we did not observe this pathway for this drug (14), ecules can only be obtained with label-free chem-
indicating the importance of in vivo studies, be- ical imaging. Supporting Online Material
www.sciencemag.org/cgi/content/full/330/6009/1368/DC1
cause the transport properties of small molecules By overcoming the challenges associated Methods
can be affected by the skin temperature, moisture with performing high-speed, epi-detected SRS Figs. S1 to S6
content, and other factors. microscopy, this powerful label-free imaging mo- Movies S1 to S4
Figure 4 shows in vivo SRS imaging of human dality can now be applied to a broad range of 1 September 2010; accepted 4 November 2010
skin. Cell layers of the viable epidermis up to a problems in whole living organisms, including 10.1126/science.1197236

REPORTS
The Role of Particle Morphology in harvesting and energy conversion applications

(2, 6). Nevertheless, there is still no clear con-
Interfacial Energy Transfer in CdSe/CdS

sensus on the microscopic nature of interfacial
energy levels and the process of carrier migra-
tion from one semiconductor to another. Very
Heterostructure Nanocrystals similar CdSe/CdS nanostructures can exhibit

properties of either a type I heterostructure, in
which both electron and positively charged hole
Nicholas J. Borys,1 Manfred J. Walter,1 Jing Huang,2 Dmitri V. Talapin,2,3 John M. Lupton1,4* are confined to one material, or a type II hetero-
structure, in which electron and hole separate
Nanoscale semiconductor heterostructures such as tetrapods can be used to mimic light-harvesting between the materials (11–14). Naturally, such
processes. We used single-particle light-harvesting action spectroscopy to probe the impact of a distinction is crucial to choosing the correct ma-
particle morphology on energy transfer and carrier relaxation across a heterojunction. The generic terial for a particular application: Light emis-
form of an action spectrum [in our experiments, photoluminescence excitation (PLE) under sion requires type I junctions, whereas charge
absorption in CdS and emission from CdSe in nanocrystal tetrapods, rods, and spheres] was controlled separation in photovoltaics requires type II (13).
by the physical shape and resulting morphological variation in the quantum confinement parameters Such knowledge of the interface is crucial for
of the nanoparticle. A correlation between single-particle PLE and physical shape as determined designing nanoparticle superstructures, such as
by scanning electron microscopy was demonstrated. Such an analysis links local structural inorganic dendrimers with a treelike branched

non-uniformities such as CdS bulbs forming around the CdSe core in CdSe/CdS nanorods to a architecture, with superior light-harvesting prop-
lower probability of manifesting excitation energy–dependent emission spectra, which in turn is erties. A first step to such a structure is the hetero-
probably related to band alignment and electron delocalization at the heterojunction interface. junction tetrapod (5).
Semiconductor nanoparticles are well suited
dvances in the synthesis of semiconductor of the physical shape and quantum confinement to microscopic optical studies, which have re-
A nanoparticles have enabled exquisite con-

trol over composition and shape, yielding
spherical, linear (1, 2), and branched structures
parameters of the semiconductor heterostruc-
tures, and showed how the energetic landscape
can inhibit complete electron transfer across
vealed a surprising diversity in electronic proper-
ties between particles that are typically masked in
ensemble-level measurements (15). Htoon et al.,
such as tetrapods (3–5). Although many semi- the CdSe/CdS interface. This single-particle clas- for example, uncovered both discrete excitonic
conductor nanoparticles originally consisted of sification highlights the sensitivity of ensemble states and a quasi-continuum in the core of
only one material, further opportunities for tai- performance, such as in light-harvesting, to mor- spherical CdSe particles capped with a ZnS shell
loring electronic functionalities are anticipated phological characteristics that affect the intrinsic (16). We probed energetic relaxation in individ-
from nanoscale semiconductor heterostructures nature of the interfaces, thereby suggesting routes ual CdSe/CdS nanoheterostructures of different
combining two or more materials (5). Indeed, a to future synthetic optimization. shapes after optical excitation of the higher-gap
host of applications has emerged from precise Large optical absorption cross sections, en- CdS. This approach, leveraging single-particle
control over nanostructure functionality, ranging hanced stability, high quantum yields, and size- PLE over a wide spectral range, illuminates the
from photovoltaics (2, 6) to commercial light- tunable electronic structure make semiconductor nature of both the high-gap material absorption
emitting devices. A microscopic understanding of nanocrystals particularly interesting for light- and the subsequent intraparticle relaxation pro-
the migration of excitation energy in heterojunc-
tions and the resulting interfacial charge transfer Fig. 1. Light-harvesting
(7) is particularly important in developing photo- action spectroscopy at 4 K A
catalytic compounds to split water (8) or reduce of two single semiconductor 1
CO2 (9). Due to only a slight mismatch in the Class A
tetrapods consisting of a low-
lattice constants of the two materials, yet well- gap CdSe core and high-gap
contrasted band gaps, the CdSe/CdS heterostruc- CdS arms. The nanoparticles
ture has evolved as one of the workhorses for are excited primarily above
relating nanoparticle synthesis and shape to spec- the CdS absorption edge, and
Emission Intensity (arb. units)
troscopic properties. Here we describe an optical emission is detected from the

classification of the quantum confinement in CdSe core. A sketch of the
complex CdSe/CdS core/shell nanostructures by assumed electron and hole CB
means of single-particle light-harvesting action probability density distribu- 0
spectroscopy, a versatile noncontact tool to measure tion within the electronic VB
electron transport or energy transfer on mesoscopic band structure is shown in B CdS CdSe
length scales (10). For a variety of nanostructure the inset in the middle. (A)
shapes, we probed the photoluminescence exci- For the first particle, the on- 1 Class B
set of absorption from the
tation (PLE) of the absorbing CdS through emis-
quantum-confined CdS exci-
sion from the lower-gap CdSe. We identified two
ton gives rise to a distinct
distinct categories of action spectra, characteristic peak that is then followed
by absorption into a continu-
1
Department of Physics and Astronomy, University of Utah, um of states at higher en-
Salt Lake City, UT 84112–0830, USA. 2Department of ergy (red, scan downward in
Chemistry, University of Chicago, Chicago, IL 60637, USA.
3 energy; blue, scan upward).
Center for Nanoscale Materials, Argonne National Labora- 0
tory, Argonne, IL 60439, USA. 4Institut für Experimentelle (B) For the second particle,
the peak associated with the 2.4 2.6 2.8 3.0
und Angewandte Physik, Universität Regensburg, D-93040
Regensburg, Germany. CdS exciton is not present, Excitation Energy (eV)
*To whom correspondence should be addressed. E-mail: and the spectrum is marked
john.lupton@physik.uni-regensburg.de by a continuum of states after the onset of CdS absorption (the black lines are a guide to the eye).

REPORTS
cess, which are both ultimately crucial in design- Although the tetrapods perform the desired gins of the distinct PLE classification. We there-
ing light-harvesting applications (6). In contrast, light-harvesting function, they do not offer the fore used two further model systems to study
most single-quantum–structure PLE spectroscopy best system in which to search for structural ori- the influence of particle form on PLE: nanorods,
(16–18) to date has been carried out over relatively
narrow spectral ranges to probe distinct excitonic
features, or else has analyzed excitation enhance- Tetrapods Rods Spheres
ment by metal nanoparticles at discrete wave-
lengths (19).
We focused our attention on CdSe/CdS tetra- 1 A C E
Class A
pods, which are inorganic light-harvesting com-
plexes with very large absorption cross sections,
Emission Intensity
which make them easily visible in single-particle
measurements. A schematic of a CdSe/CdS tetra- 0
pod nanoparticle and a suggestive band diagram
(20) are shown in the inset of Fig. 1. The struc-
tures consist of a CdSe core approximately 4 nm B D F
1
in diameter, which is surrounded by an antenna-
Class B
like CdS shell. The shell has four arms that are 80 nm

20 nm in length and 6 nm in diameter. We purified
the tetrapod solution to an ensemble composi-
tion of at least 90% tetrapods, which appeared to 0
be geometrically uniform in transmission elec-
tron microscopy (TEM) (5, 21). Previous com- 2.4 2.7 3.0 2.5 2.8 3.1 2.5 2.8 3.1
parisons of theory and experiment have indicated Excitation Energy (eV)
that, on average, the CdSe/CdS interface should Fig. 2. Classification of single-particle light-harvesting action spectra for tetrapod, rod, and near-
form a quasi–type II heterostructure, with an spherical nanoheterostructures. A total of 150, 150, and 75 raw spectra for tetrapods, rods, and spheres,
excited electron delocalizing over the CdSe and respectively, are sorted into two groups. All curves are normalized to the CdS absorption onset intensity
CdS conduction bands and the associated hole but are otherwise not manipulated. (A and B) 75 class A and 75 class B single-particle PLE spectra from
localizing on the lower-gap CdSe core (14). tetrapod structures. (C and D) 75 class A and 75 class B single-particle PLE spectra from rod structures. (E
Initial studies on the tetrapods have suggested a and F) The near-sperical structures show only class B single-particle PLE spectra. Inset in (B) to (F) are
similar electronic structure (20), although the representative TEM images of the tetrapods, rods, and spheres. Although the tetrapods appear uniform,
energy offsets are such that a range of interfacial the rod ensemble manifests two morphologies: one with a uniform diameter and the other with a
localization phenomena is conceivable (13): It pronounced bulb feature. The near-sperical structures are non-uniform in their deviations from isotropy.
is not straightforward to extrapolate band di-
agrams derived from bulk material parameters
to solution-grown nanostructures. For these het-
erostructures, at energies above the CdS funda-
mental absorption edge, the CdS extinction
dwarfs that of the CdSe, thus resulting in an
absorption spectrum dominated by the CdS. The
electronic structure, on the other hand, yields a
luminescence spectrum arising from recombi-
nation in CdSe (5, 21). The similarity of the en-
semble nanoparticle PLE and absorption spectra 80 nm
indicates that this light-harvesting process is
highly efficient (5). We expect the prominent on- A B
Emission Intensity
set of the PLE to correspond with the quantum- 1

confined exciton of the CdS, followed by the
typical continuum of higher energy states.
Such behavior was indeed observed in single-
particle light-harvesting action at 4 K in Fig. 1A.
We recorded the CdSe emission intensity while Class B
sweeping the laser excitation both down and up Class A
in energy (22). The particle exhibited a clear peak 0
in the CdS absorption that can be attributed to the
quantum-confined excitonic transition (23, 24).
We refer to PLE spectra with such behavior as 2.5 2.8 3.1 2.5 2.8 3.1
class A. A second single tetrapod showed strik- Excitation Energy (eV)
ingly different behavior depicted in Fig. 1B, Fig. 3. Correlated SEM and PLE spectra of 12 single CdSe/CdS nanorods. (A) Six class A PLE spectra
revealing particle-to-particle variations that are normalized to the CdS onset and plotted together with their corresponding SEM images, representative of
masked in ensemble measurements. In this case, rods with a uniform diameter (cartoon at bottom). (B) The SEM images of six rods that exhibit a class B PLE
there is no clear peak after the CdS absorption spectrum indicate that the spectral shape is attributable to rods that have a pronounced bulb feature and
onset. We term these PLE spectra class B. Both non-uniform diameter as sketched schematically in the cartoon. All correlated image-PLE pairs were chosen
classes were repeatedly observed in spatial solely on the basis of the quality of the PLE spectrum, without any knowledge of the corresponding SEM
proximity during the same excitation scan. image.

REPORTS
which mimic one arm and the core of the tetra- termines the symmetry of the confinement poten- basis of the quality of the PLE spectrum over up
pod; and nanospheres with a thick CdS shell. tial. Other approaches to representing the raw and down sweeps without any knowledge of the
All structures had a similar CdSe core that was data, along with quantitative thresholds for differ- corresponding image. Figure 3A shows six class
4 nm in diameter. Figure 2 shows single-particle entiating between the two classes, are discussed A PLE spectra normalized and plotted together;
spectra of 150 tetrapod, 150 rod, and 75 near- in figs. S3 and S4 (22). Fig. 3B shows six class B spectra. Inset in the
spherical nanoheterostructures, normalized to the Through its relation to the quantum confine- plot are the corresponding SEM images. A clear
CdS absorption onset. Representative TEM im- ment effect, physical shape plays a crucial role in trend that confirms the shape hypothesis is ob-
ages for each nanostructure are shown in the the optical and electronic properties of semi- servable: All of the rods in Fig. 3A exhibit a class
lower insets. The rods consist of a CdS arm 60 nm conductor nanocrystals (13, 23, 25). For the rods, A PLE spectrum, and five out of six have a uni-
in length and approximately 6 nm in diameter where quantum confinement in the CdS is inform diameter over the length of the rod. On the
surrounding the CdSe core. TEM indicated that fluenced mostly by diameter (26), the bulb struc- other hand, in Fig. 3B, all of the rods exhibit a
the sample contained two general shapes of rods: tures consist of a range of diameters that will class B PLE spectrum and have a pronounced
rods that have a constant diameter over their smear out the excitonic transition and will likely bulb feature as anticipated (see the text accom-
length and those that have a bulb feature sur- lead to particles with a class B spectrum. The panying fig. S7 for a discussion of the geometric
rounding the CdSe core, resulting in a non- same effect of shape variations in the direction of thresholds invoked). These observations are
uniform rod diameter (11, 21). Finally, the CdS radial quantum confinement could also give rise consistent with single-particle PL (fig. S8), which
shell for the near-spherical structures was a 4-nm- to exclusively class B spectra in the near-spherical demonstrates that rods with a class A PLE
thick layer surrounding the CdSe core. TEM of particles. In addition, quantum confinement in spectrum have higher-energy residual CdS emis-

these particles revealed that the thick CdS shell absorption in these particles may be weaker as sion due to their smaller diameters (stronger quan-
results in a non-uniform distribution of particles the larger particle diameters approach the Bohr tum confinement) than rods with a class B PLE
with anisotropic deviations from a perfect sphere. exciton radius in CdS, thus reducing the visibility spectrum. The basic statistical analysis presented
For the tetrapods and rods, each single-particle of excitonic transitions. in table S1 indicates that the probability of the
PLE spectrum can generally be categorized as To verify the role that shape plays in the PLE results in Fig. 3 occurring by chance is 0.3%.
class A (Fig. 2, A and C, respectively) or class B spectrum of a single nanocrystal, we correlated The similarity in PLE and emission spectra
(Fig. 2, B and D, respectively). In contrast, we PLE measurements with scanning electron mi- of rods, spheres, and tetrapods suggests that
observed exclusively class B spectra for the near- croscopy (SEM) of single rods. The correlation excessive CdS growth around the core could be
spherical structures (Fig. 2F). We conclude that process is discussed in the text accompanying responsible for the formation of two distinct spec-
the origin of the different classes is not found in figs. S6 and S7. Figure 3 shows 12 PLE spectra troscopic classes of tetrapods. In addition, varia-
the combination of constituent materials but is and the SEM images of the corresponding tions in effective arm diameter within a single
related to the shape of the nanocrystal, which de- nanorods. The pairs were selected solely on the tetrapod (20) or, quite generally, variations in
Fig. 4. Single-particle emission Column A Column B Column C

spectra as a function of excitation 1.98 2.10 1.99
energy for three individual class A
tetrapods, revealing the possible 1.96 2.08 1.97
Row i
Emission Energy (eV)
band alignment schemes. (Col-

umn A) The emission spectrum 1.94 2.06 1.95
shifts to higher energies as the 1.92 2.04 1.93
excitation energy is raised. Two-
dimensional plots of emission as a 1.98 2.10 1.99
function of excitation are shown for
the laser sweep downward (Row i) 1.96 2.08 1.97
Row ii
and upward (Row ii) in energy. The
1.94 2.06 1.95
main emission peak energy, ex-
tracted by fitting two Gaussians to 1.92 2.04 1.93
the emission spectrum, is overlaid
as white circles. (Row iii) The peak
Peak (eV)
1.96 0 320 2.09 0 1000 2.00 0 1000
Row iii
position as a function of excitation
energy shows a steplike shift of
~10 meV close to the absorption 1.94 2.07 1.98
onset of CdS at 2.6 eV. (Row iv) 2.4 2.6 2.8 3.0 2.4 2.6 2.8 3.0 2.4 2.6 2.8 3.0
This situation can be rationalized Excitation Energy (eV)
in terms of a type I band align-
ment, where a barrier to thermal- CB CB ? CB
ization of the electron from the CdS ∆E > 0 ∆E ≈ 0 ∆E < 0
Row iv
to the CdSe exists. Below 2.6 eV,

direct excitation of the CdSe core VB VB VB
occurs, whereas emission at higher
excitation energies results from an CdSe CdS CdSe CdS CdSe CdS
interfacial exciton. (Column B) No Type I Quasi-Type II Type II
spectral shift is seen with excitation Band Alignment Band Alignment Band Alignment
energy, suggesting a quasi–type II
band alignment. By analogy to column A, it is conceivable that a barrier to the CdSe core to the CdS shell. A barrier to electron transfer from CdSe to CdS
electron transfer between CdS and CdSe also exists due to, for example, lattice must exist to prevent the core electron from transferring to the shell under
strain. (Column C) The type II band alignment is characterized by a shift of excitation of the core. The black lines in the peak position plots are smoothed
emission to lower energies as the excitation energy is raised from absorption in averages that serve as a guide for the eye.

REPORTS
intraparticle CdS-CdSe coupling strength that offsets DE, so that at least two distinct exciton electron transfer between CdS and CdSe. Con-
disrupt the particle symmetry, could lead to a loss states exist: CdSe core excitons and CdSe/CdS sequently, whereas particle uniformity and the
of excitonic structure in PLE. We therefore pro- interfacial excitons. Because both states are resulting confinement may be desirable in, for
pose that the single-particle PLE can serve as a emissive and hence stable, an interfacial barrier example, light-emitting devices, nanoparticles
predictor of particle morphology and indeed pro- must be present, which prevents complete elec- with low symmetry of the confinement poten-
vides insight into the tetrapod shape, which is tron transfer to the lowest state in the conduction tial (that is, with structural variations: class B)
hard to obtain by other means. band, thereby impeding thermalization of the are more suited for light-harvesting applica-
Bulblike particle morphologies probably have emissive exciton. A barrier to electron transfer tions because of a reduced influence of interfa-
featureless PLE spectra because the CdS shell is must also exist in the situation in column C, be- cial barriers. The challenge posed to nanoparticle
not defined by one fixed diameter, and therefore a cause the excited electron does not thermalize to synthesis is to extend shape engineering to
range of excitonic transition energies exists. This the lower-level CdS conduction band under di- morphology control, so as to create samples in
morphological disorder within a single particle rect excitation of the core. By analogy, the barrier which, for example, all particles exhibit class B
also appears to affect band alignment within the to electron delocalization could also be present morphologies.
heterojunction, as revealed by spectrally resolving for tetrapods in which the absence of a shift in
tetrapod emission as a function of excitation ener- emission energy indicates conduction band align- References and Notes
gy for 50 single tetrapods. Of these tetrapods, ment. The barrier can only exist for one of the 1. X. Peng et al., Nature 404, 59 (2000).
2. W. U. Huynh, J. J. Dittmer, A. P. Alivisatos, Science 295,
26% [13 tetrapods (fig. S9)] showed a surprising carriers, because complete blocking at the inter- 2425 (2002).
dependence of the tetrapod emission energy on face would result in recombination in the CdS 3. L. Manna, D. J. Milliron, A. Meisel, E. C. Scher,

the excitation energy. Three such cases are de- arm, which is spectrally distinct (fig. S8). Noting A. P. Alivisatos, Nat. Mater. 2, 382 (2003).
tailed in Fig. 4. that our spectroscopy cannot distinguish between 4. T. Mokari, E. Rothenberg, I. Popov, R. Costi, U. Banin,
Science 304, 1787 (2004).
The particle in column A of Fig. 4 exhibits a conduction and valence band blocking, we fol- 5. D. V. Talapin et al., Nano Lett. 7, 2951 (2007).
shift in emission to higher energy as the excita- low the earlier assignment of effective hole local- 6. P. V. Kamat, J. Phys. Chem. C 112, 18737 (2008).
tion energy is raised, although the shape of the ization in the core of CdSe/CdS nanoparticles 7. A. Pandey, P. Guyot-Sionnest, Science 322, 929 (2008).
emission spectrum remains characterized by the because of a larger offset in the valence band 8. L. Amirav, A. P. Alivisatos, J. Phys. Chem. Lett. 1,
1051 (2010).
CdSe electronic transition and its phonon side- than in the conduction band (29). We speculate
9. Y. Qu et al., Nano Lett. 10, 1941 (2010).
band. It is important to consider both sweep di- that the greater effectiveness of hole transfer 10. M. J. Walter, J. M. Lupton, Phys. Rev. Lett. 103, 167401
rections of the exciting laser because nanocrystals across the interface as compared to electron trans- (2009).
exhibit random spectral diffusion (27); the spec- fer could arise from the difference in wave function 11. D. Steiner et al., Nano Lett. 8, 2954 (2008).
tral changes are reversible upon downward (row symmetry of the carrier species, or result from the 12. A. Sitt, F. Della Sala, G. Menagen, U. Banin, Nano Lett. 9,
3470 (2009).
i) and upward (row ii) sweeps. Row iii of column much larger energy-level offset. 13. A. Pandey, P. Guyot-Sionnest, J. Chem. Phys. 127,
A plots the PL peak energy, which shows a dis- The observation that excitation energies well 104710 (2007).
tinct jump at an excitation energy of 2.6 eV. The above the band gap of both CdSe and CdS (that 14. J. Müller et al., Nano Lett. 5, 2044 (2005).
position of the spectral jump corresponds to the is, at 3.1 eV) do not result in complete thermal- 15. S. A. Empedocles, R. Neuhauser, K. Shimizu,
M. G. Bawendi, Adv. Mater. 11, 1243 (1999).
onset of CdS absorption at 4 K and indicates a ization and thus emission at the lowest energy of 16. H. Htoon, P. J. Cox, V. I. Klimov, Phys. Rev. Lett. 93,
transition from direct excitation of the CdSe the system (Fig. 4, column A), which is only ob- 187402 (2004).
core to excitation of the CdS arm and sub- served under direct core excitation, indicates that 17. H. Akiyama, M. Yoshita, L. N. Pfeiffer, K. W. West,
sequent relaxation to the CdSe. In contrast, the additional excitation energy does not play a role A. Pinczuk, Appl. Phys. Lett. 82, 379 (2003).
18. T. Ihara et al., Phys. Rev. Lett. 99, 126803 (2007).
particle in column B exhibits emission indepen- in electron transfer to the core. The CdS electron 19. Y. Chen et al., Appl. Phys. Lett. 93, 053106 (2008).
dent of excitation. This case would be expected therefore cools before the CdSe/CdS interfacial 20. C. Mauser et al., Phys. Rev. B 77, 153303 (2008).
for a semiconductor heterostructure in which the exciton is formed. Our preliminary statistics on 21. E. Yoskovitz, G. Menagen, A. Sitt, E. Lachman, U. Banin,
high-energy electron-hole pair formed in the the PLE spectra (fig. S9) indicate that excitation Nano Lett. 10, 3068 (2010).
22. Materials and methods are detailed in supporting
wide-gap semiconductor (CdS) relaxes down to energy–dependent emission is much more prom-
material on Science Online.
the narrower-gap CdSe. Finally, in column C, the inent in class A tetrapods. This observation is 23. A. Efros, M. Rosen, Annu. Rev. Mater. Sci. 30, 475 (2000).
emission energy decreases with increasing ex- again consistent with the implied morphological 24. T. Vossmeyer et al., J. Phys. Chem. 98, 7665 (1994).
citation energy. As in column A, a change in the disorder of the single class B tetrapod structures, 25. M. Saba et al., Adv. Mater. 21, 4942 (2009).
emission peak is observed around excitation at because more narrowly defined (class A) energy 26. J. Planelles, F. Rajadell, J. I. Climente, J. Phys. Chem. C
114, 8337 (2010).
2.6 eV. levels should be more susceptible to band mis- 27. R. G. Neuhauser, K. T. Shimizu, W. K. Woo,
To rationalize these observations, we consider alignment at the heterojunction. S. A. Empedocles, M. G. Bawendi, Phys. Rev. Lett.
the subtleties of conduction and valence band Single-particle light-harvesting action thus 85, 3301 (2000).
offsets between CdSe and CdS in row iv of Fig. reveals distinct spectroscopic behavior that is 28. A. M. Smith, S. Nie, Acc. Chem. Res. 43, 190 (2010).
29. X. Peng, M. C. Schlamp, A. V. Kadavanich, A. P.
4. Studies of CdSe/CdS nanoparticles at the en- masked in the ensemble, providing insight into Alivisatos, J. Am. Chem. Soc. 119, 7019 (1997).
semble level have suggested that the CdSe val- the uniformity of the quantum confinement and 30. The authors thank K. van Schooten for technical
ence band is consistently higher in energy than the degree of electronic delocalization across a assistance and R. Polson, M. DeLong, and S. Rupich for
that of the CdS, although the conduction band heterojunction. Morphologies in which the help with SEM measurements and are indebted to NSF
(grants CHE-0748473 and DMR-0213745) and the
alignment of the two materials is sensitive to quantum confinement varies within the parti- U.S. Department of Defense Office of Naval Research
nanoparticle geometry and may result in a type I, cle (due to the formation of a CdS bulb around (grant N00014-10-1-0190). J.M.L. and D.V.T. are fellows
type II, or quasi–type II electronic structure (see the CdSe core, as observed in nanorods) give of the David and Lucile Packard Foundation.
sketches in Fig. 4 for definition) (11–13). Such rise to a lifting of the spectral signature of well- Supporting Online Material
band offsets will affect the emission spectrum if a defined quantum confinement by smearing out www.sciencemag.org/cgi/content/full/330/6009/1371/DC1
barrier in the conduction band inhibits electron excitonic transitions. As shown in the single- Materials and Methods
SOM Text
transfer between CdS and CdSe, which is con- paricle spectrally resolved PLE measurements, Figs. S1 to S10
ceivable given the interfacial strain due to the band misalignment that is more prevalent in Table S1
lattice mismatch of the two materials (28). We morphologically uniform particles (class A) re- References
propose that the tetrapods in columns A and C of veals nonthermalized interfacial excitons, which 21 September 2010; accepted 3 November 2010
Fig. 4 are characterized by conduction band suggest the presence of an intrinsic barrier to 10.1126/science.1198070

REPORTS
A Cryptic Sulfur Cycle in let (13), and a sulfate reducer has been isolated
from OMZ waters off the coast of Peru (14). Di-
Oxygen-Minimum–Zone Waters
rect evidence for large-scale active sulfur cycling
in OMZs, however, is lacking. When sulfide, the
product of sulfate reduction, is observed in OMZs,
off the Chilean Coast it originates in rare pockets of nitrate and nitrite-
depleted water (15) or is released from sedi-
ments (6).
Don E. Canfield,1* Frank J. Stewart,2 Bo Thamdrup,1 Loreto De Brabandere,1 Tage Dalsgaard,3 We explored the dynamics of the sulfur cy-
Edward F. Delong,2 Niels Peter Revsbech,4 Osvaldo Ulloa5 cle in the upwelling waters off Iquique, on the
northern Chilean coast, using a combination of
Nitrogen cycling is normally thought to dominate the biogeochemistry and microbial ecology geochemical and metagenomic techniques (16).
of oxygen-minimum zones in marine environments. Through a combination of molecular In general, the OMZ is well developed in this
techniques and process rate measurements, we showed that both sulfate reduction and sulfide region of Chile (17). We concentrated our efforts
oxidation contribute to energy flux and elemental cycling in oxygen-free waters off the coast of on station 3 (20°5′9.27′′S, 70°20′8.18′′W; water
northern Chile. These processes may have been overlooked because in nature, the sulfide produced depth 1050 m, 23 km from shore), which, based
by sulfate reduction immediately oxidizes back to sulfate. This cryptic sulfur cycle is linked to on preliminary survey data, was in the most bio-
anammox and other nitrogen cycling processes, suggesting that it may influence biogeochemical logically active region of the OMZ in our study

cycling in the global ocean. area. We expanded our geochemical studies to
include station 5 (20°5′9.69′′S, 70°46′5.78′′W;
xygen-minimum zones (OMZs) persist through dissimilative nitrate reduction to ammo- water depth 1500 m), located some 44 km further
O in midwater depths of the global ocean,

where large-scale circulation and the sink-
ing and decomposition of surface-derived organ-
nium. OMZs account for 33% or more of the loss
of fixed nitrogen from the oceans (10, 11), and
overall, the nitrogen cycle has been thought to 1
Institute of Biology and Nordic Center for Earth Evolution,
ics deplete oxygen as compared to higher-surface dominate the geochemistry and microbial ecology University of Southern Denmark, Campusvej 55, 5230 Odense
and deep-water oxygen concentrations (1). In some of these regions. M, Denmark. 2Department of Biological Engineering and De-
partment of Civil and Environmental Engineering, Massachu-
regions such as the eastern tropical Pacific, the The recent identification of uncultured Gam-
setts Institute of Technology, Cambridge, MA 02138, USA.
Arabian Sea, and the Benguela Current upwell- maproteobacteria, closely affiliated with sulfur- 3
National Environmental Research Institute, Aarhus University,
ing system, water column oxygen concentrations oxidizing symbionts, in OMZ waters off the Vejlsøvej 25, Post Office Box 314, DK-8600 Silkeborg,
fall below detection (2–4), prompting the devel- Chilean coast (12) suggests that sulfur cycling Denmark. 4Section of Microbiology, Department of Biological
opment of a dynamic nitrogen cycle. In these may also play an important role in oxygen-free Sciences, Aarhus University, DK-8000 Aarhus C, Denmark.
5
Departamento de Oceanografía and Centro de Investigación
zones, nitrate is actively reduced to nitrite (5, 6). nitrate-rich OMZs. A similar microbial communi- Oceanográfica en el Pacífico Sur-Oriental, Universidad de
Nitrite is further converted to N2 gas through ty with a full complement of sulfide-oxidizing and Concepción, Casilla-160-C, Concepción, Chile.
“classic” heterotrophic denitrification (7) and the nitrate-reducing genes was found in sulfide-free *To whom correspondence should be addressed. E-mail:
autotrophic anammox process (8, 9) or to NH4+ but nitrate-rich portions of the sulfidic Saanich In- dec@biology.sdu.dk
Fig. 1. Representative nutrient,

oxygen, and chlorophyll a profiles
from the OMZ off the northern
Chilean coast at station 3 (left)
and station 5 (right).

REPORTS
offshore than station 3. The water chemistry in station 5. This deep layer consists of previously ing that essentially anoxic waters define this
the northern Chilean OMZ develops within an unknown members of the cyanobacterial genus region of the eastern tropical South Pacific
eastern boundary current, and the chemical pro- Prochlorococcus (18). OMZ. There is an upper nitrite maximum re-
files are somewhat dynamic (Fig. 1). With some We observed extremely low O2 concentra- lated to aerobic processes, but nitrite accumu-
variability over time, the redoxcline at station 5 tions of <13 nM, starting from between 60 and lated as oxygen disappeared in the anoxic core
was located deeper than at station 3, and although 85 m depth (depending on station and time of of the OMZ. Nitrate reduction was the most
the surface concentrations of chlorophyll a were sampling) and continuing to >180 m (fig. S1). likely source for this nitrite. We measured with
15
higher at station 3, we frequently observed a pro- Similar low values were found off the southern N-enriched nitrite the rates and pathways of
nounced secondary chlorophyll a maximum at Peruvian coast in an earlier study (19), suggest- N2 formation, and similar to what was found in
an early study (8), anammox was the dominant
pathway (Table 1), considerably outpacing
Table 1. Summary of process rate averages. denitrification (16). The overall rates of N2 for-
mation were similar to those in previous measure-
Station 3 Station 5 ments in this region (8).
–1 –1
Process rates (nmol liter hour ) Pyrosequencing of community DNA from
Anammox 0.43 T 0.21† 0.29 T 0.10‡ below the oxycline (70 to 80 m) and in the core
Denitrification* 0.079 T 0.04† 0.042 T 0.029‡ of the anoxic zone (150 to 200 m) at station 3
Sulfate reduction 0.51 T 0.21§ 0.055 T 0.023|| suggested a substantial role for sulfur-based

Depth-integrated rates (mmol m–2 day–1) metabolic pathways in the Chilean OMZ (Fig.
Anammox 1.21 T 0.45 0.70 T 0.24 2 and figs. S4 to S7). Gene sequences matching
Denitrification 0.22 T 0.11 0.10 T 0.069 diverse sulfide-oxidizing and sulfate-reducing
Sulfate reduction 1.00 T 0.40¶ 0.28 T 0.12 taxa (table S3) constituted 6.3 to 16.2% and 2.1
Carbon oxidation in OMZ # 5.50 5.50 to 2.4% of all sequencing reads with matches
*Measured as nitrite reduction to N2. †From 65 to 183 m depth (n = 17). ‡From 73.5 to 173 m depth (n = to protein-coding genes in the National Center
16). §From 85 to 150 m depth (n = 8). ||from 85 to 300 m depth (n = 14). ¶Assuming that sulfate reduction for Biotechnology Information–nr (NCBI-nr)
stops first at 200 m. #Estimated from data in (17). database, consistent with percentages based
on 16S ribosomal RNA gene-encoding reads
(figs. S4 and S5). In contrast, sulfur-oxidizer
A Aug. 2009 Jan. 2010 B relative gene abundance and sulfate-reducer sequences represented only
% of total hits to nr 0.5 and 0.3% of the total protein-coding se-
0 2 4 6 8 0 2 4 6 8 dsrA
dsrB quences recovered in an aerobic community
dsrC from another coastal site (Monterey Bay, 10 m;
dsrE
dsrF fig. S5). The Chilean OMZ metagenomes were
dsrH
dsrJ particularly enriched in sequences matching the
dsrK genomes of sulfur-oxidizing endosymbionts
dsrL
dsrM of deep-sea clams [Candidatus Ruthia mag-
70 m 80 m dsrN nifica (Rm) and Candidatus Vesicomyosocius
dsrO
dsrP
0 2 4 6 8 0 2 4 6 8 dsrR
soxA Rate (nM h-1)

soxB
soxC 0 0.2 0.4 0.6 0.8 1
soxD 0
soxH
soxR
soxS
soxV 50
200 m 150 m soxW
soxX
soxY
soxZ
Sulfur-oxidizers (rank: SUP05,Rm,Vo) 100
Pelagibacter sp. (rank: 7211,1002,1062) aprA
Nitrosopumilus maritimus aprB
aprM
Depth (m)
Kuenenia stuttgartiensis 150

Sulfate-reducers, multiple taxa binned (see Figure S2) FCSD
α-proteobacteria (HIMB114,BAL199) SQR
Psychroflexus torquis ATCC 700755
bacterium Ellin514 200
Rhodothermus marinus DSM 4252
rpoB
Fig. 2. Taxonomic representation of protein-coding 250

genes and relative abundances of sulfur energy– 70 m 200 m 80 m 150 m
metabolism genes in OMZ metagenomic data. (A)
300 Station 3
Most abundant taxa identified from annotations of protein-coding genes (in the NCBI-nr database) in Station 5
pyrosequencing reads from genomic DNA. Reads matching multiple putative sulfate-reducer reference Station 3 corrected
taxa (fig. S1 and table S2) are binned in a single category (black bar). (B) Abundances (hit counts per Station 5 corrected
350
gene) of dissimilatory sulfur metabolism genes, shown relative to the putative single copy per organism of
RNA polymerase subunit B (rpoB). Abundances per gene are normalized to gene length but not to copy Fig. 3. Sulfide-oxidation corrected and uncor-
number variation. dsr, dissimilatory sulfite reductase gene cluster; sox, sulfur oxidation gene cluster; rected rates of sulfate reduction at stations 3 and
aprBA, adenosine 5′-phosphosulfate (APS) reductase; aprM, APS reductase membrane anchor; FCSD, 5. Standard deviations represent variability during
flavocytochrome c sulfide dehydrogenase; SQR, sulfide-quinone reductase. scintillation counting (16).

REPORTS
okutanii (Vo) (20, 21)] and the endosymbiont- the current consensus that sulfate reduction in tion 5, nitrate reduction to nitrous oxide was also
related SUP05 pelagic lineage from Saanich OMZs will be active only when other more enhanced with sulfide addition (fig. S8). At sta-
Inlet (13) (Fig. 2A). These taxa increased in thermodynamically favorable electron accept- tion 3, N2 production from both nitrite and nitrate
abundance in January 2010 (austral summer) ors, such as nitrate and nitrite, are fully util- (at 75 m depth) increased, and in general, rates of
relative to samples collected from the same site ized (27). Although not the most favorable, sulfide oxidation and subsequent rates of nitro-
in August 2009 (late winter). In the January 2010 our calculations show that sulfate reduction is gen turnover were much higher at station 3 than
samples, SUP05-like sequences dominated the still a thermodynamically favorable process in at station 5. This is consistent with the higher
identifiable protein-coding gene pool (up to these OMZ waters (16). Previous observations rates of sulfate reduction at station 3 and a more
7.5% of all hits on NCBI-nr). The SUP05 meta- of pure cultures of sulfate-reducing bacteria that active sulfur cycle.
genome (13) was represented at high coverage, actively reduce sulfate in the presence of nitrate Admittedly, our added levels of sulfide and
matching 80% of all SUP05 genes (1169 of 1456) (28, 29) also support our observations of active subsequent rates of sulfide oxidation exceed in
with relatively uniform abundances and an sulfate reduction. situ levels. Nevertheless, our results demon-
average amino acid similarity of 70% (fig. S7). Rates of sulfate reduction were much higher strate the inherent capacity for active in situ
The sulfate-reducing population contributed to a at station 3 than at station 5. Indeed, corrected coupling between the sulfur and nitrogen cy-
lower but appreciable proportion of sequencing rates at station 3 match and even exceed rates of cles in OMZ zones of the marine water column.
reads and was represented by a diverse popula- denitrification and anammox (Table 1), implying This cycling is analogous to that observed at
tion that included Desulfatibacillum, Desulfobac- that sulfate reduction is an important pathway of the sulfide/nitrate interface in other strongly
terium, Desulfococcus, Syntrophobacter, and organic carbon mineralization at this site. Depth- redox stratified marine systems (13, 31, 32)

Desulfovibrio species (figs. S4 to S6). integrated corrected rates of sulfate reduction at and demonstrates that nitrite, N2, and N2O may
The prevalence of sulfur-metabolizing taxa station 3 are equivalent to about 2 mmol of C all be products of this coupling. We speculate
was paralleled by a strong representation of oxidized m–2 day–1, assuming 2 mol of organic that other nitrate-rich oxygen-free OMZs may
sulfur energy–metabolism genes. These genes carbon oxidized per mole of sulfate reduced. Sed- also house actively coupled sulfur and nitrogen
occur in various combinations across diverse iment trap studies at coastal and offshore stations cycles.
sulfur-utilizing taxa (22). Here, genes of the dis- about 200 km south of our study site (17) reveal
similatory sulfite reductase enzyme (dsr), the about 5.50 mmol m–2 day–1 of carbon mineral- References and Notes
sulfur oxidation (sox) gene complex mediat- ization within the OMZ waters from between 65 1. K. Wyrtki, Deep-Sea Res. 9, 11 (1962).
ing thiosulfate oxidation, and the adenosine and 300 m depth. If these rates apply to station 3, 2. J. D. Cline, F. A. Richards, Limnol. Oceanogr. 17,
5′-phosphosulfate (APS) reductase (apr) were then sulfate reduction would account for about 885 (1972).
3. D. B. Olson, G. L. Hitchcock, R. A. Fine, B. A. Warren,
present throughout the OMZ (Fig. 2B). Sev- 33% of the total organic carbon mineralization in Deep Sea Res. Part II Top. Stud. Oceanogr. 40,
eral of the proteins encoded by these genes, the OMZ waters. 673 (1993).
including dsr and apr enzymes, function in both Sulfate reduction may also contribute to the 4. S. E. Calvert, N. B. Price, Deep-Sea Res. 18, 505
oxidative and reductive pathways (23, 24). Here, ammonium requirements of other indigenous (1971).
5. J. M. Morrison et al., Deep Sea Res. Part II Top. Stud.
the majority of the sequences recovered in the bacteria participating in the anammox process.
Oceanogr. 46, 1903 (1999).
OMZ matched known sulfide oxidizers, which Indeed, the source of ammonium for anammox 6. V. Brüchert et al., Geochim. Cosmochim. Acta 67,
is consistent with the high abundance of the has proven elusive because insufficient ammonium 4505 (2003).
SUP05 group. Putative sulfide-oxidizing and is liberated during organic matter decomposition 7. S. E. Bulow, J. J. Rich, H. S. Naik, A. K. Pratihary,
sulfate-reducing taxa constituted 62.0 and 2.2% by denitrification to drive measured anammox B. B. Ward, Deep Sea Res. Part I Oceanogr. Res. Pap. 57,
384 (2010).
of top hits to aprA sequences, respectively, with rates in many OMZ waters (8, 9). In a partial res- 8. B. Thamdrup et al., Limnol. Oceanogr. 51, 2145
the remainder matching aprA genes of the al- olution to this dilemma, the dissimilatory reduc- (2006).
phaproteobacterial genus Pelagibacter, whose tion of nitrate to ammonium and the heterotrophic 9. P. Lam et al., Proc. Natl. Acad. Sci. U.S.A. 106,
function in sulfide oxidation is not yet clear reduction of nitrate to nitrite have been identified 4752 (2009).
10. L. A. Codispoti et al., Sci. Mar. 65, 85 (2001).
(25) (fig. S6). Overall, the metagenomic data as significant ammonium sources in OMZ waters 11. J. N. Galloway et al., Biogeochemistry 70, 153
suggest a prevalent summer OMZ community off the Peruvian coast (9) (the latter due to the (2004).
of both oxidative and reductive sulfur-cycling ammonium liberated during mineralization of 12. H. Stevens, O. Ulloa, Environ. Microbiol. 10, 1244
bacteria. organic matter). But even these extra sources do (2008).
13. D. A. Walsh et al., Science 326, 578 (2009).
Although our metagenomic libraries sug- not account for all of the ammonium demand. 14. K. W. Finster, K. U. Kjeldsen, Antonie van Leeuwenhoek
gest an active sulfur cycle, it is cryptic, with no From our sulfate reduction rates at station 3, 97, 221 (2010).
obvious in situ chemical expression. To explore sulfate reduction produces a total of about 0.30 15. R. C. Dugdale, J. J. Goering, R. T. Barber, R. L. Smith,
the geochemical importance of the sulfur cycle mmol m–2 day–1, assuming a 6.6/1 ratio between T. T. Packard, Deep-Sea Res. 24, 601 (1977).
16. See supporting material on Science Online.
and possible links to nitrogen cycling, we mea- carbon oxidation and ammonium liberation (30).
17. R. Escribano et al., Deep Sea Res.
sured rates of sulfate reduction with 35SO42– This would contribute 22% of the ammonium Part II Top. Stud. Oceanogr. 51, 2389 (2004).
(26). We subdued the immediate reoxidation needs for anammox at station 3 (Table 1). At 18. P. Lavin, B. González, J. F. Santibáñez, D. J. Scanlan,
of sulfide produced during sulfate reduction station 5, sulfate reduction would contribute only O. Ulloa, Environ. Microbiol. Rep. 10.1111/j.1758-
by adding 10 to 13 mM unlabeled sulfide to about 8% of the ammonium needs for anammox, 2229.2010.00167.x (2010).
19. N. P. Revsbech et al., Limnol. Oceanogr. Methods
trap any radiolabeled sulfide from sulfate re- underlining the complexity of the nitrogen cycle 7, 371 (2009).
duction (16). Radiolabeled sulfate was added and the variability of ammonium sources for 20. I. L. G. Newton et al., Science 315, 998 (2007).
within 10 hours of sample collection. In some anammox (9). 21. H. Kuwahara et al., Curr. Biol. 17, 881 (2007).
cases, our added unlabeled sulfide was sub- We also explored the dynamics of sulfide 22. C. Dahl, C. G. Friedrich, Eds., Microbial Sulfur Metabolism
(Springer-Verlag, Heidelberg, 2008).
stantially oxidized during the incubations (16), oxidation in these waters and the relationship 23. B. Meyer, J. Kuever, Microbiology 153, 3478 (2007).
implying that radiolabeled sulfide must also between sulfide oxidation and the nitrogen cycle 24. B. Meyer, J. Kuever, Microbiology 153, 2026 (2007).
have been oxidized and lost as a result. After (16). In parallel with our sulfate reduction rate 25. B. Meyer, J. Kuever, Appl. Environ. Microbiol. 73,
estimating the loss of radiolabeled sulfide due determinations, we incubated OMZ water from 7664 (2007).
26. D. B. Albert, C. Taylor, C. S. Martens, Deep-Sea Res. 42,
to sulfide oxidation, we corrected the rates to two depths at both stations 3 and 5 with and with- 1239 (1995).
obtain estimates of the gross sulfate reduction out added sulfide. Sulfide oxidation was strongly 27. P. N. Froelich et al., Geochim. Cosmochim. Acta 43,
rates (16) (Fig. 3). Our findings contrast with coupled to nitrate reduction to nitrite, and at sta- 1075 (1979).

REPORTS
28. T. Dalsgaard, F. Bak, Appl. Environ. Microbiol. 60, 33. We thank the captain and crew of the Agor Vidal Supporting Online Material
291 (1994). Gormaz from the Chilean Navy for their kind support, www.sciencemag.org/cgi/content/full/science.1196889/DC1
29. R. G. L. McCready, W. D. Gould, F. D. Cook, Arch. and the Agouron Institute, the Danish National Research Materials and Methods
Microbiol. 135, 182 (1983). Foundation, the Gordon and Betty Moore Foundation, Figs. S1 to S8
30. A. C. Redfield, B. H. Ketchum, F. A. Richards, in and the Chilean Fondap Program for financial support. Tables S1 to S4
The Sea, N. M. Hill, Ed. (Academic Press, London, 1963), Additional thanks to G. Alarcón, G. Friederich, and References
vol. 2, pp. 26–77. J. Jennings for operational and experimental support.
31. M. M. Jensen, J. Petersen, T. Dalsgaard, B. Thamdrup, The genome sequence data are accessible on NCBI's 24 August 2010; accepted 28 October 2010
Mar. Chem. 113, 102 (2009). Sequence Read Archive via accession number Published online 11 November 2010;
32. G. Lavik et al., Nature 457, 581 (2009). SRA025088. 10.1126/science.1196889
Dynamical Response of the Tropical fragmented to test for any relation to persistent
solar forcing before the past millennium. Few sea
Pacific Ocean to Solar Forcing

surface temperature (SST) reconstructions from
well-placed tropical Pacific sediment cores have suf-
ficient temporal resolution to address this question.
During the Early Holocene Sediment core composite MV99-GC41/PC14
was raised from a water depth of 540 m on the
floor of Soledad Basin, which is located off the
Thomas M. Marchitto,1* Raimund Muscheler,2 Joseph D. Ortiz,3

west coast of Baja California Sur, Mexico (25.2°N,
Jose D. Carriquiry,4 Alexander van Geen5
112.7°W), and has an effective sill depth of 290 m
We present a high-resolution magnesium/calcium proxy record of Holocene sea surface temperature (8). Although this site is just outside of the tropical
(SST) from off the west coast of Baja California Sur, Mexico, a region where interannual band, modern conditions here are strongly telecon-
SST variability is dominated today by the influence of the El Niño–Southern Oscillation (ENSO). nected to ENSO. The modern annual cycle of SST
Temperatures were lowest during the early to middle Holocene, consistent with documented has an amplitude of ~8°C on average, with mini-
eastern equatorial Pacific cooling and numerical model simulations of orbital forcing into a La mum temperatures during spring, the season of
Niña–like state at that time. The early Holocene SSTs were also characterized by millennial-scale strongest coastal upwelling (9, 10). Yet interannual
fluctuations that correlate with cosmogenic nuclide proxies of solar variability, with inferred variability in SST is much more strongly depen-
solar minima corresponding to El Niño–like (warm) conditions, in apparent agreement with the dent on ENSO than on local upwelling winds. Over
theoretical “ocean dynamical thermostat” response of ENSO to exogenous radiative forcing. the 30-year period covered by satellite observa-
tions, the Niño 3 index explains 37% of the month-
he influence of solar variability on Earth’s sing tendency for the atmospheric circulation ly SST anomaly near Soledad Basin (correlation
T climate over centennial to millennial time

scales is the subject of considerable debate.
The change in total solar irradiance over recent
itself to strengthen under reduced radiative forc-
ing (7).
ENSO is a leading source of interannual climate
coefficient r = 0.61 maximum correlation with a
2-month lag), whereas the local upwelling index
explains only 2% (r = –0.16 with zero lag) (Fig. 1).
11-year sunspot cycles amounts to <0.1%, but variability over large regions of the globe, so it is ENSO is crucial for SST because the regional
greater changes at ultraviolet wavelengths (1) crucial to gain an improved understanding of its thermocline deepens during El Niño years, so that
may have substantial impacts on stratospheric ozone past responses to external forcing at various time even with vigorous local upwelling the ascending
concentrations, thereby altering both stratospheric scales. Tropical fossil corals provide the most re- waters are warmer than during La Niña or neutral
and tropospheric circulation patterns (2). Estimates liable means for reconstructing ENSO conditions years (11). Recent spring SST minima have ranged
of the secular increase in total irradiance since the from the past (5), but the record is currently too from 17°C during strong La Niñas to 20°C during
late 17th century Maunder sunspot minimum
range from ~0.05 to 0.5% (1). Values in the middle
of this range are sufficient to force the intermediate-
complexity Zebiak-Cane model of El Niño–
Southern Oscillation (ENSO) dynamics into a more
El Niño–like state during the Little Ice Age (A.D.
~1400 to 1850) (3), a response dubbed the “ocean
dynamical thermostat” because negative (or posi-
tive) radiative forcing results in dynamical ocean
warming (or cooling, respectively) of the eastern
tropical Pacific (ETP) (4). This model prediction is
supported by paleoclimatic proxy reconstructions
over the past millennium (3, 5, 6). In contrast, fully
coupled general circulation models (GCMs) lack
a robust thermostat response because of an oppo-
1
Department of Geological Sciences and Institute of Arctic and
Alpine Research, University of Colorado, Boulder, CO 80309, USA.
2
Department of Earth and Ecosystem Sciences, Lund University,
S-223 62 Lund, Sweden. 3Department of Geology, Kent State
University, Kent, OH 44242, USA. 4Department of Environ-
mental Geosciences, Universidad Autónoma de Baja California,
Ensenada, Baja California 22830, Mexico. 5Lamont-Doherty Earth Fig. 1. (A) Monthly SST anomaly for the 1° grid cell situated over Soledad Basin (black) (9), compared to
Observatory, Columbia University, Palisades, NY 10964, USA. the monthly Niño 3 SST index on the same vertical scale but lagged by 2 months (gray) (9) and (B) the
*To whom correspondence should be addressed. E-mail: local (24°N, 113°W) monthly upwelling index (offshore Ekman transport computed from wind stress)
tom.marchitto@colorado.edu anomaly (10), shown inverted.

REPORTS
El Niños. Surface warming off the coast of Baja Our Mg/Ca-based SST reconstruction indicates (21, 22), increased atmospheric baroclinicity (23),
California Sur can be further enhanced under El that early to middle Holocene [~4 to 10 thousand and/or intensification of the Asian summer mon-
Niño conditions by the northeastward expansion years ago (ka)] spring temperatures were ~1°C soon (24). Although local Baja California Sur
of subtropical surface waters that effectively block cooler, on average, than during the rest of the past upwelling-favorable winds may also respond pos-
the admixture of southward-flowing subarctic 14 ky (Fig. 2). By analogy with modern ETP dy- itively to orbital forcing, the maximum in spring
(California Current) waters (12). namics, we suggest that the cooling is best ex- insolation occurred much earlier (~15 ka) than our
An age model based on 22 calibrated accelerator plained by a shallower thermocline and a reduced observed cooling. In support of more distant
mass spectrometry radiocarbon dates (fig. S1) re- influence of subtropical surface waters. This sce- teleconnections, the shift toward warmer upwell-
veals that the composite core spans the past 13.9 nario is consistent with previous suggestions of a ing conditions just before 4 ka is close to the timing
thousand years (ky) with an average sedimentation more La Niña–like state during the early to middle of widespread evidence for an abrupt and perma-
rate of >1 m ky−1. During the Holocene, the sedi- Holocene. Mg/Ca reconstructions from the equa- nent weakening of the Asian summer monsoons
ments are laminated, indicating negligible bioturba- torial Pacific indicate an enhanced zonal SST gra- (25), which may have helped the Pacific relax into
tion under low-O2 conditions on the sea floor (8). dient at this time, with a colder eastern cold tongue a more El Niño–like state (24).
Preservation of planktonic foraminifera is excellent and warmer western warm pool (Fig. 2) (15, 16). In addition to orbital-scale changes, the Soledad
throughout the core, with glassy tests and spines At Baja California Sur, the cooling may have been Basin Mg/Ca record displays strong variance at
commonly present. We measured the SST proxy amplified by a strengthened California Current millennial time scales, as seen in the five-depth
Mg/Ca in the planktonic foraminifer Globigerina (17). In contrast, alkenone-based SST reconstruc- (nominally 200 to 300 years) running mean of the
bulloides (13), which lives at the sea surface primar- tions from both Baja California Sur (18) and the Mg/Ca data (Fig. 3). Before the data gap at ~5.9 to

ily during the spring peak upwelling season along cold tongue (19) do not exhibit a mid-Holocene 6.5 ka, sample density is high enough (48 mea-
this margin (14). Samples were nominally spaced at cooling. This disparity might be due to a summer/ surements per thousand years), and the relative
5-cm intervals and contained 30 to 60 foraminifera fall habitat for coccolithophores, resulting in an noise low enough, to give us confidence that the
each, so each measurement theoretically aver- overprinting of La Niña–like cooling by orbitally smoothed record captures a meaningful millennial-
ages 30 to 60 month-long (foraminiferal lifespan) forced seasonal radiative heating (20). scale climate history. We observe five cold inter-
upwelling-season snapshots spread over roughly Numerical models of varying complexity have vals between ~7 and 11 ka, with roughly 1-ky
a decade (1-cm sample width), with ~50-year spacing simulated a La Niña–like cooling of the ETP dur- spacing. In light of model- and proxy-based results
between samples. Although not capable of resolving ing the early to middle Holocene in response to supporting a solar influence on ENSO over the past
the typical ENSO periodicities of 2 to 7 years, this enhanced boreal summer/fall insolation. Easterly millennium (3, 6), we compare the smoothed record
sampling is sufficient to detect any multicentennial/ winds strengthen because of zonally asymmetric to cosmogenic nuclide proxies for solar activity.
millennial-scale changes in spring SSTs. heating of the tropical ocean and atmosphere For the period before the beginning of sunspot
observations in A.D. 1610, reconstructions of solar
variability are based on the cosmogenic nuclides
Fig. 2. SST reconstruc- 14
tions based on Mg/Ca in C (recorded in tree rings) (26) and 10Be (preserved
surface-dwelling plank- in polar ice cores) (27, 28). An active Sun generates
tonic foraminifera from a higher total irradiance and a stronger interplan-
the western equatorial Pa- etary magnetic field that helps to shield Earth from
cific warm pool (15) (gold the galactic cosmic rays that produce 14C and 10Be
and red), eastern equa- in the atmosphere. However, the relation between
torial Pacific cold tongue solar irradiance and cosmic-ray shielding is not well
(16) (green and pink), and understood over long time scales. In addition, at-
Soledad Basin (this study) mospheric levels of 14C may be affected by changes
(blue). Symbols denote in- in Earth’s carbon cycle, 10Be fluxes to ice sheets may
dividual measurements, and be influenced by local climate, and the production
lines trace the mean at each rates of both nuclides are modulated by long-term
depth. Along the equator, variations in Earth’s magnetic field. Nevertheless, the
G. ruber and G. sacculifer shared variance of high-pass–filtered (to correct for
are believed to represent presumed slow variations in the geomagnetic field)
mean annual conditions, 14
C and 10Be records can be taken as an indication
whereas at Soledad Basin, of fluctuations in solar activity over the Holocene.
G. bulloides reflects spring Each of the early Holocene millennial-scale
upwelling. The solid blue
coolings at Soledad Basin corresponds to an inferred
circle on the y axis denotes
millennial-scale increase in solar activity (decreased
the modern average SST
during the coldest month cosmogenic nuclides) (Fig. 3). Cross-wavelet anal-
of the year (spring peak ysis of the unsmoothed data indicates significant
upwelling) at Soledad Basin common power (in phase) between Mg/Ca and the
(9). Vertical gray dashed nuclides in the ~800- to 1000-year band (fig. S2).
1
lines bracket the early to After performing a ~250-year smoothing and 1800
middle Holocene interval of year–1 high-pass filtering of each record, Mg/Ca
increased zonal SST gradi- (before the ~5.9-6.5 ka data gap) correlates signif-
ent. BP, before the present. icantly with 14C production (r = 0.49, p = 0.02, with
50-year lag on Mg/Ca) and reasonably well with
10
Be flux (r = 0.41, p = 0.07, with 100-year lag on
Mg/Ca) (Fig. 4) (13). These correlations are based
on completely independent age models. Given the
strong link between this region and ENSO var-
iability today, we suggest that this correspondence

REPORTS
provides support for the idea that the ocean dynam- correlation between Mg/Ca and the solar proxies strengthening during early Holocene solar max-
ical thermostat (4) acts effectively at centennial- may be due to the lower sample density (less than ima, suggesting that an Asian teleconnection may
millennial time scales (3). Indeed, these early half that of the earlier interval) and/or the reduced have helped push the Pacific into a La Niña–like
Holocene oscillations between warm El Niño– amplitude of inferred solar variability, in line with state during these intervals, or vice versa. Although
like and cool La Niña–like conditions were re- the model prediction (29). the period of overlap is relatively short, the smoothed
cently predicted by solar-forcing experiments The observed sensitivity of the tropical Pacific speleothem records bear strong resemblance to the
using the Zebiak-Cane model (29). Although it is to modest radiative forcing may have been achieved Soledad Basin SST history (China: r = 0.74, p =
possible that local upwelling-favorable winds through positive feedback with other regions that 0.01; Oman: r = 0.76, p = 0.003) (Fig. 4). It is
responded directly to positive solar forcing and also responded to solar variability. La Niña has interesting to note that during the interval of greatest
amplified the cool SST signal, we argue, on the historically been associated with stronger sum- mismatch between Soledad Basin and the solar
basis of modern observations (Fig. 1), that the mer monsoons over Asia, as both are linked to proxies, the cave records agree with our SST his-
impact would have been minor without a con- strong easterlies over the tropical Pacific (30). tory: At 8.2 ka, the ETP was in an El Niño–like
comitant La Niña–like shoaling of the regional Oxygen isotopes from speleothems in southern state and the monsoons were weak, despite the in-
thermocline. Between ~2.2 and 5.9 ka, the poor China (31) and Oman (32) indicate monsoon ferred secular increase in solar activity. This appar-
ent anomaly may be attributed to the well-known
“8.2-ka event,” during which a large Laurentide
meltwater discharge is believed to have cooled
the North Atlantic and Eurasia, thereby weaken-

ing the Asian summer monsoons (33), which pos-
sibly fostered El Niño–like conditions in the ETP.
Additionally, Bond et al. (34) showed that
there was increased ice-rafted debris (IRD) de-
livery from the Labrador and Nordic Seas into
the North Atlantic during inferred Holocene solar
minima. Their stacked IRD record correlates with
Soledad Basin SSTs even more strongly than do
the solar proxies (r = 0.70, p < 0.001, with 100-
year lag on Mg/Ca) (Fig. 4). A cold North
Atlantic during solar minima may have reinforced
ETP warming through either the Asian monsoon
linkage (24) or a southward shift of the intertrop-
ical convergence zone (16). Closure of this hypo-
thetical positive feedback loop has been suggested
to occur through an El Niño–forced shift in the pre-
Fig. 3. Soledad Basin Mg/Ca record compared to solar proxies 14C and 10Be (13). (A) G. bulloides Mg/Ca mean vailing winds that deliver drift ice from the Nordic
and standard deviation at each depth (gray) with five-depth running mean (blue) and associated 2s uncertainty
Seas into the North Atlantic (29).
estimate (light blue). Open black symbols at the top of the figure denote calibrated 14C ages with 1s errors.
Persistent, decadal-scale droughts over the west-
Diamonds are from GC41, and circles are from PC14. (B) Holocene tree-ring–derived D14C (26) converted to
14
C production rate, with high values corresponding to low inferred solar activity. Data were high-pass ern United States have been linked to La Niña–like
1
filtered at 1800 year−1 to remove secular changes that are probably related to Earth’s magnetic field (gold), SST patterns in the ETP during the instrumental
1
band-pass filtered at 1800 1
to 500 year−1 as in (34) (pink), and smoothed with a 250-year running mean period (35). Tree-ring reconstructions extend this
1
before 1800 year−1 high-pass filtering (black). (C) Holocene ice core 10Be flux (27, 28) filtered as in (B), except relationship back to the Medieval Warm Period
that the gray curve is a 18001
to 501
year−1 band pass that additionally eliminates subdecadal-scale noise. (MWP, A.D. ~900 to 1300), which was seemingly
Green vertical lines indicate Soledad Basin cold intervals that correspond to times of increased solar activity. characterized by positive solar forcing, inactive
tropical volcanism, La Niña–like conditions, and
multidecadal “megadroughts” (3, 5, 6, 35). The first
high-resolution, continuous Holocene speleothem
proxy precipitation record from the southwestern
United States documents a robust connection be-
tween inferred solar-activity maxima and dry con-
ditions, which may be explained by solar forcing of
La Niña–like states (36). Taken together with our
SST record, these observations are consistent with
solar-induced dynamical cooling of the ETP and
provide predictions for millennial-scale fluctua-
tions in the hydrologic balance over the western
United States during the early Holocene.
GCMs fail to reproduce the La Niña–like nature
of the MWP because the ocean thermostat mech-
anism is either absent or dampened by atmospheric
effects in such models (6, 7). If our observations
Fig. 4. Likely teleconnected climatic and solar proxy records spanning the early Holocene, each smoothed at are supported by future SST reconstructions from
~250 years and high-pass filtered at 18001
year–1 (13). Records are Soledad Basin G. bulloides Mg/Ca (this study) the equatorial Pacific, then it is possible that the
(blue), tree-ring–derived (26) 14C production rate (gold), ice core 10Be flux (27, 28) (gray), Dongge Cave sensitivity of the climate system to solar forcing is
(southern China) stalagmite d18O (31) (light blue), Hoti Cave (Oman) stalagmite d18O (32) (green), and North underestimated by current GCMs. The nature
Atlantic stack of IRD petrologic tracers (34) (red). All records are on their independent and untuned age models. of the climate response appears to be one of

REPORTS
shifting atmosphere-ocean circulation patterns, 13. Methods are available as supporting material on Science 30. P. J. Webster et al., J. Geophys. Res. 103, 14451
with the tendency for global radiative surface Online. (1998).
14. L. R. Sautter, R. C. Thunell, Paleoceanography 6, 307 31. Y. J. Wang et al., Science 308, 854 (2005).
warming being countered by the ocean dynam- (1991). 32. U. Neff et al., Nature 411, 290 (2001).
ical thermostat. 15. L. Stott et al., Nature 431, 56 (2004). 33. R. B. Alley et al., Geology 25, 483 (1997).
16. A. Koutavas, P. B. deMenocal, G. C. Olive, J. Lynch-Stieglitz, 34. G. Bond et al., Science 294, 2130 (2001); 10.1126/
References and Notes Geology 34, 993 (2006). science.1065680.
17. J. A. Barron, D. Bukry, Palaeogeogr. Palaeoclimatol. 35. E. R. Cook, R. Seager, M. A. Cane, D. W. Stahle,
1. J. L. Lean, Geophys. Res. Lett. 27, 2425 (2000).
Palaeoecol. 248, 313 (2007). Earth Sci. Rev. 81, 93 (2007).
2. G. A. Meehl, J. M. Arblaster, K. Matthes, F. Sassi, H. van Loon,
18. T. D. Herbert et al., Science 293, 71 (2001); 10.1126/ 36. Y. Asmerom, V. Polyak, S. Burns, J. Rassmussen, Geology
Science 325, 1114 (2009).
science.1059209. 35, 1 (2007).
3. M. E. Mann, M. A. Cane, S. E. Zebiak, A. Clement, J. Clim.
19. A. Koutavas, J. P. Sachs, Paleoceanography 23, PA4205 37. We thank P. Cappa, D. Lopez, D. Weller, and C. Wolak for
18, 447 (2005).
(2008). laboratory assistance. This manuscript was improved by
4. A. C. Clement, R. Seager, M. A. Cane, S. E. Zebiak, J. Clim. 20. G. Leduc, R. Schneider, J. H. Kim, G. Lohmann, Quat. Sci.
9, 2190 (1996). comments from B. Fox-Kemper and S. Lehman. This work was
Rev. 29, 989 (2010). supported by collaborative NSF grants OCE-0214221 and
5. K. M. Cobb, C. D. Charles, H. Cheng, R. L. Edwards, Nature 21. A. C. Clement, R. Seager, M. A. Cane, Paleoceanography
424, 271 (2003). OCE-0214646. R.M. is supported by the Royal Swedish
15, 731 (2000).
6. M. E. Mann et al., Science 326, 1256 (2009). Academy of Sciences through a grant financed by the Knut
22. B. L. Otto-Bliesner, E. C. Brady, S.-I. Shin, Z. Liu, C. Shields,
7. G. A. Vecchi, A. Clement, B. J. Soden, Eos 89, 81 (2008). and Alice Wallenberg Foundation. Data are available at the
Geophys. Res. Lett. 30, 2198 (2003).
8. A. van Geen et al., Paleoceanography 18, 1098 (2003). NOAA National Climatic Data Center for Paleoclimatology.
23. A. B. G. Bush, Geophys. Res. Lett. 26, 99 (1999).
9. R. W. Reynolds, N. A. Rayner, T. M. Smith, D. C. Stokes, 24. Z. Liu, J. Kutzbach, L. Wu, Geophys. Res. Lett. 27, 2265
W. Q. Wang, J. Clim. 15, 1609 (2002). (2000). Supporting Online Material
10. F. B. Schwing, M. O’Farrell, J. M. Steger, K. Baltz, 25. C. Morrill, J. T. Overpeck, J. E. Cole, Holocene 13, 465 www.sciencemag.org/cgi/content/full/330/6009/1378/DC1

“Coastal Upwelling Indices West Coast of North America (2003). Methods
1946-96,” NOAA Tech. Memo. NOAA-TM-NMFS-SWSFC-231 26. P. J. Reimer et al., Radiocarbon 46, 1029 (2004). SOM Text
[National Oceanic and Atmospheric Administration 27. R. C. Finkel, K. Nishiizumi, J. Geophys. Res. Oceans 102, Figs. S1 to S3
(NOAA), Washington, DC, 1996]. 26699 (1997). Table S1
11. F. B. Schwing, T. Murphree, L. deWitt, P. M. Green, 28. M. Vonmoos, J. Beer, R. Muscheler, J. Geophys. Res. References
Prog. Oceanogr. 54, 459 (2002). Space Phys. 111, A10105 (2006).
12. R. Durazo, T. R. Baumgartner, Prog. Oceanogr. 54, 7 29. J. Emile-Geay, M. Cane, R. Seager, A. Kaplan, P. Almasi, 9 July 2010; accepted 29 October 2010
(2002). Paleoceanography 22, PA3210 (2007). 10.1126/science.1194887
Plasticity of Animal Genome high mutation rate per generation (m) (3). Sequence
comparisons among populations from the eastern
Architecture Unmasked by Rapid

Pacific and eastern Atlantic and within the latter
revealed low dN/dS values (dN, rate of substitutions
at nonsilent sites; dS, rate of substitutions at silent
Evolution of a Pelagic Tunicate sites) consistent with strong purifying selection, as
expected for large populations (3). In 17 of 18
France Denoeud,1,2,3 Simon Henriet,4* Sutada Mungpakdee,4* Jean-Marc Aury,1,2,3*
Corinne Da Silva,1,2,3* Henner Brinkmann,5 Jana Mikhaleva,4 Lisbeth Charlotte Olsen,4 1
Commissariat à l’Énergie Atomique, Institut de Génomique, Gen-
Claire Jubin,1,2,3 Cristian Cañestro,6,24 Jean-Marie Bouquet,4 Gemma Danks,4,7 Julie Poulain,1,2,3 oscope, Evry, France. 2CNRS, UMR 8030, Evry, France. 3Université
d’Evry, Evry, France. 4Sars International Centre for Marine Mo-
Coen Campsteijn,4 Marcin Adamski,4 Ismael Cross,8 Fekadu Yadetie,4 Matthieu Muffato,9 lecular Biology, University of Bergen, Bergen, Norway. 5Départe-
Alexandra Louis,9 Stephen Butcher,10 Georgia Tsagkogeorga,11 Anke Konrad,22 ment de Biochimie, Université de Montréal, Montréal, Canada.
Sarabdeep Singh,12 Marit Flo Jensen,4 Evelyne Huynh Cong,4 Helen Eikeseth-Otteraa,4 6
Institute of Neuroscience, University of Oregon, Eugene, OR 97403,
Benjamin Noel,1,2,3 Véronique Anthouard,1,2,3 Betina M. Porcel,1,2,3 Rym Kachouri-Lafond,13 USA. 7Bergen Center for Computational Science, University of
Bergen, Bergen, Norway. 8Laboratorio de Genética, Universidad
Atsuo Nishino,14 Matteo Ugolini,4 Pascal Chourrout,15 Hiroki Nishida,14 Rein Aasland,16 de Cádiz, Cádiz, Spain. 9Dyogen Lab, Institut de Biologie de l’ENS
Snehalata Huzurbazar,12 Eric Westhof,13 Frédéric Delsuc,11 Hans Lehrach,17 Richard Reinhardt,17 (IBENS), CNRS-UMR8197, Ecole Normale Supérieure, Paris,France.
Jean Weissenbach,1,2,3 Scott W. Roy,18 François Artiguenave,1,2,3 John H. Postlethwait,6 10
Department of Biology, University of Iowa, Iowa City, IA 52242–
J. Robert Manak,10 Eric M. Thompson,4,19 Olivier Jaillon,1,2,3 Louis Du Pasquier,20 Pierre Boudinot,21 1324, USA. 11Laboratoire de Paléontologie, Phylogénie et Paléo-
biologie, Institut des Sciences de l’Evolution, UMR 5554–CNRS,
David A. Liberles,22 Jean-Nicolas Volff,23 Hervé Philippe,5 Boris Lenhard,4,7,19 Hugues Roest Crollius,9
Université Montpellier II, Montpellier, France. 12Department of
Patrick Wincker,1,2,3† Daniel Chourrout4† Statistics, University of Wyoming, Laramie, WY 82071, USA. 13Institut
de Biologie Cellulaire et Moléculaire du CNRS, Université de
Genomes of animals as different as sponges and humans show conservation of global architecture. Strasbourg, Strasbourg, France. 14Department of Biological Sciences,
Here we show that multiple genomic features including transposon diversity, developmental gene Osaka University, Osaka, Japan. 15Centre Hospitalier d’Albi, Albi,
repertoire, physical gene order, and intron-exon organization are shattered in the tunicate France. 16Department of Molecular Biology, University of Bergen,
Bergen, Norway. 17Vertebrate Genomics, Max Planck Institute for
Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Molecular Genetics, Berlin, Germany. 18National Center for Bio-
Ancestral architecture of animal genomes can be deeply modified and may therefore be largely technology Information, National Library of Medicine, National
nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of Institutes of Health, Bethesda, MD 20894, USA. 19Department of
genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain. Biology, University of Bergen, Bergen, Norway. 20Institute of Zoology
and Evolutionary Biology, University of Basel, Basel, Switzerland.
21
Institut National de la Recherche Agronomique (INRA), Virologie et
unicates, viewed as the closest living relatives generations (2). Unique among tunicates, it has
T of vertebrates, were probably simplified from

more complex chordate ancestors (1). Lar-
vacean tunicates represent the second most abun-
separate sexes. We sequenced its genome with high-
coverage shotgun reads (14X) using males resulting
from 11 successive full-sib matings (figs. S1 and S2
Immunologie Moléculaires, Jouy-en-Josas, France. 22Department of
Molecular Biology, University of Wyoming, Laramie, WY 82071,
USA. 23Institut de Génomique Fonctionnelle de Lyon, UMR 5242–
CNRS/INRA/Université Claude Bernard Lyon 1/Ecole Normale
Supérieure, Ecole Normale Supérieure de Lyon, Lyon, France.
dant component of marine zooplankton and filter and tables S1 to S3) (3). Two distinct haplotypes 24
Departament de Genètica, Universitat de Barcelona, Spain.
small particles by their gelatinous house. Oiko- were retained, despite inbreeding. Their compar-
*These authors contributed equally to this work.
pleura dioica is the most cosmopolitan larvacean, ison yielded a high estimate of population muta- †To whom correspondence should be addressed. E-mail:
has a very short life cycle (4 days at 20°C), and tion rate (q = 4Ne m = 0.0220) that is consistent Daniel.Chourrout@sars.uib.no (D.C.); pwincker@genoscope.
can be reared in the laboratory for hundreds of with a large effective population size (Ne) and/or a cns.fr (P.W.)

REPORTS
phylogenetic trees constructed with 26 metazoan Two exceptions to global compaction are par- ulation of Oikopleura genes has relatively large
genomes and nine independent data sets, Oiko- ticularly interesting, as they may illustrate where introns and intergenic spaces (Fig. 1B). It is en-
pleura shows the fastest protein evolution, and even excessive reduction is harmful. First, a small pop- riched for developmentally regulated transcription
higher evolutionary rates are observed for mito-
chondrial genes that are heavily modified by oligo- Table 1. Minimal immune system predicted from the Oikopleura genome. Numbers of genes or domains in
dT insertions (figs. S3 to S6 and tables S4 to S6) families encoding potential immunity factors. D.m., Drosophila melanogaster; S.p., Strongylocentrotus
(3). Key components of DNA repair, especially in purpuratus; O.d., Oikopleura dioica; C.i., Ciona intestinalis; B.f., Branchiostoma floridae; P.m., Petromyzon
the nonhomologous end-joining pathway, were not marinus; H.s., Homo sapiens. TLR, Toll-like receptor; NLR, NOD-like receptor; SRCR, scavenger receptor
detected in the genome (fig. S7 and table S7) (3). cysteine-rich; PGRP, peptidoglycan recognition protein; RIG-I, retinoic acid–inducible gene–I; IgSF-ITIM,
Coincident rapid evolution of nuclear and mito- immunoglobulin superfamily domain with immunoreceptor tyrosine inhibitory motif; DEATH-TIR, DEATH
chondrial genomes may also reflect a highly muta- superfamily members with Toll/interleukin-1–receptor domain; SARM1, SAM- and ARM-containing protein
genic context at the ocean surface. 1; TIRAP, Toll/interleukin-1–receptor domain-containing adapter protein; TICAM2, Toll/interleukin-1–
receptor domain-containing adapter molecule; PLA2, phospholipase A2. ND, not determined.
At 70 megabases with 18,020 predicted genes,
the Oikopleura genome is unusually compact. Introns D.m. S.p. O.d. C.i. B.f. P.m. H.s.
are very small (peak at 47 base pairs, 2.4% > 1 kb),
as are intergenic spaces, partly because of numer- Sensors
ous operons (fig. S8 and table S8) (3). Genes outside TLR 9 222 1 3 48 21 10
operons are also densely packed (53% of intergenic NLR 0 203 0 20 92 140–220 20

distances < 1 kb). Even compared with other SRCR 14 218 1 81 270 287 81
compact genomes (4), the density of transposable PGRP 15 5 4 6 >20 ND 6
elements (TEs) is low. Most pan-animal TE super- RIG-I–like helicases 0 12 0 ND 7 ND 3
families are absent in Oikopleura, and only two C-type lectins 32 104 31 120 1215 ND 81
species-specific clades of retrotransposons (5) have IgSF-ITIM >3 ND 5 >6 >5 >3 >50
diversified. A massive purge of ancient TEs can be Adaptors
invoked, but TEs currently present in the genome MyD88-like (DEATH-TIR) 1 4 0 1 4 ND 1
show multiple signs of activity (figs. S9 to S16) (3). SARM1-like, TIRAP-like,
The low copy number of each element and the un- TICAM2-like 1 15 0 >2 12 ND 3
even genome distribution of the main TE clades sug- Potential effector
gest tight control of their proliferation (Fig. 1A) (3). PLA2 8 65 128 7 >7 ND 11
Fig. 1. Genome compaction features. (A)

Chromosome regions assembled with phys-
ical links and genetic markers. The location
of TEs is indicated with horizontal lines
(lines on the left sides, DNA transposons;
lines on right sides, short lines for long
terminal repeat–retrotransposons and long
lines for long interspersed elements). (B)
Distribution of gene models over 10% abun-
dance classes of intron size and upstream
intergenic distance for 8812 nonoperon genes
(left) and for 189 developmentally regulated
genes, mainly transcription factors (right). (C)
Conserved elements revealed in genome
alignments of Atlantic and Pacific ocean
populations of O. dioica: density of con-
served blocks (top), gene annotation (mid-
dle), and perfectly conserved elements >100
bp (bottom gray line) (blue, Norway versus
northwest America; red, Norway versus Japan).
(D) Giant Y genes and their testis expression
revealed by reverse transcription polymer-
ase chain reaction and in situ hybridization.
hpf, hours post fertilization; ctrl, control. The
arrowhead indicates the giant gene expres-
sion product.

REPORTS
factor genes that are long in other genomes be- ments of higher sequence conservation in non- Second, Mendelian analysis showed that sex in
cause of an abundance of regulatory elements (6). coding regions than in exons, suggestive of a rich Oikopleura is genetically determined (fig. S20
Regulatory-element sequences can be highly con- regulatory content (Fig. 1C and fig. S17) (3). In- and table S10) (3), and we could reconstruct large
served, though rarely across phyla, and Oikopleura terestingly, in a revolution of massive intron loss X and Y chromosomes (Fig. 1A). Seven genes on
homologs of vertebrate conserved elements were (see below), Oikopleura retained large introns more the Y chromosome, all expressed in the testis during
not detected (3). However, a comparison of genes often than small ones, and the ratio of ancestral to spermatogenesis, have giant introns (Fig. 1D). Their
encoding developmental transcription factors from newly acquired introns is highest in developmental size probably grew with the nonrecombining Y
Atlantic and Pacific O. dioica revealed short seg- transcription factor genes (figs. S18 and S19) (3). chromosome region, flaunting global compaction.
Fig. 2. Introns and in-

tron gain scenarios. (A)
Main intron logos. (B)
Transposon insertion: Du-
plicated insertion sites
(framed in blue) allow
miniature inverted repeat
transposable element
(MITE)–like insertions to

be spliced out exactly
(red, exons; black, introns).
(C) Reverse splicing: four
pairs of homologous in-
trons (black) and their
immediate exonic envi-
ronments (red).

REPORTS
Oikopleura has a rather common number of of intron loss in Oikopleura is consistent with in their host gene (table S20) (3). Competing mech-
introns per gene (4.1), but the turnover of its introns homologous recombination of reverse transcribed anisms remain possible: First, introns could be
has been extraordinarily high: Of 5589 introns mRNA (table S16) (3, 10). Among hypothetical reverse spliced into the genome itself, as can group
mapped by interspecies protein alignments, 76% mechanisms of intron gain, we provide evidence II introns (13). Some, and possibly many, introns
had positions unique to Oikopleura (newly acquired for the insertion of transposon-like elements and, of Oikopleura could originate by repair of double
introns), 17% were at ancestral positions (old introns), more remarkably for reverse splicing, a reaction in strand breaks (DSBs), as proposed for newly ac-
and 7% could not be classified (fig. S21) (3). Non- which spliced out introns can be ectopically rein- quired introns in Daphnia (14). However, for the
canonical introns, mostly GA-AG and with a very serted into transcripts (11). We identified 32 com- four mentioned intron pairs, a repair after a DSB
specific acceptor site, are unusually frequent (12%) pelling candidate introns for transposon insertion would not readily explain the systematic colocal-
(Fig. 2A and figs. S22 to S25) (3). They show (Fig. 2B and table S17) (3), those matching ization of homologous introns in the same
several peculiarities (tables S11 and S12), includ- repetitive elements containing terminal repeats at transcription unit. No feature in the sequences of
ing preferential insertion in phase 1, which is com- almost all nucleotides, with exons excluded. These those introns in pairs and their surroundings brings
patible with the current codon usage, as would be introns were usually hemizygous in genotyped particular support for this mechanism (3).
expected for the most recently gained introns (3, 7). individuals, but one individual was homozygous We explored the Oikopleura genome for genes
The most distinctive feature of newly acquired and displayed spliced transcripts (figs. S28 to S30 involved in either development or immunity. Many
introns (figs. S26 and S27 and tables S13 to S15) is and table S18) (3). We also identified four pairs of conserved immunity genes failed detection, support-
that they are more often noncanonical than old nearly identical introns (NIIs) with no or very weak ing a minimized immune system consistent with
introns (8.4 versus 2.6%) (3). Because Oikopleura similarity in flanking exons (Fig. 2C) (3), which, the short Oikopleura life history (Table 1 and table

lacks the minor spliceosome and has only one type to the best of our knowledge, represent the first S21) (3). Although frequent gene losses may have
of each spliceosomal component, we propose that reported candidates for reverse splicing (12). All affected families of developmental genes, we were
a single and permissive major spliceosome is used, animals were homozygous for NIIs and had spliced most intrigued by an unusually large number of
with U1snRNP (where snRNP is small nuclear transcripts (fig. S31 and table S19) (3). Notably, lineage-specific duplicates, thus far reported for
ribonucleoprotein) and U2AF able to recognize introns of each pair of NIIs were found within the homeobox genes only (15): 87 amplifications ac-
donor and acceptor sites (3, 8, 9). cDNA sequence same gene or the same operon, suggesting intron counting for 266 current genes (table S22) (3),
information suggests an efficient splicing for the propagation within their pre-mRNA. Many newly versus 40 amplifications in Ciona giving 106 cur-
vast majority of introns. A permissive spliceo- acquired introns of Oikopleura might have been rent genes (16). A survival analysis of early du-
some could favor intron gains by correctly propagated like these four NIIs before their se- plicates in the genome showed that duplicates are
splicing out newly acquired introns. The pattern quences diverged, because they tend to be adjacent initially lost very rapidly with less relaxed selec-
Fig. 3. Gene duplications and loss of ancestral

syntenies. (A) Early gene duplicates. (Main panel)
Histogram of binned recent duplicate pairs; a
mixture model (discrete distribution plus truncated
Weibull distributions) accommodating heteroge-
neous birth/death processes is fitted. (Inset) Non-
synonymous substitution accumulation declines with
ongoing synonymous substitution. (B) Expression of
amplified homeobox gene groups in the trunk
epithelium of larvae (red arrowheads). hD, hours
dorsal view; hL, hours lateral view; hDL, hours dorso-
lateral view. (C) Loss of ancestral gene order. Posi-
tions of orthologous genes in a given metazoan
genome (y axis) compared with ancestral chordate
linkage groups [(CLGs), x axis]. The width of CLGs
corresponds to the number of orthologs in a given
species. Amphioxus and sea anemone genome seg-
ments represent the largest 25 assembled scaffolds,
whereas Ciona, nematode, and Oikopleura segments
are chromosomes.

REPORTS
tion than in mammalian genomes (17). In contrast, pleura showed a local gene order that is in- 13. B. Cousineau et al., Cell 94, 451 (1998).
those that survive beyond 0.02 dS units are rel- distinguishable from random for distances smaller 14. W. Li, A. E. Tucker, W. Sung, W. K. Thomas, M. Lynch,
Science 326, 1260 (2009).
atively more likely to be retained (Fig. 3A, figs. S32 than 30 genes and a modest level of conserved 15. R. B. Edvardsen et al., Curr. Biol. 15, R12 (2005).
to S34, and table S23) (3). To understand how synteny at larger distances (fig. S38). 16. Y. Satou, N. Satoh, Dev. Genes Evol. 213, 211 (2003).
older developmental gene duplicates are used, we We show that multiple genome-organization 17. T. Hughes, D. A. Liberles, J. Mol. Evol. 65, 574 (2007).
focused on homeobox genes. Notably, we detected features, conserved across metazoans including 18. E. M. Thompson, T. Kallesøe, F. Spada, Dev. Biol. 238,
260 (2001).
broad expression signals in the larval trunk epithe- other tunicates and nonbilaterians, have dramatically 19. V. Katju, M. Lynch, Genetics 165, 1793 (2003).
lium for genes of most amplified groups (16 in 20), changed in the Oikopleura lineage. Despite an un- 20. N. H. Putnam et al., Nature 453, 1064 (2008).
but rarely for other groups (1 in 19) (Fig. 3B, fig. precedented genome revolution, the Oikopleura 21. N. H. Putnam et al., Science 317, 86 (2007).
S35, and table S24), likely reflecting roles in lineage preserved essential morphological features, 22. M. Srivastava et al., Nature 466, 720 (2010).
23. M. Srivastava et al., Nature 454, 955 (2008).
patterning of the house-building epithelium (18), a even maintaining the chordate body plan to the adult 24. M. Lynch, J. S. Conery, Science 302, 1401 (2003).
crucial novelty of larvaceans. A preferential reten- stage, unlike other tunicates. Evolution in this lineage 25. M. Lynch, Mol. Biol. Evol. 23, 450 (2006).
tion of duplicates for developmental genes has was rapid and probably took place in a context favor- 26. M. Lynch, Proc. Natl. Acad. Sci. U.S.A. 104 (suppl. 1),
occurred in vertebrates after whole-genome du- ing purifying selection against mildly deleterious 8597 (2007).
27. The Sars Centre budget, the Functional Genomics (FUGE)
plications. Their massive retention in Oikopleura features. Our results strengthen the view that global
Programme of the Norwegian Research Council, Genoscope,
is exceptional among invertebrates. In addition to similarities of genome architecture from sponges to and NSF grants IOS-0719577 and DBI-0743374 supported
neofunctionalization for complex innovations like humans (20–23) are not essential for the preserva- the research. This is publication ISEM-2010-123 of the
house production, another explanation may take tion of ancestral morphologies, as is widely believed Institut des Sciences de l’Evolution de Montpellier.

into consideration the general reduction of gene (24–26). GENBANK/European Molecular Biology Laboratory sequence
accession numbers are CABV01000001-CABV01005917,
size in Oikopleura. This may enhance the like- CABW01000001-CABW01006678, FN653015-FN654274,
lihood for developmental genes to escape trunca- FN654275-FN658470, FP700189-FP710243, FP710258-
References and Notes
tion after the local rearrangements that cause 1. F. Delsuc, H. Brinkmann, D. Chourrout, H. Philippe,
FP791398, and FP791400-FP884219. The sequence data
duplications (19). Other mechanisms may facili- for Capitella teleta, Daphnia pulex, Helobdella robusta
Nature 439, 965 (2006). and Lottia gigantea were produced by the U.S. Department
tate duplications or preserve developmental gene 2. J. M. Bouquet et al., J. Plankton Res. 31, 359 (2009). of Energy Joint Genome Institute (www.jgi.doe.gov/) in
duplicates in Oikopleura. 3. Supporting methods and results are available on Science collaboration with the community of users. We thank
Online.
Finally, we compared synteny relationships in I. Ahel, B. Haubold, and one anonymous reviewer for
4. T. H. Eickbush, A. V. Furano, Curr. Opin. Genet. Dev. 12,
Oikopleura and several invertebrates to ancestral 669 (2002).
generous advice. This article is dedicated to Hans Prydz
and Kåre Rommetveit for their pioneer roles in the Sars
chordate linkage groups (3, 20). Amphioxus, Ciona, 5. J. N. Volff, H. Lehrach, R. Reinhardt, D. Chourrout, Mol. Centre establishment.
Caenorhabditis, and sea anemone showed many Biol. Evol. 21, 2022 (2004).
cases of conserved chromosomal synteny (Fig. 3C, 6. A. Woolfe et al., PLoS Biol. 3, e7 (2005). Supporting Online Material
7. H. D. Nguyen, M. Yoshihama, N. Kenmochi, BMC Evol.
figs. S36 and S37, and table S25), but Oikopleura Biol. 6, 69 (2006).
www.sciencemag.org/cgi/content/full/science.1194167/DC1
Methods
orthologs showed no such conservation. We also 8. M. Marz, T. Kirsten, P. F. Stadler, J. Mol. Evol. 67, SOM Text
measured local synteny conservation between the 594 (2008). Figs. S1 to S38
same species and human (3). Amphioxus, Ciona, 9. D. A. R. Zorio, T. Blumenthal, Nature 402, 835 Tables S1 to S26
(1999).
Caenorhabditis, and sea anemone (to a much lower 10. T. Mourier, D. C. Jeffares, Science 300, 1393 (2003).
References
degree) displayed significantly higher conservation 11. S. W. Roy, W. Gilbert, Nat. Rev. Genet. 7, 211 (2006). 24 June 2010; accepted 29 October 2010
of neighborhood than expected by chance. Oiko- 12. S. W. Roy, M. Irimia, Trends Genet. 25, 67 (2009). 10.1126/science.1194167
Rewiring of Genetic Networks in systematically map genetic interaction networks

over large sets of genes in budding yeast (1–3)
and other model organisms (4, 5). Thus far, these
Response to DNA Damage networks have been constructed only under nor-
mal laboratory conditions. However, cells are con-
stantly bombarded by signals and stresses, such
Sourav Bandyopadhyay,1 Monika Mehta,2 Dwight Kuo,3 Min-Kyung Sung,4 Ryan Chuang,3
as ligands, drugs, hormones, toxins, or other
Eric J. Jaehnig,5 Bernd Bodenmiller,6 Katherine Licon,1 Wilbert Copeland,3 Michael Shales,7
Dorothea Fiedler,7,8 Janusz Dutkowski,1 Aude Guénolé,9 Haico van Attikum,9
Kevan M. Shokat,7,8 Richard D. Kolodner,5,1,10 Won-Ki Huh,4 Ruedi Aebersold,6 1
Department of Medicine, University of California, San Diego,
Michael-Christopher Keogh,2* Nevan J. Krogan,7* Trey Ideker1,3,10* La Jolla, CA 92093, USA. 2Department of Cell Biology, Albert
Einstein College of Medicine, Bronx, NY 10461, USA. 3De-
partment of Bioengineering, University of California, San Diego,
Although cellular behaviors are dynamic, the networks that govern these behaviors have been La Jolla, CA 92093, USA. 4School of Biological Sciences and
mapped primarily as static snapshots. Using an approach called differential epistasis mapping, Research Center for Functional Cellulomics, Institute of Micro-
biology, Seoul National University, 151-742 Seoul, Republic of
we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, Korea. 5Ludwig Institute for Cancer Research and Department of
and transcription factors as the cell responds to DNA damage. Differential interactions uncover Cellular and Molecular Medicine, University of California, San
many gene functions that go undetected in static conditions. They are very effective at identifying Diego, La Jolla, CA 92093, USA. 6Institute of Molecular Systems
DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 Biology, ETH Zürich, Zürich CH 8093, Switzerland, and Faculty
of Science, University of Zürich, Zürich CH 8057, Switzerland.
phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally 7
Department of Cellular and Molecular Pharmacology, Uni-
stable in response to perturbation, but the functional relations between these complexes are versity of California, San Francisco, San Francisco, CA 94158,
substantially reorganized. Differential networks chart a new type of genetic landscape that is USA. 8Howard Hughes Medical Institute, San Francisco, CA 94158,
invaluable for mapping cellular responses to stimuli. USA. 9Department of Toxicogenetics, Leiden University Medical
Center, Leiden, Netherlands. 10The Institute for Genomic Medicine,
University of California, San Diego, La Jolla, CA 92093, USA.
ne of the most basic approaches to under- occur when the phenotypic effects of one gene
O standing gene function relies on the iden-

tification of genetic interactions, which
depend on the presence of a second. Recently, a
number of technologies have been developed to
*To whom correspondence should be addressed. E-mail:
tideker@ucsd.edu (T.I.); krogan@cmp.ucsf.edu (N.J.K.); michael.
keogh@einstein.yu.edu (M.-C.K.)

REPORTS
Fig. 1. An epistasis map A B C Static
Positive
for DNA damage signaling. 80
Negative D B
T) ed
Specific Interactions (%)
Untreated
Differential
S
(A) Comparison of genetic
(U eat
70
M
A
M
interactions (positive, S ≥
r
nt
B
U
60 D
Positive
+2; negative, S ≤ −2.5) Control
E
C
A
50
uncovered in untreated 198 169 426 (UT vs UT)
40 E
or DNA damage treated D B
C
MMS
(+MMS) conditions. Con- 219 1092 262 30 A
Negative
trol represents interactions 20 E
C
from two independent ex- 631 907 795 10
periments in untreated 0
Static: Positive Negative
Differential: Positive Negative
conditions. (B) Percentage UT MMS Magnitude: Strong Weak
DNA Damage Enrichment (p-value)

of interactions (positive or
negative) identified in D E F
5
each condition that are Positive 10 Static( Untreated MMS) -25 Differential
MMS (S-Score)
specific to that condition. 2 Differential -20

0 10 DNA Damage
(C) Differences between
-2.5
the untreated and treated 10 Cell Cycle
-15
-5
maps identify differential

Negative Chromatin
-10
interactions. (D) Scatter of Differential
10
-10
S scores between untreated 10 Transcription
-5
andtreated maps and iden-

-15 Signaling
tification of positive differ- -15 -10 -5 -2.5 0 2 5
0
(− Static MMS)
ential (green, P ≤ 0.001)
MMS
Static
Static
Differential
Untreated
Differential
Untreated (S-score) -60 -40 -20
10 10 10 P-value 10-10 10-30
and negative differential in- of Enrichment
teractions (red, P ≤ 0.001). (E) Enrichment of interactions involving known DNA
repair genes, shown for static and differential networks and including both
positive and negative interactions. For the right-most bar, all differential
interactions also identified in the static network are removed. (F) Enrichment of interactions involving genes with various functions (10).
environmental conditions. Although it is clear

that some genetic interactions are condition- Known DNA Repair
dependent (6, 7), to what extent environmental Other MMS Sensitive
stresses can affect genetic interaction networks,
and the pathways they represent, is still unknown.
To gain insight into how genetic networks
are altered by stress, we assembled a large ge-
netic interactome with and without perturbation
by the DNA-damaging agent methyl methane-
sulfonate (MMS). Using the technique of ep-
istatic miniarray profiles (E-MAP) (8), genetic
interactions were interrogated among a set of 418
yeast genes selected to provide broad coverage
of the cellular signaling and transcriptional ma- SLT2
chinery, including nearly all yeast kinases, phos-
phatases, and transcription factors, as well as BCK1
known DNA repair factors (fig. S1 and table S1). FUS3

About 80,000 double-mutant strains were gen-
erated from all pairwise mutant combinations
of the 418 genes, in which mutations were com-
plete gene deletions (nonessential genes) or hy-
pomorphic alleles (essential genes) as appropriate.
Double-mutant combinations were grown with
or without 0.02% MMS, and their colony sizes
were analyzed statistically to compute a genetic Fig. 2. Identification of differential genetic interaction hubs. The scatterplot shows the number of positive
interaction score (S score) in each condition (9), and negative differential interactions associated with each gene in this study. The 30 genes whose
which indicates whether the strain was healthier deletions are the most sensitive to MMS are indicated (blue) (24), excluding those already known to
or sicker than expected (positive or negative S, function in DNA repair (red) (table S1).
respectively) (10).
From established score thresholds for positive
and negative interactions (S ≥ +2.0, S ≤ −2.5) (9) ciations with physical interaction networks of various kinase- and phosphatase-substrate pairs, as well
we identified two genetic networks: a set of 1905 kinds. For example, gene pairs with either positive as transcription factor-target pairs (fig. S2). The
interactions for the untreated condition, and a set or negative genetic interactions were highly en- correspondence to physical and functional asso-
of 2297 interactions under MMS. Analysis of riched for proteins known to physically interact. ciations reflects the predictive power of this ge-
these “static” genetic maps showed strong asso- In addition, both maps were enriched for known netic interaction data set.

REPORTS
Fig. 3. Differential genet-
ic interactions identify novel
DNA damage–dependent
pathways. (A) Full profile
of CBF1 genetic interac-
tions with the strongest
positive genetic interac-
tions in MMS highlighted.
(B) Fluorescence-activated
cell sorting (FACS) analysis
of cell cycle progression
in alpha-factor–arrested
wild-type and cbf1D cells
released into media with
or without MMS. In MMS,
cbf1D cells bypass cell cycle
checkpoints and eventual-
ly accumulate in S phase
(between 1N and 2N DNA

content). (C) gH2AX levels
for wild-type and CBF1
overexpression at times
indicated after exposure
to MMS. Pgk1 is used as a
loading control. (D) Effect
of CBF1 overexpression
on cell cycle progression
using FACS. (E) Correla-
tion coefficients between
the CBF1 genetic interac-
tion profile and that of
each gene in the epistasis
map, in MMS versus un-
treated conditions. (F) Top
changes in abundance of
phosphorylated peptides
in pph3∆ versus wild-type
cells by phospho-proteomic
profiling. (G) Histogram
of autocorrelation coeffi-
cients. For each gene, the
genetic interaction profiles
before and after MMS ex-
posure are compared by
Pearson correlation. (H)
Correlation plot as in (E)
for HTZ1. (I) Represen-
tative genetic interactions
of HTZ1, MEC1, and SWR-C
members. Clustering based
on similarity of profiles is shown. (J) The acetylation of multiple Htz1 N-terminal amount of Htz1. Rpn8 is a loading control. (K) Acetylation status of Htz1-K14 in
lysine residues (K8, K10, or K14) measured in an Htz1-3HA strain after MMS response to MMS in WT and mec1∆ backgrounds. gH2AX is a downstream
exposure in wild-type (WT) and hda1∆ backgrounds. HA represents total marker of DNA-damage signaling by Mec1.
Comparison of the genetic networks across condition (fig. S3) (10). This method identified either static condition, most likely because they
conditions revealed large differences, with more 873 differential genetic interactions at P ≤ 0.001, are too weak to detect in any single condition yet
interactions unique to each map than in common with a corresponding false-discovery rate of ~9% display a substantial change in interaction be-
(Fig. 1A). For example, more than 70% of pos- (fig. S4 and table S2). We term this approach tween conditions.
itive interactions identified under MMS were not differential epistasis mapping (dE-MAP), as it To determine whether static untreated, static
identified in the untreated sample, which reflects is based on the difference of two static networks treated, or differential genetic networks best un-
widespread DNA damage–induced epistasis (Fig. generated using the E-MAP methodology. A cover DNA damage-response pathways, we ex-
1B). To assess these changes in interaction, each total of 379 interactions were “negative differ- amined a reference set of 31 known DNA repair
gene pair was associated with its difference in S ential,” which indicated DNA damage–induced genes (table S1). We noted that static networks
score across conditions (Fig. 1C). A P value for this lethality or sickness, whereas 494 were “posi- were no more likely than random to include
difference was calculated using the null distribution tive differential,” which indicated inducible epis- interactions with genes in this reference set (Fig.
of score differences observed when comparing tasis or suppression (Fig. 1D). The majority (62%) 1E). This lack of enrichment was observed in
replicate interaction measurements from the same of differential interactions were not detectable in the untreated genetic network, as well as, sur-

REPORTS
prisingly, the static network obtained under MMS. We noted that both the static treated and un- network subtraction, however, the “housekeeping
In contrast, the differential network—obtained treated networks were dominated by interactions interactions” due to chromatin are removed, which
through the quantitative difference in interaction involving genes that function in chromatin orga- allows sensitive detection of differentially repre-
across conditions—was highly enriched for inter- nization (Fig. 1F). This strong chromatin signal sented pathways. Thus, network comparison re-
actions involving DNA damage-response genes has been previously reported in budding and fis- veals a landscape of genetic interactions particularly
such as RAD52, TEL1, and DUN1 (Fig. 1E) (10). sion yeasts and C. elegans (2, 4, 5, 11). Through tailored to the cellular response of interest.
Fig. 4. Module-based interpretation

of differential genetic interactions. (A)
MMS induces a set of positive inter-
actions between DNA damage pathways
(top right) that are not evident in un-
treated conditions (bottom left). (B) Module
map of protein complexes and pathways
connected by differential genetic interac-
tion bundles. Node color represents the
most severe single-deletion phenotype

among members of a module (table S1).
(C) Detailed view of differential genetic in-
teractions between protein complexes
corresponding to selected modules in (B)
(dotted node borders). For clarity, only
physical interactions and differential genet-
ic interactions with P < 0.01 are shown.
Thickness is scaled with increasing signifi-
cance of the P value (10).

REPORTS
Analysis of static networks has found that net- sured in the presence or absence of MMS (high damage–induced stress, many of which have not
work “hubs”, i.e., genes with many interactions, “genetic autocorrelation”) (Fig. 3G). However, been previously linked to DNA repair.
modulate a variety of cellular functions and are several genetic interaction profiles were markedly Large-scale genetic interaction networks have
more likely to be essential for viability (12). For disrupted in MMS (low autocorrelation), includ- proved extremely powerful for mapping the path-
the differential network, we found that the number ing those of RAD52, a critical factor in homol- ways that regulate essential cell functions. In this
of interactions per gene was correlated with the ogous recombination–mediated DNA repair (18), study, we have shown that differential genetic
sensitivity to MMS of the corresponding gene and HTZ1, encoding the histone variant H2A.Z, networks are comparable in size to static networks,
deletion strain (r = 0.35, P < 10−5) (fig. S5, A and whose role in DNA repair is less well understood yet access a very different set of interactions gov-
B). Differential interaction hubs were also more (19, 20). In untreated conditions, the HTZ1 pro- erning a dynamic cellular response. Given that
likely to be essential for growth under a variety of file correlated with members of the SWR com- most gene functions arise in response to changing
drug treatments and stresses (fig. S5, C and D), plex (SWR1, SWC5, VPS71, and VPS72) (11), conditions, the differential network revealed here
consistent with previous observations for static responsible for incorporating Htz1 into chroma- offers a glimpse into a much larger universe of
hubs (13). tin (21). This correlation was lost in MMS (Fig. genetic interactions that are condition-, cell type–,
Further investigation showed that many dif- 3H), which suggested a functional disassociation or tissue-specific.
ferential interaction hubs were already well known between Htz1 and the SWR-C. Conversely,
to function as key components of DNA repair HTZ1 became correlated with the DNA-damage 1. C. Boone, H. Bussey, B. J. Andrews, Nat. Rev. Genet.
pathways (Fig. 2), which led us to predict that the checkpoint kinase MEC1 upon MMS treatment 8, 437 (2007).
remaining hubs might encode this role. Two such (Fig. 3, H and I), and a mec1∆htz1∆ strain showed 2. M. Costanzo et al., Science 327, 425 (2010).

differential interaction hubs encode Slt2 and synthetic sensitivity to MMS (fig. S7), suggest- 3. X. Pan et al., Cell 124, 1069 (2006).
4. B. Lehner, C. Crombie, J. Tischler, A. Fortunato, A. G. Fraser,
Bck1, mitogen-activated protein kinases (MAPKs) ing a damage-dependent functional link between
Nat. Genet. 38, 896 (2006).
that have been implicated in the maintenance the two proteins. Htz1 is acetylated on its amino 5. A. Roguev et al., Science 322, 405 (2008).
of cell wall integrity but not yet linked to DNA terminus by histone acetyltransferase NuA4 (22) 6. J. C. Game, R. K. Mortimer, Mutat. Res. 24, 281
repair (Fig. 2). We found that Slt2 is both up- and deacetylated by histone deacetylase Hda1 (1974).
regulated and translocated to the nucleus upon (19). We found that the degree of Htz1 acetyla- 7. R. P. St Onge et al., Nat. Genet. 39, 199 (2007).
8. M. Schuldiner, S. R. Collins, J. S. Weissman, N. J. Krogan,
MMS treatment, and it is required for appropri- tion at multiple lysine residues was strongly re- Methods 40, 344 (2006).
ate regulation of ribonucleotide reductase genes duced in response to MMS (Fig. 3J). This effect 9. S. R. Collins, M. Schuldiner, N. J. Krogan, J. S. Weissman,
in response to DNA damage (fig. S6, A to D) was dependent on both Hda1 (Fig. 3J) and Mec1 Genome Biol. 7, R63 (2006).
(14). Furthermore, both MAPKs show strong ge- (Fig. 3K), which suggested that the regulation of 10. Materials and methods are available as supporting
material on Science Online.
netic interactions with DNA damage check- Htz1 acetylation contributes to the DNA damage
11. S. R. Collins et al., Nature 446, 806 (2007).
point genes (fig. S6, E and F), which suggests response. 12. H. Jeong, S. P. Mason, A. L. Barabási, Z. N. Oltvai, Nature
that they may function in a parallel signaling We next investigated the association between 411, 41 (2001).
pathway. differential genetic interactions and known yeast 13. A. Jaimovich, R. Rinott, M. Schuldiner, H. Margalit,
Another differential interaction hub not pre- pathways and protein complexes (i.e., modules) N. Friedman, Bioinformatics 26, i228 (2010).
14. S. J. Elledge, R. W. Davis, Mol. Cell. Biol. 7, 2783
viously linked to DNA repair was centromere (table S1). In contrast to static genetic inter- (1987).
binding factor 1 (CBF1) (Fig. 2), a sequence- actions, which are enriched within modules, we 15. M. Cai, R. W. Davis, Cell 61, 437 (1990).
specific transcription factor and component of found that differential genetic interactions are not 16. J. Heideker, E. T. Lis, F. E. Romesberg, Cell Cycle
the inner kinetochore (15). CBF1 gained strong (fig. S8A). Rather, differential genetic interac- 6, 3058 (2007).
17. M. B. Smolka, C. P. Albuquerque, S. H. Chen, H. Zhou,
genetic interactions upon MMS treatment (Fig. tions are much more likely to occur among pairs Proc. Natl. Acad. Sci. U.S.A. 104, 10364 (2007).
3A), which suggested an additional role in DNA of genes connecting two different modules than 18. F. Pâques, J. E. Haber, Microbiol. Mol. Biol. Rev.
repair. We found that Cbf1 is required for appro- among pairs of genes within the same module 63, 349 (1999).
priate activation of cell cycle checkpoints (Fig. (fig. S8B). These findings were corroborated by 19. Y. Y. Lin et al., Genes Dev. 22, 2062 (2008).
20. M. Papamichos-Chronakis, J. E. Krebs, C. L. Peterson,
3B) and that CBF1 overexpression interferes an alternative analysis in which modules were de- Genes Dev. 20, 2437 (2006).
with induction of the damage-dependent histone fined through hierarchical clustering of the treated 21. N. J. Krogan et al., Mol. Cell 12, 1565 (2003).
modification gH2AX (Fig. 3C) and leads to cell and untreated genetic interaction data. Genes that 22. M. C. Keogh et al., Genes Dev. 20, 660 (2006).
cycle arrest in G1 (Fig. 3D). Furthermore, MMS clustered into the same module in both conditions 23. S. Bandyopadhyay, R. Kelley, N. J. Krogan, T. Ideker,
PLOS Comput. Biol. 4, e1000065 (2008).
treatment causes the CBF1 profile of genetic were much more likely to physically interact than
24. T. J. Begley, A. S. Rosenbach, T. Ideker, L. D. Samson,
interaction scores (across all genes on the E-MAP) genes that coclustered in one condition only (fig. Mol. Cancer Res. 1, 103 (2002).
to become correlated with the profiles of TEL1 S8C). These results suggest that known protein 25. The authors thank S. Collins, H. Hombauer, A. Desai,
kinase and PPH3 phosphatase, which encode complexes tend to be stable across conditions—it S. Gasser, X. Shen, and S. Choi for helpful discussions
key proteins regulating the DNA damage check- is the genetic interactions between these modules and strains. This work was funded by NIH grants
R01-ES14811 and R01-GM084279. Additionally,
point (Fig. 3E) (16). Mass spectrometry–based that are reprogrammed in response to perturba- W-K.H. and M-K.S. were funded by the 21C Frontier
phosphoproteomics showed that Cbf1 is hyper- tion (Fig. 4A). Functional Proteomics Project (FPR08A1-060), R.A.
phosphorylated at a conserved serine-glutamine On the basis of these findings, we constructed and B.B. were supported by SystemsX.ch and the
motif (SQ145-146) in pph3∆ cells (Fig. 3F and a global map of gene modules and their dynam- Swiss National Science Foundation, and M-C.K. was
supported by the NCI (P30CA013330). N.J.K. is a Keck
table S3). As the checkpoint kinases Mec1 and ic genetic interactions in response to DNA dam- Young Investigator Fellow and a Searle Fellow. T.I. is a
Tel1 have been shown to target Cbf1 at the same age. Using an established method (23), we David and Lucille Packard Fellow. R.D.K. is a paid
SQ site (17), it is likely that Pph3 is the protein defined modules as dense clusters of physical consultant to On-Q-ity.
phosphatase that counteracts the effect of this and static genetic interactions. Module-module
phosphorylation. interactions were characterized by heavy enrich- Supporting Online Material
As another means of mapping DNA repair ment for many differential genetic interactions Materials and Methods
pathways, we identified genes with genetic inter- across the two modules (table S4) (10). The Figs. S1 to S8
action profiles that were conditionally disrupted resulting map of 56 multigene modules and 66 Tables S1 to S6
by MMS, suggesting a shift in gene function. We module-module interactions (Fig. 4, B and C) References
observed that most genes had high correlation provides a global resource of pathways and com- 26 July 2010; accepted 22 October 2010
between their genetic interaction profiles mea- plexes that are reconfigured in response to DNA 10.1126/science.1195618

REPORTS
BID, BIM, and PUMA Are Essential c efflux (6–9). Although BAX and BAK control
the mitochondrial gateway to apoptosis, how BAX
for Activation of the BAX- and

and BAK are activated, whether BAX and BAK
are activated directly or indirectly by BH3s, and
the identity of the core repertoire of activators of
BAK-Dependent Cell Death Program BAX and BAK in various tissues remain un-
settled. Two non–mutually exclusive models have
been proposed (5, 13). The direct activation mod-
Decheng Ren,1* Ho-Chou Tu,1* Hyungjin Kim,1 Gary X. Wang,1 Gregory R. Bean,1 el states that the “activator” subgroup of BH3s,
Osamu Takeuchi,2 John R. Jeffers,3 Gerard P. Zambetti,3 James J.-D. Hsieh,1 Emily H.-Y. Cheng1,4†‡ including truncated BID (tBID) and BIM, can di-
rectly induce the conformational changes of BAX
Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK (6–12, 14–16). The indirect model pro-
and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential poses that activation of BAX and BAK occurs by
role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout default as long as all the antiapoptotic BCL-2
mice showed the same developmental defects that are associated with deficiency of Bax and Bak, proteins are neutralized by BH3s, based on the
including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma observation that BAX- or BAK-dependent apo-
prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c–mediated activation ptosis proceeds in the absence of BID and BIM
of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence (17). However, PUMA appears also to function as

of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK a direct activator of BAX and BAK. In vitro trans-
at the mitochondria by a member of the BID, BIM, or PUMA family of proteins. lated PUMA protein, but not the BH3 domain
itochondria have key roles in mamma- whereas antiapoptotic BCL-2, BCL-XL, and
M lian apoptosis, a highly regulated ge-

netic program of cell suicide (1–3).
Multiple apoptotic signals release cytochrome c
MCL-1 proteins prevent cytochrome c efflux
triggered by apoptotic stimuli. The third BCL-2
subfamily of proteins, the BH3-only molecules
1
Molecular Oncology, Department of Medicine, Washington
University School of Medicine, St. Louis, MO 63110, USA. 2De-
partment of Host Defense, Research Institute for Microbial
Diseases, Osaka University, Osaka 565-0871, Japan. 3St. Jude
from the mitochondria to activate the Apaf-1 protein, (BH3s) (that is, family members with only one Children’s Research Hospital, Memphis, TN 38105, USA. 4De-
which activates caspases. The BCL-2 family of BCL-2-homology domain), constitutes the largest partment of Pathology and Immunology, Washington University
proteins integrates developmental and environ- BCL-2 subfamily with more than 10 members School of Medicine, St. Louis, MO 63110, USA.
mental cues to dictate the survival or death de- that promote apoptosis by either activating BAX *These authors contributed equally to this work.
cision of cells by regulating the integrity of the and BAK directly or inactivating BCL-2, BCL- †Present address: Human Oncology and Pathogenesis Pro-
gram, Memorial Sloan-Kettering Cancer Center, New York, NY
mitochondrial outer membrane (MOM) (1, 4, 5). XL, or MCL-1 (6–12). When apoptosis is initiated, 10065, USA.
The multidomain proapoptotic proteins BAX and BAK and BAX undergo conformational changes ‡To whom correspondence should be addressed. E-mail:
BAK mediate permeabilization of the MOM, to form homo-oligomers that mediate cytochrome chenge1@mskcc.org
Fig. 1. Triple deficiency of Bid, Bim, and Puma

phenocopies double deficiency of Bax and Bak. (A)
Bid−/−Bim−/−Puma−/− TKO mice display persistence
of interdigital webs. Ventral views of paws from WT,
Bid−/−Bim−/−, and Bid−/−Bim−/−Puma−/− mice. (B)
Bid−/−Bim−/−Puma−/− TKO mice fail to develop external
vaginal introituses. Photographs of vaginal open-
ings from WT, Bid−/−Bim−/−, and Bid−/−Bim−/−Puma−/−
mice. Arrows point to external vaginal region. (C
to G) Immunohistochemistry for cleaved caspase-
3 from cerebella of postnatal day 5 (P5) mice of
the indicated genotypes that were irradiated with
14 Gy g-irradiation. (C) WT, 18 hours after IR. (D)
Bax f/-Bak−/−Nestin-Cre+, 30 hours after IR. (E) Puma−/−,
30 hours after IR. (F) Bim−/−Puma−/−, 30 hours after
IR. (G) Bid−/−Bim−/−Puma−/−, 30 hours after IR. Arrows
denote the external granular layer of cerebellum. Data
shown are representative images of two to three inde-
pendent experiments. (H) Analyses of a whole sagittal
section of cerebellum at the same level from experi-
ments shown in (C) to (G) summarize the numbers of
neurons at the external granular layer that were
stained positive for cleaved caspase-3. Data shown are
the average of two independent experiments (n = 2).

REPORTS
peptide of PUMA, can directly activate BAX- Although the embryonic lethality caused by Bak does, we examined ionizing radiation (IR)–
and BAK-dependent permeabilization of the MOM triple deficiency of Bid, Bim, and Puma appeared induced apoptosis in cerebellar granule neu-
(11, 12, 18–20). The transmembrane domain was to be less severe than that in mice lacking Bax and rons (CGN). Because most of the Bax−/− Bak−/−
important for PUMA to induce cytochrome c ef- Bak (table S1), the viable Bid−/−Bim−/− Puma−/− mice die in embryogenesis, we used the Cre-LoxP
flux (fig. S1), which may explain why BH3 pep- mice displayed developmental defects very sim- conditional knockout strategy to delete Bax in
tides are less active (11, 19). Thus, we studied ilar to those of Bax−/−Bak−/− animals, including neurons through a Nestin-Cre construct that is
Bid−/−Bim−/−Puma−/− triple-knockout (TKO) persistent interdigital webs of skin on their feet expressed in neuronal and glial cell precursors
mice to clarify how BAX and BAK are activated and imperforate vaginas (21) (Fig. 1, A and B, (22). No activated caspase-3 was detected in
and whether BID, BIM, and PUMA represent the and table S2). To determine whether deficiency Bax f/–Bak−/−Nestin-Cre+ neurons (Fig. 1D). Puma-
core repertoire of activators of BAX and BAK of Bid, Bim, and Puma completely blocks the in- deficient neurons were less sensitive to IR than
that function downstream of other BH3s. trinsic apoptotic death as deficiency of Bax and Bax-deficient neurons (Fig. 1E and fig. S2). Al-
though deficiency of Puma greatly reduced the
activation of caspase-3, deletion of Bid, Bim, and
Fig. 2. Bid−/−Bim−/−Puma−/− Puma was required to completely block caspase-
TKO cerebellar granule neu- mediated apoptosis (Fig. 1, E to H). Similar find-
rons are completely resistant ings were also observed in IR-induced apoptosis
to potassium-deprivation– in dentate gyrus. BAD and BMF proteins were
induced apoptosis. Cerebel-
detected in CGN (fig. S3), suggesting that BAD

lar granule neurons from WT,
and BMF were unable to activate BAX- or BAK-
Bim−/−, Puma−/−, Bim−/−
Puma−/−, or Bid−/−Bim−/− dependent apoptosis in the absence of Bid, Bim,
Puma−/− mice were cultured and Puma. These data indicate that activation of
in high-K+ (K25 + S) for 7 BAX and BAK in response to IR is fully depen-
days and then transferred dent on BID, BIM, and PUMA.
to low-K+ medium (K5 + S) To exclude the possibility that BID, BIM, and
to induce apoptosis. Viability PUMA are required to activate BAX or BAK
was determined at the indi- only in response to genotoxic stress, we inves-
cated times using propidium iodide (PI) staining. Data are the mean percentage of PI-positive neurons T SD tigated potassium-deprivation–induced apoptosis
from three independent experiments. **, P < 0.01; ***, P < 0.001. of cultured CGN. CGN from early postnatal mice
Fig. 3. Bid−/−Bim−/−Puma−/− TKO T cells are re-

sistant to diverse apoptotic stimuli. (A to E) CD4+
T cells purified from the spleens of WT (n = 3),
Bid−/−Bim−/− (n = 3), or Bid−/−Bim−/−Puma−/− (n = 3)
mice at 8 to 10 weeks of age were cultured under
the following conditions: in the absence of cytokine
(A), after exposure to 2.5 Gy g-irradiation (B), in
the presence of etoposide (C), in the presence of
dexamethasone (D), or in the presence of agonistic
antibody to Fas (E). Cell death was quantified by
annexin-V staining at the indicated times. (F to I)
Thymocytes from WT (n = 3), Bid−/−Bim−/− (n = 3),
or Bid−/−Bim−/−Puma−/− (n = 3) mice at 6 to 8 weeks
of age were cultured under the following condi-
tions: in the absence of cytokine (F), after exposure to
2.5 Gy g-irradiation (G), in the presence of etoposide
(H), or in the presence of tunicamycin (I). Cell death
was quantified by annexin-V staining at the indicated
times. Data are the mean percentage of annexin-V–
positive cells T SD from three independent experi-
ments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

REPORTS
can be cultured in medium containing a high con- PUMA fails to induce BAX- and BAK-dependent Cleavage of BID was detected in thymocytes in
centration of potassium (25 mM) that provides mitochondrial permeabilization in the absence of response to DNA damage or ER stress (fig. S11).
depolarization-mediated survival. Deprivation of Bid and Bim (27). To address whether endogenous To investigate whether activation of BAX and
potassium (5 mM) induces BAX-dependent apo- PUMA can activate BAX- and BAK-dependent BAK was blocked in the absence of Bid, Bim,
ptosis that requires de novo protein synthesis (23). mitochondrial apoptosis independently of BID and and Puma, gel filtration was performed to assess
Consistent with the previous reports demonstrat- BIM, we compared the apoptotic phenotypes homo-oligomerization of BAX or BAK. In both
ing the accumulation of BIM and PUMA proteins of Bid−/−Bim−/− double-knockout (DKO) mice wild-type (WT) and Bid−/−Bim−/−Puma−/− TKO
in potassium-deprived CGN (24, 25), deficiency to Bid−/−Bim−/− Puma−/− TKO mice. The TKO cells, BAX was eluted as a 20-kD monomer from
of either Bim or Puma provided transient pro- mice had more pronounced accumulation of untreated cells (Fig. 4A). After IR or treatment of
tection, whereas deficiency of both Bim and Puma hematopoietic cells in thymus, spleen, lymph cells with tunicamycin, BAX formed higher-
conferred long-term protection against potassium- nodes, and blood than DKO mice (figs. S6 to ordered oligomers in WT but not TKO cells (Fig.
deprivation–induced apoptosis (Fig. 2 and figs. S8). Moreover, Bid−/− Bim−/−Puma−/− TKO T 4A). Similarly, higher-ordered oligomers of BAK
S4 and S5). Deletion of Bid, Bim, and Puma cells were more resistant than Bid−/−Bim−/− DKO were only detected in WT cells in response to
completely blocked apoptosis up to 3 days (Fig. 2 cells to diverse intrinsic apoptotic signals, includ- genotoxic or ER stress (Fig. 4B). Consistent with
and figs. S4 and S5). These data indicate that ing cytokine deprivation, DNA damage (IR or the lack of homo-oligomerization of BAX or BAK
BAX is not activated in the absence of Bid, Bim, etoposide), glucocorticoids (dexamethasone), detected in TKO cells, cytochrome c transloca-
and Puma, even though BAD and BMF are pres- or endoplasmic reticulum (ER) stress (tunica- tion and caspase activation were not observed in
ent (fig. S3) and the abundance of HRK (also mycin) (Fig. 3 and fig. S9). Puma-deficient cells these cells when exposed to diverse intrinsic apo-

called DP5) is increased in these neurons (26). were less resistant to apoptosis than TKO cells ptotic signals (Fig. 4, C and D, and figs. S12 and
One of the major phenotypes of Bax−/−Bak−/− (fig. S10). Indeed, deficiency of Bid, Bim, and S13). Although the majority of WT cells lost
mice is the accumulation of lymphoid and myeloid Puma appears to provide more protection against cytochrome c immunostaining within 20 hours
cells as a consequence of defective apoptosis (21). intrinsic apoptotic death than deficiency of Apaf-1 after IR, in TKO cells, cytochrome c remained in
Bid−/−Bim−/−Puma−/− animals also exhibited en- or Capase-9 (28). By analogy to Bax−/−Bak−/− T the mitochondria, exhibiting a punctate staining
larged thymi, splenomegaly, and lymphadenopathy cells (21), Bid−/−Bim−/− Puma−/− T cells were pattern (Fig. 4C). Caspase activation determined
(fig. S6). The role of PUMA as a direct activator of still sensitive to Fas-induced apoptosis because by the cleavage of poly (ADP-ribose) polymerase
BAX and BAK is debated in part because of re- the BCL-2 family does not regulate extrinsic apo- (PARP) and caspase-3 was not observed in TKO
ports showing that knockdown of Puma does not ptotic death in T cells (Fig. 3E). The proapoptotic cells treated with IR, dexamethasone, or tunica-
provide further inhibition of apoptosis in the ab- activity of BID is activated upon its cleavage by mycin (Fig. 4D). Notably, tBID, BIM, and
sence of Bid and Bim (17) and because exogenous proteases such as caspase-8 or -2 (1, 4, 5). PUMA were potent death agonists in both WT
Fig. 4. BID, BIM, and PUMA are required to activate BAX- and BAK-dependent
mitochondrial apoptosis. (A and B) Triple deficiency of Bid, Bim, and Puma prevents
activation of BAX and BAK. Thymocytes from WT or Bid−/−Bim−/−Puma−/− mice were
untreated, irradiated with 5 Gy g-irradiation (IR), or treated with tunicamycin (TC). Protein
lysates were harvested at 7 hours after IR or 20 hours after TC treatment and subjected to Superdex 200 (HR10/30) gel-filtration chromatography. Fractions were
analyzed by Western blot using antibodies to BAX (A) or BAK (B). (C) Triple deficiency of Bid, Bim, and Puma prevents the translocation of cytochrome c.
Fluorescence microscopy of WT or Bid−/−Bim−/−Puma−/− thymocytes 20 hours after exposure to 2.5 Gy g-irradiation. Red represents cytochrome c
immunostaining, and blue is Hoechst staining. White asterisks indicate apoptotic cells that have lost cytochrome c. (D) Triple deficiency of Bid, Bim, and Puma
prevents the activation of caspases. Thymocytes from WT or Bid−/−Bim−/−Puma−/− mice were untreated, treated with TC or dexamethasone (Dex), or irradiated
with 5 Gy g-irradiation (IR). After 12 hours, protein lysates were harvested and assessed by Western blot using antibodies specific for PARP, cleaved PARP, cleaved
caspase-3, or actin. Asterisk denotes a cross-reactive protein. (E) Primary mouse embryonic fibroblasts isolated from WT or Bid−/−Bim−/−Puma−/− TKO mice were
infected with retroviruses expressing the indicated genes. Cell death was quantified by annexin-V staining at 24 hours. Data are the mean percentage of annexin-
V–positive cells T SD from three independent experiments. *, P < 0.05.

REPORTS
and Bid−/− Bim−/−Puma−/− TKO cells (Fig. 4E served in Bax−/−Bak−/− DKO (21) or Bid−/−Bim−/− 19. M. Certo et al., Cancer Cell 9, 351 (2006).
and fig. S14). Puma−/− TKO mice, but not in Apaf-1–deficient 20. N. Y. Fu, S. K. Sukumaran, S. Y. Kerk, V. C. Yu,
Mol. Cell 33, 15 (2009).
The lack of strong and stable interaction be- mice (28, 30). Overall, our study reveals an es- 21. T. Lindsten et al., Mol. Cell 6, 1389 (2000).
tween BH3s and BAX/BAK was thought to sup- sential axis of activator BH3s and BAX and 22. F. Tronche et al., Nat. Genet. 23, 99 (1999).
port the indirect activation model for BAX and BAK in activating the mitochondrial death pro- 23. T. M. Miller et al., J. Cell Biol. 139, 205 (1997).
BAK. However, dynamic interactions occur be- gram, which offers common ground for ther- 24. G. V. Putcha et al., J. Cell Biol. 157, 441 (2002).
25. C. G. Besirli, E. F. Wagner, E. M. Johnson Jr., J. Cell Biol.
tween BAX and activator BH3s, including tBID, apeutic interventions. 170, 401 (2005).
BIM, and PUMA (18), which helps explain the 26. C. A. Harris, E. M. Johnson Jr., J. Biol. Chem. 276,
previous difficulty in detecting these interactions. References and Notes 37754 (2001).
Here, we demonstrate an essential role of BID, 1. A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 27. J. E. Chipuk et al., Proc. Natl. Acad. Sci. U.S.A. 105,
13, 1899 (1999). 20327 (2008).
BIM, and PUMA in activating BAX and BAK 28. V. S. Marsden et al., Nature 419, 634 (2002).
2. X. Wang, Genes Dev. 15, 2922 (2001).
and for some apoptotic events during develop- 29. T. Oltersdorf et al., Nature 435, 677 (2005).
3. D. D. Newmeyer, S. Ferguson-Miller, Cell 112, 481
ment. Our genetic study indicates that BAD is (2003). 30. H. Yoshida et al., Cell 94, 739 (1998).
unable to induce apoptosis in the absence of BID, 4. J. C. Reed, Cell Death Differ. 13, 1378 (2006). 31. We apologize to all the investigators whose research
5. R. J. Youle, A. Strasser, Nat. Rev. Mol. Cell Biol. 9, could not be appropriately cited owing to space
BIM, and PUMA, which is further supported by
47 (2008). limitation. We thank T. D. Westergard and H.-F. Chen
the observation that the BAD mimetic, ABT-737 for technical assistance. This work was supported by
6. S. Desagher et al., J. Cell Biol. 144, 891 (1999).
(29), failed to kill Bid−/−Bim−/−Puma−/− TKO 7. M. C. Wei et al., Genes Dev. 14, 2060 (2000). grants to E.H.-Y.C. from NIH (R01CA125562) and the
cells (fig. S15). These data suggest that the pro- 8. M. C. Wei et al., Science 292, 727 (2001). Searle Scholars Program, and to G.P.Z. from NIH

found block of apoptosis conferred by the triple 9. E. H. Cheng et al., Mol. Cell 8, 705 (2001). (R01GM083159 and P30CA21765)
deficiency of Bid, Bim, and Puma is not simply 10. A. Letai et al., Cancer Cell 2, 183 (2002).
11. T. Kuwana et al., Mol. Cell 17, 525 (2005). Supporting Online Material
caused by altering the ratio between antiapoptotic 12. H. Kim et al., Nat. Cell Biol. 8, 1348 (2006). www.sciencemag.org/cgi/content/full/330/6009/1390/DC1
and proapoptotic BCl-2 proteins. BH3s not only 13. H. L. Galonek, J. M. Hardwick, Nat. Cell Biol. 8, Materials and Methods
induce BAX- and BAK-dependent release of 1317 (2006). Figs. S1 to S15
cytochrome c to activate caspases but also initiate 14. T. Kuwana et al., Cell 111, 331 (2002). Tables S1 and S2
15. J. F. Lovell et al., Cell 135, 1074 (2008). References
caspase-independent mitochondrial dysfunc- 16. E. Gavathiotis et al., Nature 455, 1076 (2008).
tion (9). Accordingly, persistent interdigital webs 17. S. N. Willis et al., Science 315, 856 (2007). 30 March 2010; accepted 14 October 2010
and accumulation of hematopoietic cells were ob- 18. H. Kim et al., Mol. Cell 36, 487 (2009). 10.1126/science.1190217
Arabidopsis Type I Metacaspases nuclease, cleaved during programmed cell death

in pine, is the only in vivo type II metacaspase
Control Cell Death

substrate defined in plants (11). Although classic
animal caspases cleave after an aspartate at the P1
position, metacaspases cleave after a basic amino
Nuria S. Coll,1 Dominique Vercammen,2* Andrea Smidler,1 Charles Clover,1† acid residue such as lysine or arginine (9).
Frank Van Breusegem,2 Jeffery L. Dangl,1,3,4‡ Petra Epple1‡ LSD1 is a negative regulator of cell death ini-
tiated by localized superoxide production occurring
during the hypersensitive response (HR), a cell
Metacaspases are distant relatives of animal caspases found in protozoa, fungi, and plants. Limited death that often accompanies pathogen recog-
experimental data exist defining their function(s), despite their discovery by homology modeling a nition. HR sites form normally in lsd1 (12), but
decade ago. We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically the typical sharp boundary between dead and
control programmed cell death in Arabidopsis. AtMC1 is a positive regulator of cell death and live cells subsequently breaks down and “run-
requires conserved caspase-like putative catalytic residues for its function. AtMC2 negatively away cell death” ensues throughout the leaf (12).
regulates cell death. This function is independent of the putative catalytic residues. Manipulation of This phenotype requires proteins that are also
the Arabidopsis type I metacaspase regulatory module can nearly eliminate the hypersensitive cell necessary for pathogen recognition and salicylic
death response (HR) activated by plant intracellular immune receptors. This does not lead to acid accumulation (12–16). Hence, lsd1 is a sen-
enhanced pathogen proliferation, decoupling HR from restriction of pathogen growth. sitized genetic background for studying the con-
trol of oxidative stress–dependent cell death
rogrammed cell death is essential for plant caspases in the CD cysteine protease superfamily (17–20).
P development (1). Metacaspases in plants,

fungi, and protozoa are distant homologs of
(caspases, paracaspases, gingipains, clostripains,
legumains, and separin). These evolutionarily re-
lated proteases share the caspase-hemoglobinase
All three type I Arabidopsis metacaspases,
AtMC1 (At1g02170), AtMC2 (At4g25110), and
AtMC3 (At5g64240), possess a conserved, plant-
1
Department of Biology, 108 Coker Hall, University of North
fold, and common catalytic-site domains (2, 3). The specific LSD1-like zinc-finger N-terminal motif
Carolina (UNC), CB 3280, Chapel Hill, NC 27599–3280, USA. Arabidopsis thaliana genome encodes three type I (CxxCRxxLMYxxGASxVxCxxC) (21) (fig. S1A).
2
VIB Department of Plant Systems Biology and Department of and six type II metacaspases (AtMCs) (3). Both The LSD1 zinc-finger domain can function in
Plant Biotechnology and Genetics, Ghent University, 9052 types contain a conserved putative catalytic domain protein interactions (22). In yeast, LSD1 and AtMC1
Ghent, Belgium. 3Curriculum in Genetics and Molecular Biol-
and plausible sites for autocatalytic processing but interact via their zinc-finger domains (fig. S1, B
ogy and Department of Microbiology and Immunology, UNC,
Chapel Hill, NC 27599, USA. 4Carolina Center for Genome differ in their N-terminal domains. Despite many to D). In contrast, AtMC2 interacts only very weak-
Sciences, UNC, Chapel Hill, NC 27599, USA. reports of caspase-like activities in plants (4–9), no ly with either AtMC1 or LSD1 in this assay (fig.
*Present address: Innogenetics, Technologiepark 6, 9052 Ghent, experimental data exist defining functions or S1, B to D). We substantiated the AtMC1-LSD1
Belgium. substrates for plant type I metacaspases. Type II interaction using in vivo coimmunoprecipitation
†Present address: Department of Anesthesiology, Wake Forest metacaspase functions are nearly as enigmatic: in transgenic Arabidopsis (Fig. 1B) and in tran-
University School of Medicine, Medical Center Boulevard,
Winston-Salem, NC 27157, USA.
Recombinant plant type II metacaspases AtMC4 sient expression assays in Nicotiana benthamiana
‡To whom correspondence should be addressed. E-mail: and AtMC9 can undergo in vitro autocatalytic (23) (fig. S2A). The predicted N-terminal prodo-
pepple@email.unc.edu (P.E.); dangl@email.unc.edu ( J.L.D.) processing (10), and the Tudor staphylococcal main of AtMC1 is required for interaction with

REPORTS
LSD1 (fig. S2A). AtMC2 does not coimmunopre- AtMC1 is a positive mediator of lsd1 runaway cell We monitored AtMC1 or AtMC2 expression in
cipitate with either LSD1 (fig. S2B) or AtMC1 death, and AtMC2 acts genetically as a negative transgenic plants carrying promoter b-glucuronidase
(fig. S2C) under these conditions. regulator of AtMC1. (GUS) reporter genes (pAtMC1::GUS and
We obtained transfer DNA (T-DNA) insertion- Hemagglutinin (HA)–epitope-tagged AtMC1 and pAtMC2::GUS, respectively) in either Col-0 or
al mutant alleles of atmc1, atmc2, and lsd1 in the AtMC2 constructs controlled by their native promot- lsd1. AtMC1 expression was confined to the leaf
Col-0 accession (Fig. 1A and fig. S1B). atmc1, ers (pAtMC1::AtMC1-HA and pAtMC2::AtMC2-HA) veins. However, 24 hours after BTH treatment,
atmc2, and the atmc1 atmc2 double mutant exhib- complemented the respective mutant phenotypes AtMC1 was expressed in a narrow zone of sev-
ited no signs of enhanced production of reactive for BTH-induced ion leakage in lsd1 (Fig. 2B). eral cells adjacent to cell death sites in lsd1 (fig.
oxygen species and had no obvious phenotypes (fig. AtMC1-HA protein was detectable 1 day after S4A). Cells expressing AtMC1 are destined to die
S3). We generated lsd1 atmc1 and lsd1 atmc2 dou- BTH spray, before plants displayed any visible as runaway cell death expands over time (15).
ble mutants, and the lsd1 atmc1 atmc2 triple mutant sign of cell death, and accumulated thereafter (Fig. AtMC2, in contrast, was expressed at low levels,
to determine whether atmc1 or atmc2 modifies the 2C). AtMC2-HAwas undetectable until 2 days after except for a halo of nonstained cells outlining
lsd1 cell death phenotype. lsd1 runaway cell death BTH treatment and accumulated to lower levels than cells that are committed to die (fig. S4B).
can be induced with benzo(1,2,3)thiadiazole-7- AtMC1-HA (Fig. 2C). We observed no HA-tagged We infected Col-0 pAtMC1::GUS and pAtMC2::
carbothioic acid S-methyl ester (BTH), a sal- low-molecular-weight products, suggesting either GUS transgenic plants with either the obligate bio-
icylic acid analog (16). lsd1 exhibited maximum a lack of processing during activation or instability trophic oomycete Hyaloperonospora arabidopsidis
ion leakage (a cell death proxy) 96 hours after BTH of a putative C-terminal HA-tagged fragment. (Hpa; isolate Emwa1), or the hemibiotrophic bacte-
treatment. The Col-0 wild type, atmc1, atmc2, and

atmc1 atmc2 did not display significant increases in
ion leakage (Fig. 2A). lsd1 atmc1 exhibited sup-
pressed BTH-induced ion leakage (Fig. 2A, left).
In contrast, lsd1 atmc2 displayed accelerated
BTH-induced ion leakage (Fig. 2A, right). Ion
leakage in lsd1 atmc1 atmc2 was similar to that in
lsd1 atmc1 (Fig. 2A, right). Consistent with these
data, morphological lsd1 phenotypes were abol-
ished in the absence of AtMC1, and more pro-
nounced in the lsd1 atmc2 mutant (fig. S3). Hence,
Fig. 1. AtMC1 interacts with LSD1. (A) Scheme of

AtMC1 and AtMC2 proteins (not to scale). Z1, LSD1-
like zinc finger; PP, proline-rich region; p20 and p10,
putative caspase-like catalytic domains (2); numbered
H and C, putative catalytic residues (2); atmc1 and
atmc2, T-DNA insertion sites. (B) AtMC1 and LSD1
coimmunoprecipitate. Protein extracts from leaves of
lsd1 atmc1 [pLSD1::LSD1-myc] × lsd1 atmc1 [Dex-
AtMC1-HA] F1 plants were immunoprecipitated with
anti-myc–coupled magnetic beads. Crude extract (in- Fig. 2. AtMC1 and AtMC2 antagonistically control lsd1 runaway cell death. (A and B) Four-week-old
put) and eluate (elution) were analyzed by SDS– homozygous transgenic plants (genotypes, right) were sprayed with 150 mM BTH, and conductivity was
polyacrylamide gel electrophoresis (SDS-PAGE) with measured at the time points indicated. Repeated three times in (A) and once in (B). Error bars indicate two
anti-HA (top) or anti-myc (bottom) immunoblot. The times the standard error, calculated from four replicate measurements per genotype and data point.
eluate is 8× concentrated as compared to the input. Letters a to d at right represent groups with significant differences [P < 0.05, Tukey’s honest significant
The asterisk denotes a nonspecific cross-reacting band; difference (HSD) test]. hpt, hours post treatment. (C) Four-week-old plants of the indicated genotypes
arrowheads are ~34 kD, the expected apparent mo- were sprayed with 150 mM BTH. Tissue was harvested at the indicated time points, and 50 mg of total
lecular mass of LSD1-myc. WB, Western blot. protein were analyzed by SDS-PAGE and anti-HA immunoblot.

REPORTS
ria Pseudomonas syringae pv. tomato [Pto; strain yeast (27). In Trypanosoma brucei metacaspase expressing full-length AtMC2-HA (fig. S6, A and
DC3000(avrRpm1)] to trigger HR via the spe- TbMCA4, a second, metacaspase-specific cys- B). Cell death suppression was nearly complete
cific intracellular Toll–interleukin-1 receptor (TIR) teine residue was catalytic, and the corresponding in AtMC2-DN-HA–expressing lines (Fig. 4A and
and coiled-coil (CC)–nucleotide-binding–leucine- residue compensated for mutation of the classic figs. S6C and S7). Surprisingly, the predicted cat-
rich repeat (NB-LRR) immune receptors RPP4 cysteine residue in AtMC9 (28, 29). We gener- alytic cysteines were not required for cell death
(24) and RPM1 (25), respectively. AtMC1 was ac- ated Col-0 transgenic lines expressing high or suppression by either AtMC2 or AtMC2-DN-HA
tivated upon NB-LRR–based pathogen recogni- low levels of either a conditional AtMC1 expres- (Fig. 4A and fig. S6B).
tion and subsequently expressed in a spatially sion construct containing a cysteine-to-alanine To assess whether AtMC2 suppressed NB-
restricted manner in cells destined to undergo HR mutation (AtMC1-C220A-HA) or a double mu- LRR–mediated HR, we infected Col-0, atmc1,
(fig. S5). In contrast, AtMC2 was expressed in a tation including the potentially compensatory atmc2, atmc1 atmc2, and transgenic plants ex-
relatively more diffuse zone around infection sites cysteine (AtMC1-C99A-C220A-HA) (Fig. 3B). pressing AtMC2-DN-HA or AtMC2-DN-C135A-
(fig. S5). Conditional overexpression of AtMC1-HA re- C256A-HA with Pto DC3000(avrRpm1). We
We generated transgenic plants conditionally sulted in a dose-dependent increase in ion leakage, observed enhancement of RPM1-mediated HR
expressing either a full-length or an N-terminal whereas overexpression of either AtMC1-C220A- in atmc2 (Fig. 4B). Conversely, we observed
truncated version of AtMC1 lacking the LSD1- HA or AtMC1-C99A-C220A-HA did not (Fig. 3C). suppression of RPM1-mediated HR in atmc1
like extension (AtMC1-HA and AtMC1-DN-HA, Hence, AtMC1-C220 is required for function, and and in atmc1 atmc2 at low bacterial inocula
respectively). High levels of AtMC1-HA accu- AtMC1-C99 cannot compensate for its loss. resembling natural infections (Fig. 4B), but not at
mulation resulted in cell death in Col-0 trans- Overexpression of AtMC1 did not induce cell artificially high doses. Conditional expression of

genics; lower levels did not (Fig. 3A). Col-0 death in cotyledons before infection. However, either AtMC2-DN (Fig. 4C and fig. S6F) or full-
AtMC1-DN-HA plants accumulating relatively massive cell death occurred after infection with length AtMC2 (fig. S6, D and E) suppressed
low levels of protein nevertheless exhibited Hpa and activation of RPP4 (Fig. 3, D and E). RPM1-mediated HR, phenocopying atmc1.The
enhanced cell death (Fig. 3A). We failed to Hence, AtMC1 activation in seedlings, in contrast suppression of HR did not result in increased
recover transgenics expressing AtMC1-DN-HA in to adult plants (Fig. 3A), requires an additional susceptibility to Pto DC3000(avrRpm1) (Fig. 4,
lsd1 either by direct transformation or from signal, which is consistent with the fact that lsd1 D and E). RPP4-mediated HR was also sup-
crosses of two independent Col-0 AtMC1-DN- runaway cell death also does not occur in pressed by conditional expression of AtMC2-DN
HA-expressing transgenic lines to lsd1 [see cotyledons. and in atmc1 (Fig. 4F). Thus, AtMC2 inhibits at
methods in supporting online material (SOM)]. We generated similar transgenics in lsd1 and least RPM1- and RPP4- mediated HR, via direct
These results suggest that the LSD1-like putative Col-0 that conditionally expressed full-length or indirect regulation of AtMC1. The putative
prodomain of AtMC1 negatively regulates its AtMC2 (AtMC2-HA), an analogous N-terminal catalytic sites of AtMC2 are not required for HR
pro–cell death activity. truncated version of AtMC2 (AtMC2-DN-HA) and suppression (Fig. 4, B, C, and F, and fig. S6E).
Typical caspase active sites include a histidine- either AtMC2 or AtMC2-DN with cysteine-to- atmc2 did not exhibit increased RPP4-mediated
cysteine catalytic dyad, also found in metacaspases alanine mutations in the predicted catalytic (C256) HR (30). Although Hpa Emwa1 is an obligate
(3). The predicted catalytic cysteine residue was and potentially compensatory (C135) cysteine biotrophic oomycete, AtMC2-DN–mediated sup-
essential for autoprocessing of the pine metacaspase residues (AtMC2-C135A-C256A-HA and AtMC2- pression of HR did not abolish RPP4-dependent
mcII-Pa (26), of Arabidopsis AtMC4 and AtMC9 DN-C135A-C256A-HA). BTH-induced lsd1 run- pathogen growth restriction (Fig. 4F). These
in bacteria (10), and of AtMC1 and AtMC5 in away cell death was suppressed in transgenic lines surprising observations are consistent with pre-
Fig. 3. AtMC1 function is negatively regulated by

its LSD1-like putative prodomain and requires
predicted metacaspase catalytic residues. (A)
Three-week-old plants from sets of three inde-
pendent homozygous transgenics of indicated
genotypes in Col-0 were sprayed with 20 mM
dexamethasone (Dex). Tissue was harvested 1 day
after treatment; total protein was analyzed by SDS-
PAGE with anti-HA immunoblot (upper panel).
Dex-treated plants photographed 3 days after
treatment are shown (lower panel). (B) As in (A), but using two independent transgenics of the genotypes listed above the blot. (C) AtMC1-mediated ion
leakage requires the putative catalytic cysteine residue C220. Plants treated as described in (A) were harvested at 46 hpt and processed as described in the SOM.
Error bars represent two times the standard error. Letters a to c represent experimental groups with significant differences (P < 0.05, Tukey’s HSD test). The
experiment was repeated twice. (D and E) AtMC1 function in cotyledons requires additional signals. Ten-day-old seedlings were pre-treated with 20 mM Dex
(+Dex). One day later, half of the seedlings were inoculated with 50,000 spores/ml of Hpa isolate Emwa1 (Dex + Hpa) to activate RPP4. All seedlings were
harvested 3 days after inoculation and stained with Trypan blue to visualize cell death. (D) Pictures of representative Trypan blue–stained cotyledons. (E)
20 cotyledons were evaluated per genotype and treatment. CD, cell death; <50% of cot., extensive cell death covering less than 50% of cotyledon; >50%
of cot., extensive cell death covering more than 50% of cotyledon.

REPORTS
vious results showing that HR can occur in the Our data establish AtMC1 as a pro-death are enhanced by removal of this domain and, at
absence of pathogen growth restriction and that caspase-like protein required for both superoxide- least for AtMC1, this domain is required for
HR can be inhibited by caspase inhibitors (6, 31). dependent cell death in a reactive oxygen–sensitized interaction with LSD1. In the absence of LSD1,
The pathogen-stress marker PR1 is not induced state and for full HR mediated by intracellular AtMC1-dependent cell death requires addition-
by AtMC2-DN overexpression, suggesting that NB-LRR immune receptor proteins. AtMC2 anal, unknown signals and is developmentally
these results are not a general consequence of tagonizes these functions. AtMC1 and AtMC2 regulated. Hence, activation of AtMC1 is com-
stress. Finally, AtMC2 control of AtMC1 is post- contain a plant-specific N terminus shared with plex and probably context-dependent. The in-
transcriptional (fig. S7). LSD1. The functions of both AtMC1 and AtMC2 hibitory function of AtMC2 does not require
classic cysteine catalytic residues. The AtMC1-
AtMC2 antagonism is reminiscent of animal
caspase-12, which negatively regulates caspase-1
to dampen the inflammatory response to bacte-
ria and in colitis-associated colorectal cancer
(32, 33); these caspase-12 functions do not re-
quire catalytic function (33). Caspase-12 also
inhibits NOD-like receptor-mediated innate im-
munity independent of caspase-1 (34), provid-
ing a striking parallel to our observation that

AtMC2 inhibits AtMC1-dependent cell death
controlled by analogous plant NB-LRR innate
immune receptors. These results suggest an an-
cient link between cell death control by divergent
metacaspase/caspase proteases and innate im-
mune receptor function governed by NB-LRR or
NLR proteins.

1. E. Lam, Nat. Rev. Mol. Cell Biol. 5, 305 (2004).
2. L. Aravind, E. V. Koonin, Proteins 46, 355 (2002).
3. A. G. Uren et al., Mol. Cell 6, 961 (2000).
4. N. V. Chichkova et al., EMBO J. 29, 1149 (2010).
5. N. Hatsugai et al., Genes Dev. 23, 2496 (2009).
6. N. Hatsugai et al., Science 305, 855 (2004).
7. E. Lam, O. del Pozo, Plant Mol. Biol. 44, 417 (2000).
8. E. Rojo et al., Curr. Biol. 14, 1897 (2004).
9. D. Vercammen, W. Declercq, P. Vandenabeele,
F. Van Breusegem, J. Cell Biol. 179, 375 (2007).
10. D. Vercammen et al., J. Biol. Chem. 279, 45329 (2004).
11. J. F. Sundström et al., Nat. Cell Biol. 11, 1347 (2009).
12. R. A. Dietrich et al., Cell 77, 565 (1994).
13. R. A. Dietrich, M. H. Richberg, R. Schmidt, C. Dean,
J. L. Dangl, Cell 88, 685 (1997).
14. D. H. Aviv et al., Plant J. 29, 381 (2002).
15. T. Jabs, R. A. Dietrich, J. L. Dangl, Science 273,
1853 (1996).
16. C. Rustérucci, D. H. Aviv, B. F. Holt 3rd, J. L. Dangl,
J. E. Parker, Plant Cell 13, 2211 (2001).
17. A. Mateo et al., Plant Physiol. 136, 2818 (2004).
18. P. Mühlenbock, M. Plaszczyca, M. Plaszczyca, E. Mellerowicz,
S. Karpinski, Plant Cell 19, 3819 (2007).
19. P. Mühlenbock et al., Plant Cell 20, 2339 (2008).
20. M. A. Torres, J. D. Jones, J. L. Dangl, Nat. Genet. 37,
1130 (2005).
Fig. 4. AtMC2 negatively regulates AtMC1. Seedlings of the depicted genotypes were sprayed with 1 mM 21. Single-letter abbreviations for the amino acid residues
Dex 6, 11, and 16 days after germination or left unsprayed. CACA denotes constructs where both putative are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;
catalytically active cysteine residues (C135A C256A for AtMC2) were mutated to alanine. (A) For ion leakage G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro;
Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; and
measurements, plants were sprayed with 150 mM BTH 1 day after the last treatment with Dex. Tissue was x, any amino acid.
harvested at 43 hpt and processed as described in the SOM. Blue lines, Dex-treated genotypes; yellow lines, 22. H. Kaminaka et al., EMBO J. 25, 4400 (2006).
non–Dex-treated controls. Error bars represent two times the standard error. Letters a and b at right 23. Materials and methods are available as supporting
represent experimental groups with significant differences (P <0.05, Tukey’s HSD test). The experiment was material on Science Online.
repeated twice. (B) Plants were vacuum-infiltrated with 250,000 colony-forming units (CFU)/ml of Pto 24. E. A. van der Biezen, C. T. Freddie, K. Kahn, J. E. Parker,
J. D. Jones, Plant J. 29, 439 (2002).
DC3000(avrRpm1). Twelve hours later, plants were harvested and stained with Trypan blue to visualize cell
25. M. R. Grant et al., Science 269, 843 (1995).
death. To quantify cell death, all dead cells in one field of vision (10× magnification) were counted. Average 26. P. V. Bozhkov et al., Proc. Natl. Acad. Sci. U.S.A. 102,
and two times the standard error were calculated from 20 leaves per genotype and treatment. The 14463 (2005).
experiment was repeated. (C) Ten hours after the last treatment with Dex, plants of indicated genotypes 27. N. Watanabe, E. Lam, J. Biol. Chem. 280, 14691 (2005).
were treated as in (B). (D) Seedlings were dip-infiltrated with Pto DC3000(avrRpm1) at 2.5 × 107 CFU/ml. 28. B. Belenghi et al., J. Biol. Chem. 282, 1352 (2007).
The average and two times the standard error were calculated from four samples per genotype. The 29. A. Szallies, B. K. Kubata, M. Duszenko, FEBS Lett. 517,
144 (2002).
experiment is representative of three independent replicates. (E) Three hours after the last Dex spray, plants
30. Because this assay counts interaction sites, the number of
were treated as in (D). (F) One day after the last Dex treatment, plants were inoculated with 50,000 spores/ spores that infect each leaf determines the maximum
ml of Hpa Emwa1. Three days later, plants were stained with Trypan blue, and interaction sites per field of number of interactions that can occur per leaf. This cannot
vision were counted. The experiment was repeated three times. TN, trailing necrosis; FH, free hyphae. be altered by an increase in RPP4-dependent responses.

REPORTS
31. K. S. Century, E. B. Holub, B. J. Staskawicz, Proc. Natl. Fellowship (PBEZA-115173) to N.S.C.; and the Research Supporting Online Material
Acad. Sci. U.S.A. 92, 6597 (1995). Fund, Ghent University (grant 12051403), and Fonds www.sciencemag.org/cgi/content/full/science.1194980/DC1
32. J. Dupaul-Chicoine et al., Immunity 32, 367 (2010). voor Wetenschappelijk Onderzoek-Vlaanderen to Materials and Methods
33. M. Saleh et al., Nature 440, 1064 (2006). F.V.B. and D.V., respectively. We thank D. Baltrus for Figs. S1 to S7
34. P. M. LeBlanc et al., Cell Host Microbe 3, 146 (2008). statistical analysis, T. Perdue of the UNC Biology References
35. We thank J. McDowell (Virginia Tech, Blacksburg, VA) Microscope Facility for patient and expert assistance,
and M. Nishimura (UNC) for critical reading of the E. Washington and T. Eitas for pMDC7 vectors, B. van de 12 July 2010; accepted 19 October 2010
manuscript. This work was funded by NIH RO1 Cotte for technical assistance, and K. Overmyer for Published online 18 November 2010;
GM057171 to J.L.D.; a Swiss National Science Foundation early contributions to this work. 10.1126/science.1194980
An Antagonistic Pair of FT Homologs to cold temperature and is subsequently induced

by other inductive pathways (1). Vernalization
of the winter-annual Arabidopsis and other mem-
Mediates the Control of Flowering bers of the Brassicaceae family involve several
steps that culminate in the repression of the major
Time in Sugar Beet floral inhibitor gene FLOWERING LOCUS C
(FLC) (2–5). In cereals, cold temperature stimuli
are integrated by the activation of the FRUIT-
Pierre A. Pin,1,2 Reyes Benlloch,1 Dominique Bonnet,3 Elisabeth Wremerth-Weich,2 FULL (FUL)/APETALA1 (AP1) homolog VRN1

Thomas Kraft,2 Jan J. L. Gielen,3 Ove Nilsson1* (6), which represses the CONSTANS, CONSTANS-
like, and TIMING OF CAB EXPRESSION 1
Cultivated beets (Beta vulgaris ssp. vulgaris) are unable to form reproductive shoots during the
(CCT)-domain transcription factor VRN2 (7, 8).
first year of their life cycle. Flowering only occurs if plants get vernalized, that is, pass through
The phosphatidylethanolamine-binding protein
the winter, and are subsequently exposed to an increasing day length (photoperiod) in spring. Here, we
(PEBP) gene family, found in both angiosperms
show that the regulation of flowering time in beets is controlled by the interplay of two paralogs of
and gymnosperms (9), has evolved both activa-
the FLOWERING LOCUS T (FT) gene in Arabidopsis that have evolved antagonistic functions. BvFT2 is
tors and repressors of flowering. The Arabidopsis
functionally conserved with FT and essential for flowering. In contrast, BvFT1 represses flowering and its
down-regulation is crucial for the vernalization response in beets. These data suggest that the beet
1
has evolved a different strategy relative to Arabidopsis and cereals to regulate vernalization. Umeå Plant Science Centre, Department of Forest Genetics
and Plant Physiology, Swedish University of Agricultural Sci-
ences, 901-83 Umeå, Sweden. 2Syngenta Seeds AB, Box 302,
n flowering plants, the timing of the tran- genous stimuli, such as changes in day length
I sition from the vegetative to the reproductive

stage is determined by interactions between
the developmental state of the plant and exo-
and temperature. Vernalization is a checkpoint
through which many plants must pass, where
flowering is conditional on prolonged exposure
261-23 Landskrona, Sweden. 3Syngenta Seeds SAS, 12 Chemin
de l’Hôbit, 31790 Saint-Sauveur, France.
Ove.Nilsson@genfys.slu.se
Fig. 1. Expression pattern of BvFT1 and BvFT2 in

beets (15). (A) Expression of BvFT1 and BvFT2 in
various tissues, including se., seed; hy., hypocotyl;
co., cotyledon; le., leaf; ap., apex; ro., root; st., stem;
bu., floral bud, and fl., flower. Samples were har-
vested in LDs at zeitgeber time (ZT) 8 (i.e., 8 hours
after lights on). Error bars, mean T SE (n = 3). (B)
Expression of BvFT1 and BvFT2 in leaf samples in
LDs across different developmental stages in an-
nuals and biennials. Samples were harvested at
ZT6. Error bars, mean T SE (n = 3). (C and D) Diurnal
rhythms of BvFT1 and BvFT2 in annual, biennial, and
vernalized-biennial beets in SDs (C) and LDs (D). Error
bars, mean T SE (n = 5). (E) Leaf number at time of
bolting in biennial, vernalized-biennial, and annual
plants grown under the different photoperiodic
conditions described in (C) and (D). Error bars,
mean T SD (n = 6). (F) BvFT1 transcript accumula-
tion in biennials in response to vernalization. Leaf
samples were harvested at ZT6. Error bars, mean T
SE (n = 3).

REPORTS
Fig. 3. Simplified flowering models in beets, Ara-

bidopsis, and barley, showing the key integrators
of the vernalization pathway, which mediate tran-
scriptional regulation of the FT orthologs.
shift mutation in an FT-like gene, ensuing in

the HaFT1 gene acquiring a role in delaying
flowering through a dominant-negative interfer-
ence with an activating paralog, HaFT4 (18)
(fig. S6). This does not appear to be the case for
BvFT1, which acts as a true repressor on the basis

of the Arabidopsis overexpression phenotypes
(fig. S3, J and K), the differences between the
BvFT2 RNAi and BvFT1-ox phenotypes in beets
(Fig. 2 and fig. S5), and the fact that expression
of BvFT1 and BvFT2 is mutually exclusive rather
than overlapping (Fig. 1, A, C, and D).
As for all previously characterized FT-like
genes, transcripts of both BvFT1 and BvFT2 were
mainly detected in leaves (Fig. 1A), but their
respective temporal expression patterns were very
different. Whereas BvFT1 was expressed in the
leaves (Fig. 1A) of plants that were not compe-
tent to flower, that is, plants grown in short days
(SDs) or nonvernalized biennials (Fig. 1, C and
Fig. 2. Misexpression of BvFT1 or BvFT2 control bolting in sugar beet. (A to C) Nonbolting phenotype D), BvFT2 expression always correlated with
observed in BvFT2 RNAi annual (A), BvFT1-ox annual (B), and vernalized BvFT1-ox biennial (C) plants when flower induction and reproductively mature plants
grown in LDs for 6 weeks. (D to F) BvFT1 and BvFT2 expression in BvFT2 RNAi (D), BvFT1-ox (E), and vernalized (Fig. 1, A to D). These observations suggest
BvFT-ox (F) plants (15). Expression levels were set to unity in wild type (WT). Error bar, mean T SE (n = 3). that neofunctionalization of the pair of Beta FT
paralogs occurred in terms of both expression pro-
PEBP gene FLOWERING LOCUS T (FT) (10, 11) and gene expression analysis showed that BvFT2 files and protein function. Expression of BvFT1
is a major output of the photoperiod pathway is the functional FT ortholog in beets. Transgenic and BvFT2 displays diurnal oscillations (Fig. 1,
and controls the floral transition in response to expression of BvFT2 in both Arabidopsis and C and D), suggesting that both genes might be
changes in day length, whereas its close ho- sugar beet (fig. S3, B and G, and fig. S4) strong- under the control of the circadian clock. However,
molog TERMINAL FLOWER 1 (TFL1) acts as a ly promoted flowering. Furthermore, BvFT2 ex- BvFT1 is preferentially expressed in the morn-
strong floral repressor (10–12). pression levels were correlated with the initiation ing in SDs (Fig. 1C), whereas BvFT2 is expressed
Cultivated sugar beet (Beta vulgaris ssp. of flowering in both annual and biennial beets in the evening and in LDs (Fig. 1D). In annuals,
vulgaris) is a biennial crop that requires vernal- (Fig. 1B). When BvFT2 expression was sup- expression of both genes responded quickly to
ization to enter the generative phase. The vernal- pressed by RNA interference (RNAi) in annual changes in photoperiod, but in opposing ways
ization period typically needs to be followed plants, a continuous vegetative growth was ob- (fig. S7). Transcription of BvFT1 was not re-
by exposure to long days (LDs). Without LDs, served in LDs (Fig. 2A and fig. S5A), suggesting pressed in LDs in biennials before vernalization,
floral induction does not occur in beets, despite that BvFT2 is essential for flowering. Unexpected- and BvFT2 expression was not induced (Fig. 1D).
the fact that they become competent to flower ly, BvFT1 repressed flowering when ectopically This suggests that biennials might be insensitive to
after vernalization (13). One of the main factors expressed in transgenic sugar beet and Arabi- the photoperiodic signal before vernalization and
controlling flowering time in beets is the bolting dopsis plants (Fig. 2, B and C, fig. S5, B and C, that a vernalization-dependent factor acts to restore
gene B, a dominant promoter of flowering that and fig. S3, C and G). These data suggest that the photoperiodic regulation of BvFT1 and BvFT2.
overrides the need for vernalization (14). Plants an FT-like gene, evolutionarily more related to Consequently, these observations suggest that
carrying the dominant B allele behave as an- FT than to TFL1/CEN (figs. S1A and S6), has BvFT1 and BvFT2 regulation is dependent on
nuals and proceed to bolting and flowering as a evolved into a true flowering repressor. BvFT1 the presence of the dominant allele B.
direct response to LDs. expression was not affected in the BvFT2 RNAi Vernalization and photoperiod pathways are
To determine whether FT function is con- plants (Fig. 2D), whereas BvFT2 expression was connected by the coordinated repression of BvFT1.
served between beets and Arabidopsis, we ini- dramatically reduced compared with wild type BvFT1 transcription in biennials was both re-
tiated a first characterization of the PEBP genes in BvFT1-ox plants, both before (Fig. 2E) and duced during vernalization (Fig. 1F) and main-
in Beta (15), isolating two genes grouped within after (Fig. 2F) vernalization. tained after winter at low levels if plants were
the FT-like clade [BvFT1 and BvFT2 (fig. S1A)]. In sunflower, neofunctionalization (the evo- grown in LDs. These data indicate that cold treat-
Genetic mapping showed that none of the PEBP lution of new function and/or expression of a ment induces BvFT1 repression and also affects
homologs maps in vicinity to B (fig. S2) on chro- duplicated gene) (17) of FT function was pos- the vernalization-dependent factors that maintain
mosome II (16). Complementation experiments tulated to have occurred as a result of a frame- BvFT1 repression in LDs. Given that a transient

REPORTS
completely revert its repressing function to pro-
moting function (Fig. 4C and table S1). This
suggests that BvFT1 was initially a promoter of
flowering but that mutations within the external
loop of its protein resulted in a shift in function
to flowering repression. We are currently inves-
tigating which of the three amino acids are crit-
ical for the activator versus repressor function.
To get further insights into the evolution of
the BvFT1 repressor function, we isolated BvFT1-
and BvFT2-like sequences from other Beta
species, as well as other members of the Amaran-
thaceae family. BvFT2-like genes showed high
homology across the Amaranthaceae (fig. S10,
A and B). In contrast, we could only isolate
BvFT1-like sequences from Beta species, sug-
gesting that FT1 arose through gene duplication
during the diversification of the genus Beta. We

cannot exclude that FT1 may be present in other
Amaranthaceae, but given the high level of con-
servation of FT-like genes, our results suggest
that FT1 is likely to be restricted to Beta. Gene
expression analysis showed that in LDs FT1
was expressed in all cultivated varieties of B.
vulgaris, but not in wild Beta species, except in
some B. vulgaris ssp. maritima accessions from
northern latitudes (fig. S10C). In contrast, FT2
Fig. 4. Characterization of important regions of BvFT1 and BvFT2 involved in their antagonistic was expressed in these species, consistent with
functions. (A and B) Folding prediction of BvFT1 and BvFT2. The structures are shown as solid three- their flowering behavior. Indeed, B. macrocarpa,
dimensional traces with an alignment diversity color setting. (C) Chimeric FT proteins containing domains B. precumbens, and B. vulgaris ssp. maritima
of BvFT1 and BvFT2 show antagonistic function when expressed in Arabidopsis. accessions from southern latitudes bolt and flower
quickly without exposure to cold temperature,
exposure to SDs directly after vernalization [so BvFT1 itself is likely to regulate downstream while maritima accessions from northern latitudes
called “devernalization,” a condition known to bolting and flowering promoters. Such a scenario and all cultivated beets require vernalization to
abolish flowering in Beta (19)] resulted in the res- suggests that in beets an FT-like gene represses enter the generative phase (22). These results are
toration of BvFT1 expression (fig. S8), unfavorable bolting and flowering as part of the vernaliza- consistent with our findings in sugar beet, clearly
conditions for flowering appear to be coupled to tion response (Fig. 3). In Arabidopsis, FLC in- linking the BvFT1 expression in LDs to the bien-
expression of BvFT1, which suggests that BvFT1 hibits flowering, whereas in cereals, VRN2 (a nial growth behavior. Interestingly, domesticated
is important for the devernalization phenomenon. member of the CCT gene family) performs this beets (ssp. vulgaris) most likely originated from
On the basis of these results, we propose a role (Fig. 3). This suggests that the vernalization B. vulgaris ssp. maritima plants (23).
model whereby BvFT1 prevents flowering dur- response has developed differently not only in Because BvFT1 overexpression leads to the
ing SDs and before vernalization by repressing monocots and eudicots but also in the two eudicot prevention of bolting and flowering, even after
the expression of BvFT2 (Fig. 3). Biennials, lack- families Brassicaceae and Amaranthaceae (for- a prolonged vernalization period (Fig. 2C and
ing B, would be unable to sense the long-day merly Chenopodiaceae) within the evolution- fig. S5C), it may have interesting applications in
signal before vernalization because of high lev- arily diverged eudicot groups of the Rosids and breeding. Our BvFT1-ox plants may fulfill the
els of BvFT1 expression. During vernalization, Caryophyllales, respectively. Sequence compar- desire for a “winter beet” that can be planted in
BvFT1 expression decreases and the plant be- ison between the two proteins showed that both the fall without bolting during the following
comes competent to respond to LDs, which is BvFT1 and BvFT2 carry the functionally im- spring to provide a prolonged growing season
required to maintain low levels of BvFT1 expres- portant FT signatures, Tyr85(Y) and Gln140(Q) and harvesting campaign with increased yields.
sion. This appears to be necessary to induce flow- (fig. S1C) (20). However, the proteins differ with- This demonstrates how extending our understand-
ering. However, formally, we cannot exclude in the segment B of the fourth exon [encoding ing of the molecular determinants of vernaliza-
that other factors might also contribute to the an external loop of PEBP (21)] (Fig. 4, A and tion has direct applications in agriculture.
vernalization response and the regulation of B, and fig. S1C), which is important for FT ver-
BvFT2 (Fig. 3, dotted arrow). sus TFL1 function in Arabidopsis (21). BvFT2 References and Notes
1. S. Sung, R. M. Amasino, Curr. Opin. Plant Biol. 7, 4 (2004).
Unexpectedly, BvFT2 RNAi plants responded is identical in this region to most other FTs 2. S. D. Michaels, R. M. Amasino, Plant Cell 11, 949 (1999).
to vernalization and bolted after cold treatment (consistent with its role in promoting flowering), 3. C. C. Sheldon et al., Plant Cell 11, 445 (1999).
(fig. S9A), indicating that additional mechanisms whereas BvFT1 has three substitutions out of 14 4. M. Tadege et al., Plant J. 28, 545 (2001).
are acting to promote bolting in beets in paral- amino acids (Fig. 4A and fig. S1C). By swap- 5. R. Wang et al., Nature 459, 423 (2009).
6. L. Yan et al., Proc. Natl. Acad. Sci. U.S.A. 100, 6263 (2003).
lel to BvFT2. Although BvFT2 RNAi plants pro- ping regions between BvFT1 and BvFT2 (Fig. 7. L. Yan et al., Science 303, 1640 (2004).
ceeded to bolting, flowering was completely 4C and table S1), we demonstrated that diver- 8. M. N. Hemming, W. J. Peacock, E. S. Dennis, B. Trevaskis,
abolished or initiated at very late stages (fig. gence within these three amino acids of the ex- Plant Physiol. 147, 355 (2008).
S9, B and C), suggesting that BvFT2 is needed ternal loop is the major cause of their antagonistic 9. N. Gyllenstrand, D. Clapham, T. Källman, U. Lagercrantz,
Plant Physiol. 144, 248 (2007).
for a normal flower initiation. In contrast, be- functions. Remarkably, expression of BvFT1B2 10. I. Kardailsky et al., Science 286, 1962 (1999).
cause BvFT1-ox plants do not respond to vernal- (chimeric BvFT1 carrying the external loop of 11. Y. Kobayashi, H. Kaya, K. Goto, M. Iwabuchi, T. Araki,
ization followed by LDs (Fig. 2C and fig. S5C), BvFT2 with the three altered amino acids) can Science 286, 1960 (1999).

REPORTS
12. D. Bradley, O. Ratcliffe, C. Vincent, R. Carpenter, E. Coen, 22. J. P. W. Letschert, thesis, Beta Section Beta: Nucleotide sequences have been deposited with GenBank
Science 275, 80 (1997). Biogeographical Patterns of Variation and Taxonomy under accession numbers HM448909 to HM448918
13. G. D. H. Bell, A. B. Bauer, J. Agric. Sci. 32, 112 (1942). (Wageningen Agricultural University Papers, 93-1, and HQ148107 to HQ148132. The use of BvFT1 and
14. F. A. Abegg, J. Agric. Res. 53, 493 (1936). Wageningen, Netherlands 1993). BvFT2 in control of sugar beet flowering is the subject
15. Materials and methods are available as supporting 23. C. Jung, K. Pillen, L. Frese, S. Fähr, A. E. Melchinger, of the patent application WO2010/025888.
material on Science Online. Theor. Appl. Genet. 86, 449 (1993).
16. P. Boudry et al., Theor. Appl. Genet. 88, 852 (1994). 24. We thank F. Domène, J. Bensefelt, Å. Karlsson, R. Sant,
Supporting Online Material
17. S. Ohno, Evolution by Gene Duplication (Springer-Verlag, P. van Roggen, and A.-M. Nilsson for technical assistance, www.sciencemag.org/cgi/content/full/330/6009/1397/DC1
Materials and Methods
New York, ed. 1, 1970) and Y. Fischer for help in preparing the manuscript.
SOM Text
18. B. K. Blackman, J. L. Strasburg, A. R. Raduski, We also thank C. Bellini for critical reading of the
S. D. Michaels, L. H. Rieseberg, Curr. Biol. 20, 629 (2010). manuscript. This work was supported by the Swedish Figs. S1 to S10
Tables S1 to S3
19. H. Van Dijk, J. Exp. Bot. 60, 3143 (2009). Research Council and the Swedish Governmental Agency
20. Y. Hanzawa, T. Money, D. Bradley, Proc. Natl. Acad. Sci. for Innovation Systems to O.N. and T.K., as well as by References
U.S.A. 102, 7748 (2005). Südzucker. Phylogenetic data have been deposited with 26 August 2010; accepted 22 October 2010
21. J. H. Ahn et al., EMBO J. 25, 605 (2006). Dryad Digital Repository (DOI: 10.5061/dryad.4930). 10.1126/science.1197004
Alleviating Neuropathic Pain plasticity is the major cellular model used for un-
derstanding these mechanisms. In addition to
Hypersensitivity by Inhibiting PKMz

numerous studies on synaptic mechanisms for
learning and memory, these same synaptic mech-

anisms may be responsible for pathological pain
in the Anterior Cingulate Cortex conditions (2–4). For example, nerve injury trig-
gers long-term plastic changes along sensory path-
ways, from the peripheral sensory terminals to
Xiang-Yao Li,1,2* Hyoung-Gon Ko,3* Tao Chen,1,2* Giannina Descalzi,1 Kohei Koga,1
the spinal cord dorsal horn, amygdala, and cortex
Hansen Wang,1 Susan S. Kim,1 Yuze Shang,1 Chuljung Kwak,3 Soo-Won Park,3 Jaehoon Shim,2,3
(3–7). These plastic changes contribute to pain
Kyungmin Lee,3,4 Graham L. Collingridge,2,5 Bong-Kiun Kaang,2,3† Min Zhuo1,2†
perception, fear, and memory. The persistent nature
Synaptic plasticity is a key mechanism for chronic pain. It occurs at different levels of the central 1
Department of Physiology, Faculty of Medicine, Center for
nervous system, including spinal cord and cortex. Studies have mainly focused on signaling proteins the Study of Pain, University of Toronto, 1 King's College Circle,
that trigger these plastic changes, whereas few have addressed the maintenance of plastic changes Toronto, Ontario M5S 1A8, Canada. 2Department of Brain and
related to chronic pain. We found that protein kinase M zeta (PKMz) maintains pain-induced persistent Cognitive Sciences, Seoul National University, Seoul 151 747,
Korea. 3National Creative Research Initiative Center for Mem-
changes in the mouse anterior cingulate cortex (ACC). Peripheral nerve injury caused activation of ory, Department of Biological Sciences, College of Natural Sci-
PKMz in the ACC, and inhibiting PKMz by a selective inhibitor, z-pseudosubstrate inhibitory peptide ences, Seoul National University, San 56-1 Silim-dong, Gwanak-gu,
(ZIP), erased synaptic potentiation. Microinjection of ZIP into the ACC blocked behavioral sensitization. Seoul 151-747, Korea. 4Department of Anatomy, School of Med-
These results suggest that PKMz in the ACC acts to maintain neuropathic pain. PKMz could thus be icine, Kyungpook National University, 2-101 Dongin-Dong,
Daegu 700-422, Korea. 5Medical Research Council (MRC) Centre
a new therapeutic target for treating chronic pain. for Synaptic Plasticity, School of Physiology and Pharmacology,
University of Bristol, Bristol BS8 1TD, UK.
ne key function of the brain is to learn and Investigations into the molecular mechanisms of
O memorize sensory experiences and then

adapt subsequent behavioral responses.
such processes provide information for our basic
understanding of brain functions (1). Synaptic
†To whom correspondence should be addressed. E-mail:
kaang@snu.ac.kr (B.-K.K.); min.zhuo@utoronto.ca (M.Z.)
Fig. 1. Expression and phosphorylation of PKMz in the ACC during neuropathic

pain. (A) Mechanical allodynia was tested in the sham and nerve injury groups
from wild-type (WT) and AC1−/− animals 3 days after nerve injury. * indicates P <
0.05; error bars indicate SEMs. (B) Western blots for PKMz and p-PKMz in the ACC
obtained between 3 days and 2 weeks after nerve injury. (The ACC of mice from the
sham and nerve injury groups were used for Western blot analysis 90 min after the allodynia test.) (C) Level of PKMz in the ACC of mice from the sham and nerve
injury groups. Level of PKMz increased significantly 3 days after nerve injury compared with PKMz in the sham group. (D) Level of p-PKMz increased significantly 3
days after nerve injury. This effect lasted over 2 weeks after nerve injury compared with p-PKMz in the sham group. (E) Peripheral nerve injury did not increase the
levels of PKMz and p-PKMz in the ACC of AC1−/− mice. (F) Forskolin incubation increased PKMz (gray) and p-PKMz (blue) levels in the ACC of WT mice.

REPORTS
of chronic pain results in resistance to commonly tenance of pain-related long-term synaptic plasticity Multiple protein kinases are thought to con-
used analgesics (6). Therefore, revealing the mo- changes is critical for developing effective chronic tribute to the induction of long-term potentiation
lecular mechanisms that are involved in the main- pain treatments. (LTP) and initial consolidation of information
storage. Among them, only protein kinase M zeta
(PKMz) maintains persistent synaptic changes
(8–10). PKMz, an atypical isoform of protein
kinase C (PKC), has been detected in many re-
gions of the brain, including the frontal cortex
(11). In the hippocampus, LTP induction triggers
the synthesis of PKMz, and activation of PKMz
is critical for late-phase LTP (L-LTP) and mem-
ory consolidation (8, 12–15).
The anterior cingulate cortex (ACC) is a key
cortical area involved in chronic pain (2, 16, 17).
We first examined whether, in the ACC, periph-
eral nerve injury causes changes in PKMz (18).
As reported previously (17, 19), behavioral allodynic
response was increased 3 days after nerve injury

when compared with the response in sham-treated
mice (Fig. 1A). The levels of PKMz in the ACC
were significantly increased after nerve injury
(sham-operated versus nerve injury group; n = 9
mice for each group, unpaired t test, t16 = –2.51,
P < 0.05; Fig. 1, B and C). Because PKMz is
activated by phosphorylation, we also conducted
experiments to detect possible changes in the
level of phosphorylated PKMz (p-PKMz). Con-
sistently, the level of p-PKMz was also signifi-
cantly increased (n = 8 for each group, unpaired t
test, t14 = –2.26, P < 0.05; Fig. 1, B and D). These
data suggest that peripheral nerve injury increases
PKMz activity in the ACC.
To determine whether such changes are long-
lasting, we examined PKMz and p-PKMz levels
7 and 14 days after nerve injury. Although be-
havioral allodynia persisted at these time points,
the protein levels of PKMz returned to baseline
(Fig. 1C), indicating that the regulation of the
amount of PKMz is short-lasting. However, the
p-PKMz level remained increased [two-way
analysis of variance (ANOVA), F1,28 = 6.10,
P < 0.05; Fig. 1D], suggesting that PKMz activ-
ity could contribute to the maintenance of neuro-
pathic pain. To investigate whether the changes
in PKMz protein levels or activity were a gener-
alized phenomenon in the central nervous sys-
tem, we also examined the levels of PKMz and
p-PKMz in the hippocampus and spinal dorsal
horn 3 days after nerve injury. PKMz and p-PKMz
levels in the hippocampus and spinal cord did
Fig. 2. Bilateral microinjections of ZIP into the ACC decreased allodynia in mice with peripheral nerve injury. (A) The not change after nerve injury (fig. S1), suggesting
schematic view of the microinjection experiments. (B) Location of microinjection sites in the ACC. Solid circles represent that changes in PKMz are affected in a regionally
ZIP injection sites, whereas open circles represent saline injection sites. The right image shows an example of the specific manner in response to peripheral nerve
location of the cannula tip in one ACC slice with hematoxylin and eosin staining. The injection site is indicated by an injury.
asterisk. (C) Microinjection of ZIP (blue) into the ACC decreased the positive response rate in the nerve injury animal,
What could be the possible second messen-
whereas saline microinjection (black) into the ACC had no effect on the response level. Open gray circles represent the
summary control data of positive response level tested from the saline and ZIP group. The red arrows indicated the gers that trigger the activation and synthesis of
time point of ZIP injection. *P < 0.05; error bars, SEMs. (D) Two hours after injection, ZIP still has an analgesic effect. PKMz in the ACC? In the ACC, calmodulin-
However, the allodynia response recovered to preinjection level 24 hours after injection. (E) ZIP has no effect on the stimulated adenylyl cyclase 1 (AC1) is critical for
traveled distance in the open field test. (F) The location of microinjection sites in the ACC in the conditioned place ACC LTP and behavioral sensitization caused by
preference experiments. Solid or open circles indicate the location of the injection that did or did not induce place peripheral nerve injury (16, 20). Thus, we exam-
preference, respectively. (G) The changed time in the ZIP paired chamber was significantly different from that of the ined whether AC1 acts upstream of PKMz in
saline paired chamber. *P < 0.05. (H) The effect of ZIP infusion into the ACC on contextual fear memory. the ACC after nerve injury by using AC1 gene
Representative brain slice shows the location of the injection site. ZIP was infused into the ACC 2 hours before memory knockout mice (AC1−/−). AC1−/− mice did not
retrieval, which was tested 3, 7, and 18 days after conditioning. (I) ZIP did not change the response latency in the tail- show any significant behavioral sensitization 3
flick (TF) test. (J) Microinjection of ZIP into the ACC had no effect on the paw-withdrawal threshold in normal mice. days after nerve injury (Fig. 1A). Similar amounts

REPORTS
of PKMz and p-PKMz were found in sham- and regionally selective analgesic effect on nerve receptor-mediated excitatory postsynaptic currents
operated and nerve-injured AC1−/− mice 3 days injury–related tonic pain. (EPSCs) (23). Because glutamatergic synaptic trans-
after nerve injury (n = 9 for each group, unpaired t What is likely to be the synaptic mechanism mission in the ACC is increased after nerve injury
test, PKMz, t16 = –0.99, p-PKMz, t16 = –0.67, P > responsible for the analgesic effects produced by (17), we speculated that PKMz may contribute to
0.05, Fig. 1E). To further examine the link be- ZIP in neuropathic pain? PKMz can potentiate the maintenance of enhanced synaptic transmis-
tween the AC1 and PKMz, we performed brain postsynaptically the amplitude of a-amino-3- sion induced by nerve injury. First, we recorded
slice experiments. We used bath application of hydroxy-5-methyl-4-isoxazole-propionate (AMPA) AMPA receptor-mediated EPSCs in layers II and
forskolin (5 mM), a cell-permeable activator of
AC. Forskolin treatment increased the amount
of PKMz and p-PKMz in a time-dependent man-
ner (Fig. 1F). The increase in PKMz was rapid;
the amount of PKMz was increased within 5 min
after forskolin (5 mM) treatment (one-way ANOVA,
F3,15 = 4.52, P < 0.05, n = 7 per group; Fig. 1F).
This suggests that adenosine 3´,5´-monophosphate
(cAMP) signaling increases PKMz levels in a
transcription-independent manner, because tran-
scription of PKMz pre-mRNA is likely to take

more than 30 min (12, 21).
So what could be the function of increased
PKMz activity in the ACC? We wondered whether
this increase contributes to behavioral sensitiza-
tion after nerve injury. We performed intra-ACC
microinjections of z-pseudosubstrate inhibitory
peptide (ZIP) (10 nmol/ml, 0.5 ml per side), a se-
lective cell-permeable PKMz inhibitor, in mice 3
days after nerve injury. Bilateral microinjections
of ZIP into the ACC significantly reduced the al-
lodynia (n = 8, paired t test, t7 = 8.78, P < 0.001;
Fig. 2, A to C) unlike saline-injected controls (n =
6, paired t test, t 5 = 1.84, P > 0.05; Fig. 2C). Similar
analgesic effects of ZIP were detected 7 days after
nerve injury (n = 7, paired t test, t6 = 2.89, P <
0.05; Fig. 2C). The analgesic effects lasted for at
least 2 hours, but there was recovery at 24 hours
after the injection (n = 5, Fig. 2D). To determine
whether the effect of ZIP is regionally selective,
we microinjected ZIP into the primary somato-
sensory cortex. The same amount of ZIP did not
produce any significant analgesic effect (paired
t test, t3 = –2.30, P > 0.05, n = 4, fig. S2, A and
B). To determine whether the effects of ZIP in ACC
are sensory-selective, we examined the effects of
this inhibitor in the open-field test and found no
differences in the distance traveled between mice Fig. 3. Inhibition of PKMz selectively decreased the amplitude of eEPSCs in ACC neurons of mice with
microinjected with ZIP or saline (Fig. 2E). neuropathic pain by reducing the number of active AMPARs. (A and B) Samples showed the effect of ZIP (5
Nerve injury can induce tonic pain (22). By mM) on the amplitude of eEPSCs in the ACC neurons of animals from the nerve injury [black circles in (A)]
using an injury-induced, conditioned place pref- and sham group [black circles in (B)]. The gray open circles represent the change of membrane resistance
erence paradigm (22), we evaluated the effects of during recording. Black traces in the upper part of (A) and (B) indicate the averaged response at baseline,
ZIP on nerve injury–related tonic pain. Micro- and blue traces indicate the average of 2 min responses collected 10 min after ZIP application. (C) Pooled
injection of ZIP into the ACC also produced sig- data of effects of ZIP on the eEPSCs recorded from the ACC of mice in the sham (open) and nerve injury
nificant analgesic effects (n = 6, paired t test, t5 = (solid) groups 10 min after ZIP application. *P < 0.05; error bars, SEMs. (D) The variance-current
–3.19, P < 0.05; Fig. 2, F and G). In addition, we relationships of the eEPSCs recorded with (red) or without (black) CNQX (0.5 mM) for a representative
neuron. (E) Voltage dependence of the single channel currents. Open gray circles indicate the individual
examined whether ZIP infusion caused any
data collected from nine neurons. (F) Example of peak-scaled NSFA performed on the eEPSCs before and
memory impairment. When we infused the ACC
after ZIP application from a neuron in a mouse with nerve injury. (G) ZIP reduced the numbers of active
with ZIP 2 hours before memory retrieval on 3, 7, channels selectively in mice with nerve injury. The white and red bars represent the baseline of the sham
or 18 days after contextual fear conditioning, we and nerve injury groups, respectively. The blank blue and solid blue bars indicate the percent change in the
did not find any obvious defects in memory number of active channels in sham and nerve injury, respectively, groups 10 min after bath application of
retrieval [n = 7 to 11 per group, unpaired t test, P > ZIP. (H) Digitized photomicrograph showing neurons in laminae II of the spinal cord, which were recorded
0.05 in day 3 (t20 = 1.92), 7 (t16 = 1.56) or 18 (t13 = by stimulating dorsal roots. (I) Example eEPSCs from neurons in lamina II of spinal cord after Ad or C fiber
–1.75); Fig. 2H]. Moreover, microinjecting ZIP stimulation. (J) The amplitudes of eEPSCs recorded in nerve injury mice were significantly larger than those
into the ACC did not affect the tail-flick reflex and in control mice at 1.5 (medium) and 2 times (high) the threshold (low)-stimulating intensity (sham, n = 11;
the paw-withdrawal threshold in normal animals nerve injury, n = 14; one-way ANOVA, F1,97 = 10.0, P < 0.01, Tukey test was used for the multiple
(Fig. 2, I and J). These data show that micro- comparisons). (K) Pooled data showed that ZIP had similar effects on EPSCs recorded in the spinal cord of
injection of ZIP into the ACC produces a modality nerve injury and sham mice.

REPORTS
III of the ACC 3 or 7 days after nerve injury (24). 0.05; Fig. 3D) and the change of single-channel neurons in lamina II of the spinal cord after dorsal
After obtaining a stable baseline, we bath-applied current amplitude after an alteration in membrane root stimulation. We found that peripheral nerve
ZIP (5 mM) to block PKMz activity. The evoked potential (n = 6 per holding potential; Fig. 3E). injury significantly increased the amplitudes of
EPSCs (eEPSCs) were significantly decreased in NSFA was performed on five of seven neurons eEPSCs at 1.5 and 2 times of the threshold-
six of seven experiments, and total mean re- where ZIP produced substantial inhibition of the stimulating intensity (two-way ANOVA, F1,97 =
sponses were reduced to 83 T 6% (mean T SEM) eEPSCs (mean 76 T 5%); we found that the 10.00, P < 0.01; Fig. 3, H to J). In contrast to the
of baseline after 10 min of bath application of ZIP number of active channels decreased to 65 T 11% ACC, bath application of ZIP (5 mM) to the spinal
(n = 7; Fig. 3, A and C). In contrast, ZIP did not (n = 5, paired t test, t4 = 6.59, P < 0.05) of the cord did not distinguish between EPSCs recorded
affect the amplitude of eEPSCs recorded in baseline (Fig. 3, F and G), whereas g was un- from sham and nerve injury mice (Fig. 3K, two-
neurons from sham-operated mice (n = 6; Fig. 3, affected. This effect was not observed in slices way ANOVA, F1,68 = 1.02, P > 0.05). In both
B and C). obtained from sham-operated mice (Fig. 3G). These cases, there was a reduction in sensory transmis-
The ZIP-induced reduction of eEPSCs may results suggest PKMz may exert its effect by in- sion (Fig. 3K), which may be due to a constitutive
be due to a decrease in either the AMPA receptor creasing the number of AMPA receptors (AMPARs) activity of PKMz in spinal sensory neurons. Con-
unitary conductance (g) or number of active chan- at synapses in the ACC. A biochemical assay also sistent with these findings, intrathecal injection of
nels. Peak-scaled nonstationary fluctuation anal- showed that ZIP infusion reduced postsynaptic ZIP (2 nmol/ml, 5 ml per animal) did not produce
ysis (NSFA) has been developed to distinguish GluA1, one component of AMPARs, selectively any analgesic effect (n = 4, paired t test, t3 = 0.52,
between these parameters (23, 25, 26). As a con- in the nerve injury group (n = 4 per group, un- P > 0.05).
trol, we performed manipulations that should af- paired t test, t6 = 7.06, P < 0.001; fig. S3). The ACC is a heterogeneous cortical area, in

fect either the number of active channels or the Sensory synaptic transmission in the spinal which not all neurons would be expected to
single-channel current amplitude but not the cord dorsal horn plays important roles in chronic respond to neuropathic pain. We therefore used
single-channel conductance. Similar to previous pain and so could be an additional site of action transgenic mice in which the expression of green
reports (26), NSFA was able to detect a reduc- of ZIP, although PKMz and p-PKMz levels in the fluorescent protein (GFP) is controlled by the
tion in the number of active channels induced by spinal cord did not change in response to nerve promoter of the c-fos gene (27, 28), because c-fos
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) injury. To examine the effects of ZIP on spinal is known to be activated in sensory neurons after
(0.5 mM) (n = 4, paired t test, t3 = 6.60, P < sensory transmission, we recorded eEPSCs from injury (29). In these mice, we found that many
Fig. 4. PKMz activity maintains L-LTP in the ACC. (A) Fos-immunopositive

cells were found in layers II, III, V, and VI in the ACC of mice 7 days after
nerve injury. (B) NeuN immunostaining in the ACC, with dashed line
showing the structure of the ACC. The framed area in (A) is magnified in
(a). (b) is the magnified image of (B). (c) is the merged result of (a) and (b).
Yellow color in (c) represents the Fos/NeuN double-labeled neurons. Scale
bar indicates 100 mm in (A) and (B) and 30 mm in (a) to (c). (C) The total
number of Fos-positive neurons were counted in the layers II and III on
both sides of the ACC (n = 5 mice in each group). *P < 0.001; error bars
indicate SEMs. (D) An example of the recorded FosGFP-negative neuron
from the ACC of the sham-treated mice. (E) An example showing the
double-patched FosGFP-negative (1) and -positive (2) neurons from the
ACC of the nerve injury group. (a) images show the expression of FosGFP;
(b) images indicate the recorded neurons that were labeled by Alexa fluor
594. Yellow color in (Eb) indicates the overlap of GFP and Alexa fluor 594.
Scale bar, 20 mm. (F) Pooled data showing the input-output curves of
FosGFP-positive neurons from nerve injury mice (dark red) are different
from FosGFP-negative neurons of nerve injury (red) and sham-treated
(black) mice. Representative eEPSCs recorded from FosGFP-negative neu-
rons (a) of sham-treated or nerve injury (b) mice or FosGFP-positive (c)
neurons of nerve injury mice. (G and H) Pooled data showing the effects of
ZIP (5 mM) on FosGFP-negative [black and (a)] neurons of sham-treated
mice and FosGFP-negative [light blue and (b)] and FosGFP-positive [dark
blue and (c)] neurons of nerve injury mice. Representative waveforms
indicated eEPSCs before (red) and after ZIP (blue) application. *P < 0.05.
(I) L-LTP of the field EPSP induced by the TBS in the ACC can last for at
least 5 hours. L-LTP from naïve (n = 6) and sham-operated mice (n = 5)
were similar, and data were pooled together (black). Blocking of the
activity of PKMz by bath application of ZIP (5 mM) for 1 hour can reverse
the synaptic potentiation (blue). L-LTP was occluded in nerve injury mice
(red). Sample traces showing the field EPSP recorded at 0.5 hours before
TBS conditioning (a) and 2.5 hours (b) and 4.5 hours (c, 0.5 hours after
ZIP incubation) after TBS conditioning in the control, ZIP treatment, and
nerve injury groups. The blue bar indicates the time scale of ZIP ap-
plication. (J) Summary data showing ACC L-LTP 4 hours after the
induction with ZIP treatment or nerve injury. White, control with TBS
conditioning; blue, TBS conditioning with ZIP incubation; red, nerve
injury with TBS conditioning. *P < 0.05.

REPORTS
ACC neurons were activated 3 and 7 days after may be required for maintaining L-LTP in the ACC, 14. E. Pastalkova et al., Science 313, 1141 (2006).
nerve injury (Fig. 4, A to C). We performed whole- we recorded LTP in ACC slices. We induced L- 15. R. Shema, S. Hazvi, T. C. Sacktor, Y. Dudai, Learn. Mem.
16, 122 (2009).
cell patch-clamp recordings from FosGFP-positive LTP by using a theta burst stimulation (TBS) pro- 16. F. Wei et al., Neuron 36, 713 (2002).
pyramidal cells (“green” cells) that were acti- tocol. The slope of the excitatory postsynaptic 17. H. Xu et al., J. Neurosci. 28, 7445 (2008).
vated by allodynic pain and compared these with potential (EPSP) was significantly greater 3 hours 18. Materials and methods are available as supporting
nonactivated neighboring neurons and neurons after induction (n = 11, Fig. 4I). Bath application material on Science Online.
19. K. I. Vadakkan, Y. H. Jia, M. Zhuo, J. Pain 6, 747 (2005).
in sham-operated FosGFP mice (Fig. 4, D and of ZIP (5 mM) 3 hours after induction significantly 20. J. Liauw, L. J. Wu, M. Zhuo, J. Neurophysiol. 94, 878
E). The amplitudes of eEPSCs stimulated at the reduced the potentiation (n = 6; Fig. 4, I and J), (2005).
same intensity in the FosGFP-positive neurons suggesting that PKMz is required for the mainte- 21. M. T. Kelly, J. F. Crary, T. C. Sacktor, J. Neurosci. 27,
were significantly greater than those of FosGFP- nance of L-LTP in the ACC. To test whether nerve 3439 (2007).
22. T. King et al., Nat. Neurosci. 12, 1364 (2009).
negative neurons in the ACC of nerve injury and injury–triggered plasticity shares some common 23. D. S. Ling, L. S. Benardo, T. C. Sacktor, Hippocampus 16,
sham-operated mice (two-way ANOVA, F2,59 = mechanism with L-LTP in the ACC, we performed 443 (2006).
17.25, P < 0.05, Fig. 4F). ZIP (5 mM) signifi- occlusion experiments. Indeed, synaptic potentia- 24. L. J. Wu, M. G. Zhao, H. Toyoda, S. W. Ko, M. Zhuo,
cantly reduced the amplitude of eEPSCs (n = 7, tion was significantly reduced in slices of nerve J. Neurophysiol. 94, 1805 (2005).
25. T. A. Benke, A. Lüthi, J. T. Isaac, G. L. Collingridge, Nature
paired t test, t6 = 2.85, P < 0.05; Fig. 4, G and H) injury mice as compared with those of control mice
393, 793 (1998).
in FosGFP-positive pyramidal cells but had no (unpaired t test, t12 = 3.52, P < 0.05; Fig. 4, I and J). 26. S. F. Traynelis, R. A. Silver, S. G. Cull-Candy, Neuron 11,
effect on eEPSCs in either the FosGFP-negative We found that PKMz is critical for the main- 279 (1993).
neurons from the same nerve injury group or in tenance of long-term plasticity in the ACC and 27. R. L. Clem, T. Celikel, A. L. Barth, Science 319, 101 (2008).

neurons from the sham group (n = 5, nerve contributes to neuropathic pain by modulating ex- 28. A. L. Barth, R. C. Gerkin, K. L. Dean, J. Neurosci. 24,
6466 (2004).
injury, t4 = –0.63; n = 6, sham, t5 = –1.45; paired citatory synaptic transmission within this cortical 29. S. P. Hunt, A. Pini, G. Evan, Nature 328, 632 (1987).
t test, P > 0.05; Fig. 4, G and H). Furthermore, structure. Its role in maintaining synaptic potenti- 30. This work was supported by grants from the EJLB-CIHR
ZIP had no effect on the eEPSCs recorded on the ation is a critical part in allodynia resulting from Michael Smith Chair in Neurosciences and Mental Health,
FosGFP-positive neurons from the primary somato- nerve injury. Therefore, PKMz in ACC synapses Canada Research Chair, NeuroCanada, and Canadian
Institute for Health Research operating grants (CIHR66975
sensory cortex of mice with nerve injury (n = 6, provides a potential new therapeutic target for the and CIHR84256) (M.Z.). M.Z., B.-K.K., and G.L.C. are also
paired t test, t5 = –0.55, P > 0.05; fig. S2C). In treatment of chronic pain. supported by the World-Class University (WCU) program
contrast to PKMz, cAMP-dependent protein of the Ministry of Education, Science and Technology
kinase (PKA) is not required for the maintenance References and Notes in Korea through National Research Foundation (R32-10142).
1. E. R. Kandel, Science 294, 1030 (2001). X.-Y.L., T.C., and H.W. are supported by postdoctoral
of enhanced EPSCs in the FosGFP-positive neu- fellowships from Fragile X Research Foundation of
rons, because the PKA inhibitor KT5720 (1 mM) 2. M. Zhuo, Trends Neurosci. 31, 199 (2008).
Canada. B.-K.K. is supported by the National Creative
3. A. V. Apkarian, M. N. Baliki, P. Y. Geha, Prog. Neurobiol.
did not affect EPSCs (n = 5, paired t test, t4 = 87, 81 (2009). Research Initiative Program of the Korean Ministry of
–0.32, P > 0.05; fig. S4). These data suggest that 4. M. Costigan, J. Scholz, C. J. Woolf, Annu. Rev. Neurosci. Science and Technology, Korea. H.-G.K. and C.K. are
supported by a BK21 fellowship, Korea. G.L.C. receives
neurons in the ACC that are affected by neuro- 32, 1 (2009).
5. H. Ikeda et al., Science 312, 1659 (2006). support from the MRC (UK).
pathic pain have enhanced excitatory synaptic trans-
6. A. Latremoliere, C. J. Woolf, J. Pain 10, 895 (2009).
mission and that this is expressed by a mechanism 7. F. Wei, P. Li, M. Zhuo, J. Neurosci. 19, 9346 (1999). Supporting Online Material
that is independent of PKA but involves persistent 8. T. C. Sacktor, Prog. Brain Res. 169, 27 (2008). www.sciencemag.org/cgi/content/full/330/6009/1400/DC1
activation of PKMz. 9. E. A. Drier et al., Nat. Neurosci. 5, 316 (2002). Materials and Methods
Cortical LTP has been proposed as a cellular 10. P. V. Migues et al., Nat. Neurosci. 13, 630 (2010). Figs. S1 to S4
11. M. U. Naik et al., J. Comp. Neurol. 426, 243 (2000). References
model for chronic pain (2). One key function of 12. A. I. Hernandez et al., J. Biol. Chem. 278, 40305 (2003).
PKMz is to maintain cortical potentiation at in- 13. T. V. Bliss, G. L. Collingridge, S. Laroche, Science 313, 3 May 2010; accepted 18 October 2010
dividual synapses (23). To test whether PKMz 1058 (2006). 10.1126/science.1191792
Micro-Optical Sectioning Tomography has made great progress in brain studies at both
the system and cellular levels, our empirical knowl-
to Obtain a High-Resolution Atlas

edge of neuroanatomical connectivity remains in-
adequate, limiting the progress of brain studies
(2). Thus, it is necessary to gain new insights into
of the Mouse Brain the morphology, localization, and interconnectiv-
ity of neural circuits throughout the whole brain
at an appropriate resolution. Individual synapses
Anan Li,* Hui Gong,* Bin Zhang,* Qingdi Wang, Cheng Yan, Jingpeng Wu, Qian Liu, are the finest functional element in circuits, but it
Shaoqun Zeng, Qingming Luo† is currently not technologically feasible to study
the brainwide connectivity of complex vertebrate
The neuroanatomical architecture is considered to be the basis for understanding brain function organisms (e.g., mice) at the synaptic level. In con-
and dysfunction. However, existing imaging tools have limitations for brainwide mapping of neural trast, mesoscale techniques are currently more
circuits at a mesoscale level. We developed a micro-optical sectioning tomography (MOST) system feasible and applicable for understanding specific
that can provide micrometer-scale tomography of a centimeter-sized whole mouse brain. Using neural functions (2).
MOST, we obtained a three-dimensional structural data set of a Golgi-stained whole mouse brain at Light microscopy has remained a key tool for
the neurite level. The morphology and spatial locations of neurons and traces of neurites could be neuroscientists to observe cellular properties at
clearly distinguished. We found that neighboring Purkinje cells stick to each other.
Britton Chance Center for Biomedical Photonics, Wuhan Na-
tional Laboratory for Optoelectronics–Huazhong University of
ne of the most important aims in neuro- of the brain, but the complex functions of the
O science is to obtain an interconnection

diagram of the whole brain. In mammals,
individual neurons are considered the basic units
brain depend more on the fine anatomical archi-
tecture of a very large number of neurons and their
connections (1). Although modern neuroscience
Science and Technology, Wuhan 430074, P. R. China.
†To whom correspondence should be addressed. E-mail:
qluo@mail.hust.edu.cn

REPORTS
the mesoscopic level (3). A whole mouse brain is While working, the microtome slices the speci- The total amount of uncompressed volume data
centimeter-sized, however, and is beyond the field men into ribbons about 450 mm in width. Once exceeded 8 terabytes, and the average acquisition
of view of modern light microscopy techniques separated from the specimen block, the ribbons rate was about 0.11 ms per voxel, or 0.001 mm3/s.
such as confocal and multiphoton microscopy are imaged immediately. The light microscope is For optimal visualization of the highly com-
(4, 5). Before microscopic imaging, histological a reflected bright-field microscope in which the plicated neuronal architecture, the raw mass data
sections are usually prepared for observation of the illumination beam is perpendicular to the rake should be preprocessed to solve problems with
internal microstructure of large specimens (6–8), face of the knife and coincides with the imaging periodic noise and nonuniform brightness. The pre-
but it is difficult to perform serial ultrathin sec- beam. As the ribbons are being imaged in motion, processed data can then be reconstructed and the
tioning on large specimens by means of a tradi- a time-delay integration line-scan charge-coupled volume rendered in three dimensions with the use
tional microtome. In the past decade, several new device (CCD) is used to improve the sensitivity of of the program Amira 5.2.2 to show the whole
techniques have been developed to explore the image recording. All of these design features, es- anatomical sections, small regions of the brain,
neural circuits of the whole brain (9–14). A com- pecially the optical design, can effectively reduce and individual neurons (15).
mon characteristic of the techniques is the use of the system complexity to ensure automation sta- Figure 2 shows a series of coronal images
various kinds of automated sectioning or sectioning- bility and high resolution during time-consuming reconstructed along the anterior-posterior axis of
like approaches to increase the detection depth of data acquisition without interruption. the entire mouse brain with a spacing of 1 mm.
light microscopes. Some methods start by build- We used MOST to image a whole brain of an We could also use the original coronal images to
ing section libraries automatically and then imaging adult Kunming (KM) mouse. The brain was from reconstruct virtual sagittal images; Fig. 3A shows
these libraries with light or electron microscopy a 5-week-old male with a body weight of 27.57 g the reconstruction of such sagittal images. The se-

(12, 13). Another method is to perform imaging and a body length of 173 mm. During specimen lected location of the sagittal plane and the ab-
and sectioning simultaneously without collecting preparation (15), staining was performed before breviations used in Fig. 3A conform to those in
sections, which is a faster, more automated ap- embedding; we used a modified Golgi-Cox ap- Franklin and Paxinos’ atlas (16), and almost all
proach (14). Although these techniques have been proach for staining and Spurr resin for embedding. major regions of the brain can be seen. Details of
successfully applied in specific fields, they have The brain was kept intact throughout specimen the cerebral cortex, hippocampus, and cerebel-
not provided a brainwide architecture atlas at the preparation. The three anatomical axes of the lum in Fig. 3A are shown in Fig. 3, B to D. The
neurite level (3). brain—left-right, dorsal-ventral, and anterior- original raw images and additional reconstruc-
We developed an automatic micro-optical sec- posterior—corresponded to the three movement tions of cross sections are shown in fig. S5 and
tioning tomography (MOST) instrument to deter- directions of the specimen stage, X, Y, and Z. figs. S7 to S10.
mine the neuronal connectivity of the whole mouse Throughout data acquisition, the section thick- MOST also allows the 3D imaging of neurons
brain at the mesoscopic level. The MOST system ness was 1.0 mm, and a water-immersion objec- and neuronal processes (Fig. 4). Among our sam-
is composed of a microtome, light microscope, tive (40×, numerical aperture 0.8) was used for pled reconstructions of the neurons, we found that
and image recorder, and it performs imaging and imaging. Uninterrupted data acquisition lasted for the neighboring Purkinje cells are close to each
sectioning simultaneously (Fig. 1A and fig. S1). about 242 hours, covering 15,380 coronal sections. other (Fig. 4D and movie S4), which is different
Fig. 1. (A) Schematic representation of the MOST system.

The specimen is mounted in a chamber, the motion of which is
controlled by a series of mechanical translation stages that
can move in three directions (the chamber and stages are not
shown). Slicing is performed by moving the specimen along
the x axis to generate ribbons, and each ribbon is simulta-
neously imaged. The illuminating beam passes through the
beam splitter, mirror, and objective and irradiates the ribbon.
After it passes through the mirror, beam splitter, and tube
lens, the imaging beam collected by the objective is then
recorded by a line-scan CCD. (B) Schematic representation of
slicing. The slicing produces ribbons that glide forward along
the knife face. The illuminating and imaging areas are indi-
cated by a circle and a red line, respectively. To expand the
detection range, we performed slicing with a lateral and an
axial scan (15). (C) An image stack acquired using MOST. The
stack is composed of many subimages. The subimages aligned
along the x axis were produced from a ribbon. These image
sequences reconstitute the entire cross section of the specimen
along the y axis. A subimage of the cortex, hippocampus, and
corpus callosum is also indicated.

REPORTS
Fig. 2. (A) Locations of
images shown in (B) to (L),
spaced 1 mm apart, from the
olfactory bulb to the cerebel-
lum. (B to L) A series of im-
ages reconstructed from a
stack of 100 coronal sections
(total thickness of 0.1 mm).
The horizontal stripes, caused
by refractive index mismatch
between specimen and am-
bient medium, lead to little
information loss. The raw data
from MOST are 8-bit grayscale
images, and the resulting re-
construction uses a green
pseudocolor to enhance their
visible effects; dorsal-ventral
and left-right axes are in-

dicated at lower left. The
dashed box in (H) is described
in Fig. 4B.
Fig. 3. (A) A sagittal im-

age reconstructed from a
stack of 100 virtual sagit-
tal sections (total thick-
ness of 0.1 mm). These
sections were transformed
from the original coronal
sections. The sagittal
image was located in the
right hemisphere about
0.4 mm lateral to the mid-
dle. Dorsal-ventral and
anterior-posterior axes
are indicated. Almost all
major regions of the
brain can be seen in this
image: OB, olfactory bulb;
Cx, cerebral cortex; Hc, hip-
pocampus; f, fornix; ac,
anterior commissure; T,
thalamus; Cb, cerebellum;
Mb, midbrain; P, pons;
Md, medulla; cc, corpus
callosum; SC, superior
colliculus; IC, inferior col-
liculus; Ht, hypothala-
mus; Po, preoptic area;
ox, optic chiasm; 4V, 4th
ventricle; arabic numerals 2 to 10, nine lobules of the cerebellum; Sk, spinal cord. (D). In the reconstruction of sagittal images, no dislocation was observed along the
The three regions inside the dashed rectangles are the positions of (B), (C), and (D), dorsal-ventral axis; that is, the coronal sections are inherently aligned along the
from left to right. (B to D) The cerebral cortex (B), hippocampus (C), and cerebellum anterior-posterior axis. Arrow in (D) indicates the image described in Fig. 4D.

REPORTS

Fig. 4. (A) A large volumetric reconstruction of a partial hippocampus of the Three-dimensional reconstruction of three neighboring pyramidal cells in
mouse brain. The cube volume is 1.4 mm by 1.5 mm by 0.9 mm. The multi- layer 5 of the ectorhinal cortex as in (B). These three cells are distinguished by
layered structure of the hippocampus and a large number of transverse fibers different colors; other cells, neurites, and blood vessels that densely cover the
of the corpus callosum are clearly visible. Dorsal-ventral, anterior-posterior, three cells are not shown. (D) Three-dimensional reconstruction of a pair of
and left-medial axes are indicated. (B) We traced the neurites of 17 neighboring Purkinje cells in the ninth lobule of the cerebellum. The Purkinje
neurons in the ectorhinal cortex, indicated by a dashed box in Fig. 2H. (C) cell in green can also be seen in Fig. 3D, indicated by an arrow.
from the corresponding description in Gray’s Anat- during data acquisition. If we stack these subim- tems and in understanding and treating various
omy (17). We also used the program V3D (18) to ages in three-dimensional space, a digital whole kinds of nervous system diseases.
trace the neurites in the ectorhinal cortex (Fig. 4B mouse brain with a voxel size of 0.33 mm by
and movie S5). 0.33 mm by 1.0 mm can be reconstructed. References and Notes
One of the advantages of MOST is that no The Golgi method stains a limited number of 1. E. R. Kandel, in Principles of Neural Science, E. R. Kandel,
additional registration is needed because of the cells at random in their entirety (15). It is likely J. H. Schwartz, T. M. Jessell, Eds. (McGraw-Hill, New York,
ed. 4, 2000), pp. 19–35.
accurate spatial positioning of the images (15). that individual neurons or typical structures of 2. J. W. Bohland et al., PLOS Comput. Biol. 5, e1000334 (2009).
Most mechanical slicing methods suffer from se- the whole brain will be recognized in a complex 3. B. A. Wilt et al., Annu. Rev. Neurosci. 32, 435 (2009).
rious deformation, which can be overcome when background and that it will be possible to analyze 4. J. B. Pawley, Handbook of Biological Confocal Microscopy
using MOST. Figure 3 shows the smooth contours architectural features with limited computing re- (Plenum, New York, ed. 3, 2006).
5. W. Denk, J. H. Strickler, W. W. Webb, Science 248,
of the brain tissue surface, and Fig. 4A shows a sources. MOST is capable of fluorescence imaging. 73 (1990).
fine inner structure of the mouse brain; both dem- Combined with new developments in specimen 6. M. H. Chin et al., Physiol. Genomics 30, 313 (2007).
onstrate that no registration is needed for the preparation techniques—for example, the multi- 7. R. W. Williams, in Mouse Brain Development, A. M. Goffinet,
MOST data set. Even at the finer levels, the com- labeled transgenic animal models (19)—the MOST P. Rakic, Eds. (Springer-Verlag, New York, 2000), pp. 21–50.
8. E. S. Lein et al., Nature 445, 168 (2007).
plex distribution of neurites is shown (e.g., fig. S7). system will help us to obtain a better connectivity 9. P. S. Tsai et al., Neuron 39, 27 (2003).
The images are saved with a unified dimension, map of the entire brain. Such studies will play an 10. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt,
and their spatial position information is recorded important role in functional studies of neural sys- E. H. K. Stelzer, Science 305, 1007 (2004).

REPORTS
11. H. U. Dodt et al., Nat. Methods 4, 331 (2007). 18. H. C. Peng, Z. C. Ruan, F. H. Long, J. H. Simpson, Supporting Online Material
12. K. D. Micheva, S. J. Smith, Neuron 55, 25 (2007). E. W. Myers, Nat. Biotechnol. 28, 348 (2010). www.sciencemag.org/cgi/content/full/science.1191776/DC1
13. K. J. Hayworth, N. Kasthuri, R. Schalek, J. W. Lichtman, 19. G. Feng et al., Neuron 28, 41 (2000). Materials and Methods
Microsc. Microanal. 12 (S02), 86 (2006). 20. We thank P. Wu, J. B. Wu, and L. Wu for their early SOM Text
14. D. Mayerich, L. Abbott, B. McCormick, J. Microsc. 231, work in MOST; L. Fu, T. H. Xu, and W. Zhou for helpful Figs. S1 to S10
134 (2008). discussions; Z. Dong and Z. Feng for assistance in Movies S1 to S5
15. See supporting material on Science Online. experiments; J. Zhou for the nanowire sample; and References
16. K. B. J. Franklin, G. Paxinos, The Mouse Brain in Stereotaxic Diatome AG, Switzerland, for the diamond knife.
Coordinates (Academic Press, San Diego, CA, ed. 3, 2007). Supported by the National Natural Science Foundation
17. S. Standring, Gray’s Anatomy: The Anatomical Basis of of China (grants 30727002, 60025514, 30700214, 3 May 2010; accepted 23 September 2010
Clinical Practice (Churchill Livingstone, London, ed. 39, and 30925013) and the Program for Changjiang Published online 4 November 2010;
2004). Scholars and Innovative Research Team in University. 10.1126/science.1191776
Paradoxical False Memory for tinguish the novel from the repeated object. Animals
and human subjects with damage to the perirhinal
Objects After Brain Damage

cortex are impaired on this task when a sufficiently
long delay is interposed between study and test
(2–4); indeed, much work has established that
Stephanie M. McTighe,1,2 Rosemary A. Cowell,3 Boyer D. Winters,4 damage to the perirhinal cortex alone is sufficient

Timothy J. Bussey,1,2 Lisa M. Saksida1,2* to cause severe impairments in object recognition
(2, 5–8). When the standard task procedure is
Poor memory after brain damage is usually considered to be a result of information being lost or used, the requirement that two items be simulta-
rendered inaccessible. It is assumed that such memory impairment must be due to the incorrect neously compared precludes determination of
interpretation of previously encountered information as being novel. In object recognition memory whether the novel item is viewed as familiar, or
experiments with rats, we found that memory impairment can take the opposite form: a tendency vice versa, because the presence of one item
to treat novel experiences as familiar. This impairment could be rescued with the use of a visual- affects how much the other is explored and how
restriction procedure that reduces interference. Such a pattern of data can be explained in terms of it is evaluated.
a recent representational-hierarchical view of cognition. We therefore devised a method of assessing
object recognition that decouples the subjects’
evaluation of novel and repeated objects (Fig. 1)
lthough individuals with some types of terval can be greatly compromised or nonexistent (9). Consistent with previous studies, intact rats
A brain damage—for example, to structures

in the medial temporal lobe—are able to
remember new information immediately after it
(1). It would seem to follow that the experiences
of such individuals must be lost rapidly or are
inaccessible, such that when a novel object is
explored in the novel-object condition more than
in the repeated-object condition, thus showing
intact memory for the repeated object. Also con-
is learned, their recall after even a very short in- encountered and later reexperienced, it is as if the sistent with previous studies, rats with perirhinal
object had never before been seen. We explored cortex damage explored novel and repeated ob-
1
Department of Experimental Psychology, University of Cam- this assumption with an animal model of object jects equally, indicating an inability to distin-
bridge, Cambridge CB2 3EB, UK. 2MRC and Wellcome Trust recognition memory that has been widely used as guish between them (2). Paradoxically, however,
Behavioural and Clinical Neuroscience Institute, University of a model of memory impairment (2). this inability to distinguish novel from repeated
Cambridge, Cambridge CB2 3EB, UK. 3Department of Psychol- The standard object recognition procedure in- objects was characterized not by an increase in
ogy, University of California, San Diego, La Jolla, CA 92093,
USA. 4Department of Psychology, University of Guelph, Guelph, volves exposure to a study object, followed (after exploration of the repeated object—which would
Ontario N1G 2W1, Canada. a delay) by a test phase in which the study object have indicated that they judged the repeated ob-
*To whom correspondence should be addressed. E-mail: is again presented, along with a perceptually dis- ject as novel—but by a decrease in their explo-
lms42@cam.ac.uk tinct novel object. The participant’s task is to dis- ration of the novel object, indicating that they
Fig. 1. A modified version of the

spontaneous object recognition task,
in which exploration of the repeated
object is decoupled from exploration
of the novel object. In the novel-object
condition, an animal received a study
exposure to two copies of object A for
3 min. Then the animal was put into
an individual holding cage (standard Stage 2: Delay
D
condition) or a visually restricted en- Holding cage OR
Stage 3: Test
vironment (reduced-interference con- Visual Restriction
Repeated OR Novel
dition) for a delay of 1 hour. After the 1 hour
3 minutes
delay, the animal received a test ex-
posure of 3 min to two copies of a Stage 1: Study
novel object, object B. In the repeated- 3 minutes
object condition, the animal received a
study exposure of 3 min to two copies
of object A. Then, as before, the animal
was put into an individual holding cage
or a visually restricted environment for
a delay of 1 hour. After the delay, the
animal received a test exposure of 3 min to two copies of the familiar object, object A.

REPORTS

Fig. 2. Performance of rats with perirhinal cortex damage on the object rec- ANOVA showed a significant effect of condition (F1,19 = 18.33, P < 0.001), no
ognition task. (A) Standard condition, with normal interference-filled delay. effect of lesion (F < 1), and no interaction (F < 1), indicating that both groups
Repeated-measures analysis of variance (ANOVA) showed a significant effect of discriminated the objects after visual restriction. (C) Repetition of the standard
condition (F1,19 = 15.38, P < 0.001), an effect of lesion (F1,19 = 5.39, P < 0.05), condition. Repeated-measures ANOVA showed a significant effect of condition
and a significant interaction (F1,19 = 13.58, P < 0.005). Post hoc t tests using (F1,18 = 33.97, P < 0.001), a significant effect of lesion (F1,18 = 14.59, P < 0.005),
Bonferroni correction showed a significant difference in the novel condition (t19 = and a significant interaction (F1,18 = 16.17, P < 0.005). Post hoc t tests using
3.88, P < 0.001) and no difference in the repeated-object condition (t19 = 0.76, Bonferroni correction showed a significant difference in the novel condition (t18 =
P > 0.05), indicating that lesioned animals treated the novel object as though it 4.62, P < 0.001) and no difference in the repeated condition (t18 = 0.33, P >
were familiar. (B) Reduced-interference condition in which animals were placed 0.05), showing that the impairment returned when normal levels of interference
into a visually restricted environment during the delay. Repeated-measures were reinstated. ***P < 0.001.
treated the novel object as though it were familiar

(Fig. 2A) (9).
Our previous work has suggested that impair-
ments in this task may be a result of interference
during the delay (10) and has shown that the ex-
plicit addition of interfering objects during the de-
lay can impair memory in animals with perirhinal
cortex damage (11). Accordingly, we reduced in-
terference during the delay by putting the animals
into a dark environment between the study and test
phases (9, 12). This treatment completely rescued
the performance of the lesioned animals (Fig. 2B)
(9). To ensure that the rescue of the impairment
was not due to brain reorganization or recovery,
we retested the animals under the original experi-
mental conditions, and the impairment returned
(Fig. 2C).
The finding that the object recognition mem-
ory impairment in this model was characterized
by animals judging novel objects as familiar seems
counterintuitive. It is difficult to reconcile with
the assumption that all information presented dur-
ing the study phase is lost or inaccessible, because
Fig. 3. The representational-hierarchical view. Representations of visual stimulus features are organized
this assumption would suggest that animals should
hierarchically in increasingly complex conjunctions as information flows from posterior to anterior regions
judge repeated objects as novel. An alternative of the ventral visual stream (18, 19). The representation of a particular object is distributed throughout
view (13–15), however, suggests that memory loss the system, but it is represented differently at different points: as features early in the ventral visual
after brain damage may be better understood, not in stream, as conjunctions of features in intermediate regions in the ventral visual stream, and as highly
terms of loss of a system dedicated to a specific type complex conjunctions of features—shown here at the level of object wholes—in the perirhinal cortex. The
of memory—for example, long- versus short-term traditional multiple memory systems view suggests that structures within the medial temporal lobe
memory, or memory processes such as encoding, subserve exclusively mnemonic function, whereas structures in the ventral visual stream are important for
storage/consolidation, or retrieval (16)—but in perceptual functions. In contrast, the representational-hierarchical view suggests that stimulus
terms of the stimulus representations that the dif- representations throughout the ventral-visual-perirhinal-hippocampal stream are useful for any cognitive
ferent regions contain (Fig. 3) (17). For visual function that requires them.

REPORTS
input, stimulus representations are organized hi- the representation of an object is affected by the 6. A. Ennaceur, N. Neave, J. P. Aggleton, Behav. Brain Res.
erarchically, with conjunctive representations of lesion is indicated by the lack of impairment 80, 9 (1996).
7. R. E. Clark, L. R. Squire, Neuropsychologia 48, 2234 (2010).
relatively complex stimuli in anterior brain re- when lesioned animals spent the delay in a vis- 8. D. G. Mumby, Behav. Brain Res. 127, 159 (2001).
gions, and with representations of relatively sim- ually restricted environment. This intact perform- 9. See supporting material on Science Online.
ple features in more posterior regions (18, 19). ance must be supported by representations 10. R. A. Cowell, T. J. Bussey, L. M. Saksida, J. Neurosci. 26,
We have suggested that this representational hi- outside the perirhinal cortex, as predicted by 12186 (2006).
11. S. J. Bartko, R. A. Cowell, B. D. Winters, T. J. Bussey,
erarchy may extend into structures within the me- the representational-hierarchical view (9). This L. M. Saksida, Neuropsychologia 48, 2987 (2010).
dial temporal lobe, and that it is the highly complex shows that object recognition impairments after 12. N. Cowan, N. Beschin, S. Della Sala, Brain 127, 825
conjunctive representations of stimuli that are main- perirhinal cortex damage cannot be understood in (2004).
tained in regions such as the perirhinal cortex the usual way—that is, as the result of damage to 13. T. J. Bussey, L. M. Saksida, Hippocampus 17, 898 (2007).
14. E. A. Murray, T. J. Bussey, L. M. Saksida, Annu. Rev.
(13–15). According to this view, individuals with all or part of a dedicated memory system. The Neurosci. 30, 99 (2007).
damage to such regions should be impaired not present results also add to a growing body of 15. L. M. Saksida, T. J. Bussey, Neuropsychologia 48, 2370
just on a particular type of memory function, but evidence suggesting that interference may be an (2010).
also on any cognitive function that requires the important factor in memory impairments after 16. B. D. Winters, T. J. Bussey, J. Neurosci. 25, 52 (2005).
17. L. M. Saksida, Science 325, 40 (2009).
complex stimulus representations that are damaged. brain damage (12, 20–26).
18. D. J. Felleman, D. C. Van Essen, Cereb. Cortex 1,
This representational-hierarchical view ac- The combination of perirhinal lesions and 1 (1991).
counts for the seemingly paradoxical findings of object recognition used in our study models the 19. L. G. Ungerleider, M. Mishkin, in Analysis of Visual
the present study. Indeed, the pattern of results is disturbances in the recognition of objects and Behavior, D. J. Ingle, M. A. Goodale, R. J. W. Mansfield,

a central prediction of the model, as illustrated by people that are critical components of amnesia. It Eds. (MIT Press, Cambridge, MA, 1982), pp. 549–586.
20. E. K. Warrington, L. Weiskrantz, Nature 228, 628 (1970).
simulations using a computational model (9, 10). does not, however, model all forms of memory 21. J. T. Wixted, Annu. Rev. Psychol. 55, 235 (2004).
In these simulations, it is assumed that during the impairment; for example, it is not a complete 22. S. Della Sala, N. Cowan, N. Beschin, M. Perini, Memory
delay between study and test, the subject will be model of the dense, global amnesic syndrome 13, 435 (2005).
exposed to other, non-experimental visual mate- observed in people with extensive damage to me- 23. M. Dewar, Y. F. Garcia, N. Cowan, S. Della Sala,
Neuropsychology 23, 627 (2009).
rial that occurs naturally in the environment. This dial temporal lobe structures including the hip- 24. M. T. Dewar, N. Cowan, S. D. Sala, Cortex 43, 616 (2007).
material will almost certainly share some features pocampus. Nonetheless, these findings raise the 25. E. K. Warrington, L. Weiskrantz, Neuropsychologia 12,
in common with the novel object. This can lead possibility that the false memory reported here 419 (1974).
to interference, because as a result of this ex- may be a more general phenomenon. If this turns 26. E. K. Warrington, L. Weiskrantz, Neuropsychologia 16,
169 (1978).
posure, features in the novel object will now be out to be the case, we may need to reevaluate our 27. Supported by a grant from the BBSRC (L.M.S., T.J.B.,
familiar. However, because it is very unlikely that current conceptions of memory impairment and B.D.W.), and a 3-year MRC-funded Ph.D. studentship
the specific complex object presented during the amnesia. at the University of Cambridge, MRC/Wellcome Trust
test phase will have been experienced by chance Behavioral and Clinical Neuroscience Institute (S.M.M.).
during the delay, the unique, object-level repre- References and Notes
1. A. D. Baddeley, E. K. Warrington, J. Verbal Learn. Verbal Supporting Online Material
sentations in anterior regions of the system will www.sciencemag.org/cgi/content/full/330/6009/1408/DC1
Behav. 9, 176 (1970).
not be familiar and therefore can protect the sys- 2. B. D. Winters, L. M. Saksida, T. J. Bussey, Materials and Methods
tem from this interference. If these conjunctive Neuropsychologia 48, 2251 (2010). Figs. S1 to S3
representations of complex stimuli are damaged, 3. P. Alvarez, S. Zola-Morgan, L. R. Squire, Proc. Natl. Acad. Tables S1 to S13
Sci. U.S.A. 91, 5637 (1994). References
however, then the subject will have to rely on the
4. D. G. Mumby, J. P. Pinel, Behav. Neurosci. 108, 11 (1994).
simpler, feature-based memory that is highly sus-
5. T. J. Bussey, J. L. Muir, J. P. Aggleton, J. Neurosci. 19, 7 July 2010; accepted 19 October 2010
ceptible to interference. From this theoretical frame- 495 (1999). 10.1126/science.1194780
work, we obtain the perhaps counterintuitive
prediction that brain damage can produce impair-
Frequent Mutation of BAP1 in

ments in visual recognition memory tasks not
because the repeated object looks novel, but be-
cause the novel object looks familiar (fig. S3A).
The clear corollary of this prediction is that re-
ducing interference in the delay should improve Metastasizing Uveal Melanomas
performance in memory-impaired subjects (fig.
S3B), which is what we found (Fig. 2B). J. William Harbour,1,3* Michael D. Onken,1 Elisha D. O. Roberson,2 Shenghui Duan,2 Li Cao,2
These simulations and the current results sug- Lori A. Worley,1 M. Laurin Council,2 Katie A. Matatall,1 Cynthia Helms,2 Anne M. Bowcock2,3*
gest that object recognition memory impairments
may not be due to damage to a dedicated memory Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related
system from which all information presented in death, yet the genetics of metastasis are poorly understood. We used exome capture coupled with
the study phase is lost or inaccessible. Instead, massively parallel sequencing to search for metastasis-related mutations in highly metastatic uveal
brain damage that leads to such impairments com- melanomas of the eye. Inactivating somatic mutations were identified in the gene encoding BRCA1-
promises only a very specific type of complex associated protein 1 (BAP1) on chromosome 3p21.1 in 26 of 31 (84%) metastasizing tumors, including
stimulus representation. Other, simpler feature- 15 mutations causing premature protein termination and 5 affecting its ubiquitin carboxyl terminal
level representations of the repeated item remain. hydrolase domain. One tumor harbored a frameshift mutation that was germline in origin, thus
Because these remaining, relatively simple stim- representing a susceptibility allele. These findings implicate loss of BAP1 in uveal melanoma metastasis
ulus features tend to be repeated across objects and suggest that the BAP1 pathway may be a valuable therapeutic target.
and situations, their representations do not pro-
vide a unique signal of prior occurrence of an veal melanoma (UM) is the most common gene clinical prognostic assay included in the TNM
object. As a result, the disruption to the encoding
of complex stimulus representations has the knock-
on effect of making the system very susceptible
U primary cancer of the eye and has a strong
propensity for fatal metastasis (1). UMs are
divided into class 1 (low metastatic risk) and class 2
classification system (2, 3). However, the genetic
basis of metastasis remains unclear. Oncogenic
mutations in the Gaq stimulatory subunit GNAQ
to interference. The fact that only a portion of (high metastatic risk) based on a validated multi- are common in UM (4), but these mutations occur

REPORTS
early in tumorigenesis and are not correlated with Using exome capture followed by massively than BAP1 contained deleterious somatic muta-
molecular class or metastasis (5, 6). On the other parallel sequencing (8–10), we analyzed two class tions that were present in both tumors (table S3).
hand, class 2 tumors are strongly associated with 2 tumors that were monosomic for chromosome 3 BAP1 encodes a nuclear ubiquitin carboxy-
monosomy 3 (7), suggesting that loss of one copy (MM 56 and MM 70) and matching normal DNA terminal hydrolase (UCH), one of several classes
of chromosome 3 may unmask a mutant gene on from peripheral blood lymphocytes. Both tumors of deubiquitinating enzymes (11). In addition to
the remaining copy that promotes metastasis. contained inactivating mutations in BAP1, located the UCH catalytic domain, BAP1 contains a
at chromosome 3p21.1 (Fig. 1A) (10). MM 56 UCH37-like domain (ULD) (12), binding domains
1
Department of Ophthalmology and Visual Sciences, Wash- contained a C/G to T/A transition that created a for BRCA1 and BARD1, which form a tumor
ington University School of Medicine, St. Louis, MO 63110, premature termination codon (p.W196X). MM 70 suppressor heterodimeric complex (13), and a
USA. 2Department of Genetics, Washington University School contained a deletion of 11 base pairs in exon 11, binding domain for HCFC1, which interacts with
of Medicine, St. Louis, MO 63110, USA. 3Siteman Cancer Cen- leading to a frameshift and premature termination histone-modifying complexes during cell division
ter, Washington University School of Medicine, St. Louis, MO
63110, USA. of the BAP1 protein (p.Q322fsX100). The matched (12, 14, 15). BAP1 also interacts with ASXL1 to
normal DNA samples did not contain these mu- form the Polycomb group repressive deubiquitinase
bowcock@genetics.wustl.edu (A.M.B.); harbour@vision. tations, indicating that they were likely to be so- complex (PR-DUB), which is involved in stem cell
wustl.edu ( J.W.H.) matic in origin. No gene on chromosome 3 other pluripotency and other developmental processes
Fig. 1. Inactivating mutations in BAP1 occur frequently in uveal melanomas. (A) 598 to 729 (11). (C) Location of BAP1 missense mutations in the UCH domain Downloaded from www.sciencemag.org on December 2, 2010
Sanger sequence traces of MM 056 and MM 070 at the sites of the mutations. aligned to the crystal structure of UCH-L3 (21). Three-dimensional structure of
Location of mutated base in MM 056 and the start of the deletion of MM 070 are UCH-L3 was visualized with MMDB software (22). The small molecule near C91G,
indicated (arrows). The noncoding BAP1 strand is shown for MM 070. (B) Map of H169Q, and S172R represents a suicide inhibitor, illustrating the critical location
BAP1 gene and location of BAP1 mutations. BAP1 contains 17 exons (shaded of these mutations for catalytic activity. (D) BAP1 mRNA levels measured by
boxes) that encode a 728–amino acid protein. Introns are not to scale. Mutations quantitative RT-PCR in 9 nonmetastasizing class 1 UMs and 28 metastasizing
are shown below the gene figure as indicated. The UCH domain [amino acids (aa) class 2 UMs. (E) Relationship between BAP1 mRNA levels (measured by quan-
1 to 188] and UCH37-like domain (ULD) (aa 635 to 693) are indicated (12, 13). titative RT-PCR) and type of BAP1 mutation in 9 UMs with nonsense and other
The critical Q, C, H, and D residues of the active site (Gln85, Cys91, His169, and truncating mutations, 10 UMs with missense mutations (together with small in-
Asp184) are indicated with asterisks. The catalytic cysteine is indicated with a frame deletions, splice acceptor, and stop codon read-through mutations), and
circle. Also shown are the NHNY consensus sequence for interaction with HCFC1 4 class 2 UMs in which no BAP1 mutations were detected. Single-letter
(aa 363 to 365, exon 11), nuclear localization signals (NLS) at aa 656 to 661 abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp;
(exon 15) and aa 717 to 722 (exon 17), the BARD1 binding domain within the E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;
region bounded by aa 182 to 240 (13), and the BRCA1 binding domain within aa R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

REPORTS
(16, 17). BAP1 exhibits tumor suppressor activ- cytogenetic data were available, consistent with tion, we performed a preliminary analysis of DNA
ity in cancer cells (11, 13), and BAP1 mutations chromosome 3 loss uncovering recessive BAP1 methylation of BAP1. This did not reveal a con-
have been reported in a small number of breast mutations (10). Normal DNA from 20 patients vincing difference between class 1 and class 2
and lung cancer samples (11, 18). with BAP1-mutant class 2 primary tumors and tumors (10). However, analysis of the BAP1 pro-
To further investigate BAP1, genomic DNA the two with metastatic tumors was available and moter was limited by an unusually complex CpG
from 29 additional class 2 UMs and 26 class did not contain a BAP1 mutation, indicating that island that will require further work to resolve.
1 UMs were subjected to Sanger resequencing of the mutations were somatic in origin. However, Thus, we cannot rule out a role for methylation in
all BAP1 exons. Altogether, BAP1 mutations were we detected one germline mutation (p.E402fsX2; class 2 tumors in which BAP1 mutations were not
identified in 26 of 31 (84%) class 2 tumors, in- c.1318-1319insA) in the patient with tumor MM found. However, with almost 85% of class 2 tumors
cluding 13 out-of-frame deletions and two nonsense 087 (table S2), suggesting that germline alter- harboring mutations, we do not expect that methyl-
mutations leading to premature protein termina- ations in BAP1 can predispose to UM. GNAQ ation will be a major mechanism of BAP1 inactiva-
tion, six missense mutations, four in-frame dele- mutation status was available in 15 cases. GNAQ tion. An alternative explanation is that these tumors
tions, and one mutation predicted to produce an mutations were present in 4 of 9 BAP1 mutant may contain very large deletions of the BAP1 locus
abnormally extended BAP1 polypeptide (Fig. 1, tumors and 3 of 6 BAP1 wild-type tumors, indi- not detectable by our sequencing method.
A to C) (10). Three of the missense mutations af- cating that there was no correlation between Immunofluorescence revealed abundant nu-
fected catalytic residues of the UCH active site GNAQ and BAP1 mutation status. clear BAP1 protein in two class 1 tumors but vir-
(C91 and H169), two occurred elsewhere in the Quantitative reverse transcription polymerase tually none in four BAP1 mutant class 2 tumors
UCH domain, and one affected the ULD (Fig. 1, chain reaction (RT-PCR) showed that BAP1 mRNA (fig. S3). This was expected for the two tumors

B and C). All BAP1 missense mutations and in- levels were significantly lower in class 2 tumors with mutations expected to cause premature pro-
frame deletions affected phylogenetically con- compared with class 1 tumors (P < 0.0001) (Fig. tein terminations (MM 091 and MM 100), but it
served amino acids (fig. S1). Only one of 26 class 1D). Truncating mutations were associated with was surprising for the two tumors with missense
1 tumors contained a BAP1 mutation (NB101) (10). significantly lower mRNA levels than missense mutations (MM 071 and MM 135) and suggests
This case may represent a transition state in which mutations (P = 0.001) (Fig. 1E), consistent with that these mutations lead to protein instability.
the tumor has sustained a BAP1 mutation but has nonsense-mediated mRNA decay in the former RNA interference (RNAi)–mediated knock
not yet converted to class 2, suggesting that BAP1 group. Class 2 tumors in which BAP1 mutations down of BAP1 in 92.1 UM cells, which did not
mutations may precede the emergence of the were not identified expressed very low levels of harbor a detectable BAP1 mutation, recapitulated
class 2 signature. Somatic BAP1 mutations were BAP1 mRNA (Fig. 1E). many characteristics of the dedifferentiated class
also detected in two of three metastatic tumors (10). To determine whether the low BAP1 mRNA 2 UM phenotype (19). Cells transfected with con-
One copy of chromosome 3 was missing in levels in class 2 tumors without detectable BAP1 trol small interfering RNA (siRNA) exhibited typ-
all 17 BAP1-mutant class 2 tumors for which mutations may be explained by DNA methyla- ical melanocytic morphology, including dendritic
Fig. 2. UM cells de-

pleted of BAP1 acquire
properties that are typi-
cal of metastasizing class
2 tumor cells. (A) Phase-
contrast photomicrographs
of 92.1 uveal melanoma
cells transfected with
BAP1 or control siRNA at
the indicated days. Bot-
tom panels show repre-
sentative examples of
class 1 and class 2 uveal
melanoma cells obtained
from patient biopsy sam-
ples (Papanicolaou stain).
Scale bars, 10 mm. (B)
92.1 cells transfected
with BAP1 siRNA and
evaluated after 5 days.
BAP1 protein levels were
efficiently depleted to less
than 95% of control lev-
els (see Western blot).
Upper panel depicts prin-
cipal component analy-
sis to show the effect of
BAP1 knockdown on gene
expression signature. The
small spheres represent
the training set of known
class 1 (blue) and class 2
(red) tumors. Large spheres represent the control-transfected (gray) and BAP1 siRNA–transfected (red) cells. The lower panel depicts mRNA levels measured by
quantitative RT-PCR of a panel of melanocyte lineage genes, presented as relative change (fold change) in BAP1 siRNA/control siRNA–transfected cells. Results are
representative of three independent experiments.

REPORTS
projections and cytoplasmic melanosomes (Fig. ior. Thus, simultaneous targeting of both genetic 16. A. G. de Ayala Alonso et al., Genetics 176, 2099
2A), whereas cells transfected with BAP1 siRNA alterations might have synergistic therapeutic ef- (2007).
17. J. C. Scheuermann et al., Nature 465, 243 (2010).
lost these features, developed a rounded epithe- fects. One potential strategy to counteract the ef- 18. L. D. Wood et al., Science 318, 1108 (2007).
lioid morphology, and grew as multicellular non- fects of BAP1 mutation would be to inhibit the 19. M. D. Onken et al., Cancer Res. 66, 4602 (2006).
adherent spheroids, strikingly similar to the features RING1 ubiquitinating activity that normally op- 20. M. D. Onken, L. A. Worley, J. P. Ehlers, J. W. Harbour,
of class 2 clinical biopsy samples (Fig. 2A). Mi- poses the deubiquitinating activity of BAP1 (16). Cancer Res. 64, 7205 (2004).
21. S. Misaghi et al., J. Biol. Chem. 280, 1512 (2005).
croarray gene expression profiling of 92.1 UM Our findings strongly implicate mutational inac- 22. Y. Wang et al., Nucleic Acids Res. 35, D298 (2007).
cells transfected with control versus BAP1 siRNA tivation of BAP1 as a key event in the acquisition 23. This work was supported by grants to J.W.H. from the
showed that most of the top genes that discrim- of metastatic competence in UM, and they ex- National Cancer Institute (R01 CA125970), Barnes-Jewish
inate between class 1 and class 2 tumors shifted pand the role of BAP1 and other deubiquitinating Hospital Foundation, Kling Family Foundation, Tumori
Foundation, Horncrest Foundation, and a Research to
in the class 2 direction in BAP1-depleted cells enzymes as potential therapeutic targets in cancer. Prevent Blindness David F. Weeks Professorship, and by
compared with control cells (fig. S4). Similarly, awards to the Department of Ophthalmology and Visual
depletion of BAP1 shifted the gene expression References and Notes Sciences at Washington University from a Research to
profile of the multi-gene clinical prognostic assay 1. S. Landreville, O. A. Agapova, J. W. Harbour, Future Prevent Blindness, Inc., unrestricted grant, and the
Oncol. 4, 629 (2008). NIH Vision Core Grant P30 EY02687c. E.D.O.R. was
toward the class 2 signature (Fig. 2B). BAP1 de-
2. M. D. Onken, L. A. Worley, M. D. Tuscan, J. W. Harbour, supported by NIH National Institute of Arthritis and
pletion caused a reduction in mRNA levels of Musculoskeletal and Skin Diseases training grant
J. Mol. Diagn. 12, 461 (2010).
neural crest migration genes (ROBO1), melano- 3. P. T. Finger; 7th Edition, AJCC-UICC Ophthalmic Oncology AR007279-31A1. We thank J. Hoisington-Lopez from
cyte differentiation genes (CTNNB1, EDNRB, and Task Force, Arch. Pathol. Lab. Med. 133, 1197 (2009). the Center for Genome Sciences (Washington University

SOX10), and other genes that are down-regulated 4. C. D. Van Raamsdonk et al., Nature 457, 599 (2009). School of Medicine) for running Solexa paired end
sequences and M. Lovett for comments on the
in class 2 tumors (LMCD1 and LTA4H) (19). In 5. M. D. Onken et al., Invest. Ophthalmol. Vis. Sci. 49,
manuscript. J.W.H. and Washington University may
5230 (2008).
contrast, BAP1 depletion caused an increase in 6. J. Bauer et al., Br. J. Cancer 101, 813 (2009). receive income based on a license of related technology
mRNA levels of CDH1 and the proto-oncogene 7. L. A. Worley et al., Clin. Cancer Res. 13, 1466 (2007). by the university to Castle Biosciences, Inc.
KIT, which are highly expressed in class 2 tumors 8. S. Bashiardes et al., Nat. Methods 2, 63 (2005).
(20). Similar results were seen in other UM cell 9. S. B. Ng et al., Nat. Genet. 42, 30 (2010). Supporting Online Material
10. Materials and methods are available as supporting www.sciencemag.org/cgi/content/full/science.1194472/DC1
lines and with an independent BAP1 siRNA (10). material on Science Online. Materials and Methods
GNAQ mutations occur early in UM and are 11. D. E. Jensen et al., Oncogene 16, 1097 (1998). Figs. S1 to S5
not sufficient for malignant transformation (4), 12. S. Misaghi et al., Mol. Cell. Biol. 29, 2181 (2009). Tables S1 to S3
but they may create a dependency of the tumor 13. H. Nishikawa et al., Cancer Res. 69, 111 (2009). References
14. Y. J. Machida, Y. Machida, A. A. Vashisht, J. A. Wohlschlegel,
cells on constitutive GNAQ activity. In contrast, A. Dutta, J. Biol. Chem. 284, 34179 (2009). 30 June 2010; accepted 25 October 2010
BAP1 mutations occur later in UM progression 15. S. Tyagi, A. L. Chabes, J. Wysocka, W. Herr, Mol. Cell 27, Published online 4 November 2010;
and coincide with the onset of metastatic behav- 107 (2007). 10.1126/science.1194472
Direct Exchange of Electrons Within the methanogen Methanobacterium ruminantium,

which consumed hydrogen with the reduction of
Aggregates of an Evolved Syntrophic

carbon dioxide to methane (Eq. 2) (6):
CH3 CH2 OH þ H2 O →
Coculture of Anaerobic Bacteria CH3 COO− þ 2H2 þ Hþ ð1Þ
Zarath M. Summers,1 Heather E. Fogarty,1 Ching Leang,1 Ashley E. Franks,1 4H2 þ CO2 → CH4 þ 2H2 O ð2Þ
Nikhil S. Malvankar,1,2 Derek R. Lovley1*
The S organism is no longer available in cul-
Microbial consortia that cooperatively exchange electrons play a key role in the anaerobic ture, but Pelobacter carbinolicus functions
processing of organic matter. Interspecies hydrogen transfer is a well-documented strategy similarly (7). P. carbinolicus evolved from Fe(III)-
for electron exchange in dispersed laboratory cultures, but cooperative partners in natural reducing members of the Geobacteraceae family
environments often form multispecies aggregates. We found that laboratory evolution of a to grow as an ethanol-oxidizing syntroph (8).
coculture of Geobacter metallireducens and Geobacter sulfurreducens metabolizing ethanol When Fe(III)-reducing microorganisms deplete
favored the formation of aggregates that were electrically conductive. Sequencing aggregate Fe(III) in anaerobic soils and sediments, it is
DNA revealed selection for a mutation that enhances the production of a c-type cytochrome advantageous for them to form syntrophic as-
involved in extracellular electron transfer and accelerates the formation of aggregates. sociations with microorganisms that can accept
Aggregate formation was also much faster in mutants that were deficient in interspecies the electrons that were formerly transferred to
hydrogen transfer, further suggesting direct interspecies electron transfer. Fe(III).
To investigate how Fe(III) reducers in the Geo-
nterspecies exchange of metabolites is cru- is interspecies hydrogen transfer, in which two bacteraceae family switch to syntrophic growth,
I cial to the balanced functioning of many

microbial communities (1–5). The canon-
ical example of an essential microbial exchange
cell types, neither of which is independently ca-
pable of anaerobically oxidizing an organic com-
pound, cooperatively exchange electrons through
we initiated nine replicate cocultures (9) with
Geobacter metallireducens, an ethanol-oxidizing
Fe(III) reducer closely related to P. carbinolicus
the production and consumption of hydrogen in (10). Geobacter sulfurreducens (11), which can-
1 order to degrade the substrate (4, 5). The con- not metabolize ethanol, was added as a hydrogen-
Department of Microbiology, University of Massachusetts,
Amherst, MA 01003, USA. 2Department of Physics, University cept of interspecies hydrogen transfer was devel- consuming partner with fumarate as the electron
of Massachusetts, Amherst, MA 01003, USA. oped from studies of Methanobacillus omelianskii, acceptor, which G. metallireducens cannot use.
*To whom correspondence should be addressed. E-mail: a coculture of the “S organism” that converted In such a coculture, G. sulfurreducens could
dlovley@microbio.umass.edu ethanol to acetate and hydrogen gas (Eq. 1), and presumably make ethanol metabolism by G.

REPORTS
metallireducens possible by consuming the hy-
drogen produced by G. metallireducens with
the reduction of fumarate to succinate (Eq. 3);
G. sulfurreducens would also consume the ace-
tate that G. metallireducens produced from eth-
anol (Eq. 4):
H2 þ C4 H2 O4 2− → C4 H4 O4 2− ð3Þ
CH3 COO− þ 4C4 H2 O4 2− þ 2H2 O þ Hþ →
2CO2 þ 4C4 H4 O4 2− ð4Þ
Initially the coculture grew very slowly and
required about 30 days to metabolize ~70% of
the ethanol provided. During this phase, mea-
sured hydrogen concentrations in the culture
were ~10 parts per million (ppm). With con-
tinued transfer (1% inoculum) into fresh medi-
um after the cocultures had metabolized ~70%

Fig. 1. Time required for sequential transfers of each of the individual cocultures of G. metallireducens of the ethanol, the coculture adapted to consume
and G. sulfurreducens to metabolize at least 70% of the ethanol provided. at least 70% of the ethanol within 4 days (Fig.
1). This increase in metabolic rate was accom-
Fig. 2. Aggregates in panied by the formation of large spherical ag-
evolved coculture of gregates (diameter ~1 to 2 mm) that began to
G. metallireducens and appear as small flocks by transfer 12 and had
G. sulfurreducens. (A)
formed large, tight spherical associations by
Aggregates in culture
transfer 30 (Fig. 2A). Hydrogen concentrations
bottle. (B) Scanning elec-
tron micrograph of a typ- were ~6 ppm at this stage. The aggregates were
ical aggregate. (C) FISH morphologically similar to those that typically
of a semi-thin section of form during anaerobic treatment of wastewater
an aggregate treated (12) with channels to promote exchange of ma-
with green-fluorescing terials with the bulk environment (Fig. 2B).
G. metallireducens probes Quantitative polymerase chain reaction tar-
and red-fluorescing G. geting 16S rRNA gene sequences indicated that
sulfurreducens probe. (D) G. metallireducens accounted for ~15% of the
FISH analysis at higher cells in the aggregates (fig. S1). Examination of
magnification. thin sections of the aggregates by fluorescence
in situ hybridization (FISH) with species-specific
oligonucleotide probes revealed that the two
species formed distinct clusters within the aggre-
gates (Fig. 2, C and D).
To evaluate the role of interspecies hydrogen
transfer within the aggregates, we initiated co-
cultures with a strain of G. sulfurreducens in which
the gene hyb was deleted. This gene encodes a
hydrogenase subunit, and previous studies have
shown that the strain with hyb deleted is unable
C Aggregates with Evolved WT cells

4
Aggregates with Evolved WT cells
Aggregates with G. sulfurreducens ∆hyb
Aggregates with G. sulfurreducens ∆hyb
Control
2
Current (µA)
-2
Fig. 3. Localization of the c-type cytochrome OmcS in aggregates and aggregate con-
ductivity. (A) Transmission electron micrograph of an aggregate thin section successively
-4
labeled with rabbit polyclonal antibodies to OmcS and antibody to rabbit immunoglobulin G
conjugated with 10-nm gold secondary antibody. (B) Transmission electron micrograph of a -0.4 -0.2 0.0 0.2 0.4
whole-cell mount from an aggregate treated with antibodies as in (A). (C) Linear ohmic Voltage (Volts)
response of aggregates spanning two gold electrodes, and lack of response in media controls.

REPORTS
to consume hydrogen (13). The coculture meta- the intercellular matrix (Fig. 3A) and was asso- (26, 27), suggests that interspecies hydrogen trans-
bolized ethanol (fig. S2). Furthermore, it formed ciated with pili (Fig. 3B). Heme staining and fer is not the primary mechanism for electron
large aggregates within 21 days, rather than the Western blot analysis indicated that OmcS was exchange. Rapid direct electron exchange is con-
7 months required for the cocultures containing the most abundant c-type cytochrome in the sistent with the findings in those studies, and in
wild-type G. sulfurreducens. Interspecies formate aggregates and that OmcS was much more fact, extracellular c-type cytochromes may play
transfer is a potential alternative to interspecies abundant in the evolved aggregates than in mono- an important role in aggregates catalyzing syn-
hydrogen transfer for electron exchange between cultures of G. sulfurreducens (fig. S5). Deletion trophic methane oxidation (28).
some organisms (5), but this was not a possibil- of the genes encoding OmcS or PilA, the struc-
ity in the Geobacter coculture because G. sulfur- tural pilin protein, prevented any visible growth References and Notes
reducens is unable to use formate as an electron even after long-term incubation (>9 months). 1. C. J. Marx, Science 324, 1150 (2009).
2. K. L. Hillesland, D. A. Stahl, Proc. Natl. Acad. Sci. U.S.A.
donor (11), and aggregates were incapable of These results show that OmcS is essential 107, 2124 (2010).
formate-dependent fumarate reduction (fig. S3). for effective syntrophic electron exchange and 3. K. Knittel, A. Boetius, Annu. Rev. Microbiol. 63, 311 (2009).
These results suggest that an alternative mecha- that selective pressure for syntrophic growth 4. M. J. McInerney, J. R. Sieber, R. P. Gunsalus, Curr. Opin.
nism of electron transfer between the two Geo- selected for a mutation that enhanced OmcS pro- Biotechnol. 20, 623 (2009).
5. A. J. Stams, C. M. Plugge, Nat. Rev. Microbiol. 7, 568
bacter species, enhanced by close cell association, duction. The lack of detailed study of G. metal-
(2009).
confers a growth advantage when interspecies lireducens and the lack of homology between 6. M. P. Bryant, E. A. Wolin, M. J. Wolin, R. S. Wolfe, Arch.
hydrogen transfer is no longer possible. outer-surface cytochromes in the Geobacter spe- Mikrobiol. 59, 20 (1967).
Sequencing of genomic DNA extracted from cies (20) has precluded definitive verification of 7. B. Schink, in The Prokaryotes, M. Dworkin, S. Falkow,

E. Rosenberg, K. H. Schleifer, E. Stackebrandt, Eds.
one of the aggregate cultures evolved from wild-type the mechanisms for extracellular electron trans-
(Springer, New York, 2006), vol. 7, pp. 5–11.
cells after the 15th transfer (~100 cell generations) fer in G. metallireducens, but it is expected that 8. J. E. Butler, N. D. Young, D. R. Lovley, BMC Genomics 10,
revealed a single mutation in G. sulfurreducens G. metallireducens also uses a network of con- 103 (2009).
and none in G. metallireducens. The mutation in ductive pili and cytochromes, similar to the closely 9. See supporting material on Science Online.
G. sulfurreducens, which was also present in the related G. sulfurreducens. Thus, a likely model 10. D. R. Lovley et al., Arch. Microbiol. 159, 336 (1993).
11. F. Caccavo Jr., et al., Appl. Environ. Microbiol. 60,
other eight replicate cocultures but was not defor electron exchange between G. sulfurreducens 3752 (1994).
tected in the common inoculum, was a deletion and G. metallireducens is that OmcS of G. sulfur- 12. J. E. Schmidt, B. K. Ahring, Biotechnol. Bioeng. 49,
of a single base pair in the gene encoding PilR, reducens can accept electrons from outer-surface 229 (1996).
which functions as an RpoN-dependent enhancer- c-type cytochromes of G. metallireducens that 13. M. V. Coppi, R. A. O’Neil, D. R. Lovley, J. Bacteriol. 186,
3022 (2004).
binding protein and regulates the expression of a are either localized on the cell or along pili. This 14. K. Juárez et al., J. Mol. Microbiol. Biotechnol. 16, 146
variety of genes in G. sulfurreducens (14). This direct exchange of electrons would alleviate the (2009).
deletion resulted in a frame shift changing the need for interspecies hydrogen transfer and 15. T. Mehta, M. V. Coppi, S. E. Childers, D. R. Lovley,
amino acids starting at position 337 and intro- would explain the effective syntrophy between Appl. Environ. Microbiol. 71, 8634 (2005).
16. D. E. Holmes et al., Environ. Microbiol. 8, 1805 (2006).
duced a stop codon at position 342, resulting in G. metallireducens and G. sulfurreducens, even
17. C. Leang, X. Qian, T. Mester, D. R. Lovley, Appl. Environ.
a truncated PilR protein that lacked the helix- when G. sulfurreducens was unable to consume Microbiol. 76, 4080 (2010).
turn-helix domain required for DNA binding hydrogen. Indeed, the aggregates exhibited ohmic 18. G. Reguera et al., Nature 435, 1098 (2005).
(fig. S4). Although this mutation was not de- conductance when placed between two gold elec- 19. G. Reguera et al., Appl. Environ. Microbiol. 72, 7345
(2006).
tected in the common inoculum used to initiate trodes (Fig. 3C and fig. S6).
20. J. E. Butler, N. D. Young, D. R. Lovley, BMC Genomics 11,
the cocultures, it is conceivable that it was a rare Our results suggest that selective pressure for 40 (2010).
variant in that culture. Furthermore, genome re- effective electron exchange in this coculture fa- 21. Y. A. Gorby et al., Proc. Natl. Acad. Sci. U.S.A. 103,
sequencing may fail to detect transposition or vored direct electron transfer between consorti- 11358 (2006).
other genome rearrangements. Therefore, to de- um members, rather than interspecies hydrogen 22. S. S. Oztürk, B. Ø. Palsson, J. H. Thiele, Biotechnol. Bioeng.
33, 745 (1989).
termine whether a mutation in pilR was sufficient transfer. This is consistent with previous specu- 23. B. Kartal, J. G. Kuenen, M. C. van Loosdrecht, Science
to promote rapid syntrophic metabolism, we ini- lation on the possibility of direct electron trans- 328, 702 (2010).
tiated the cocultures with a strain of G. sulfur- fer between cells (18, 21), but in the absence of 24. M. J. Alperin, T. M. Hoehler, Am. J. Sci. 309, 869 (2009).
reducens in which pilR was deleted. Within 21 experimental evidence, this possibility has been 25. B. Orcutt, C. Meile, Biogeosciences 5, 1587 (2008).
26. K. B. Sørensen, K. Finster, N. B. Ramsing, Microb. Ecol.
days, all three replicate cocultures formed aggre- met with skepticism (4, 5), especially consider- 42, 1 (2001).
gates in the initial culture tubes, which suggests ing the preponderance of evidence from studies 27. K. Nauhaus, A. Boetius, M. Krüger, F. Widdel, Environ.
that inactivation of PilR was sufficient to pro- with defined cocultures in which interspecies Microbiol. 4, 296 (2002).
mote aggregate formation. transfer of hydrogen or formate predominates. 28. A. Meyerdierks et al., Environ. Microbiol. 12, 422 (2010).
29. We thank P. Brown, C. Barrett, K. Zengler, and Y. Qiu
One of the primary impacts of deleting pilR Previous coculture studies, however, typically ex- for sequence analysis; L. Raboin for SEM assistance;
in G. sulfurreducens is enhanced expression amined dispersed (i.e., not aggregated) syntrophic and K. Rubin for photography assistance. Supported
of the gene encoding OmcS (14), a multiheme partners (22). The lack of cell aggregation dic- by the Office of Science (Office of Biological and
c-type cytochrome that promotes electron trans- tates that interspecies hydrogen or formate trans- Environmental Research), U.S. Department of Energy,
Cooperative Agreement DE-FC02-02ER63446 and
fer to insoluble Fe(III) oxides (15) and electrodes fer predominates over direct cell-to-cell electron
Award DE-FC-0004485.
(16). OmcS is primarily associated with pili (17). transfer under those conditions. In contrast, ag-
The multiple hemes in OmcS are thought to gregation of cells involved in syntrophic anaer- Supporting Online Material
facilitate the transfer of electrons exported from obic metabolism appears to be the rule in most
Materials and Methods
the cell along the pili, which are electrically con- environments (1, 3, 23) and is key to the suc- Figs. S1 to S5
ductive (18, 19), onto electron acceptors, such as cessful anaerobic treatment of wastewater (12). References
Fe(III) oxide (17). Immunogold labeling of OmcS Modeling of electron transfer (22, 24–26) in 16 August 2010; accepted 22 October 2010
revealed that OmcS was dispersed throughout such aggregates, as well as experimental evidence 10.1126/science.1196526

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Edge Plates enable researchers to obtain quality data from automated FlexiPERM reusable silicone inserts can be used to subdivide slides and
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sults to remain more true to the population phenotype for more efficient, ate for use with 60 mm dishes. FlexiPERM conA and conB inserts are
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www.sciencemag.org/products 1419
POSITIONS OPEN POSITIONS OPEN
online @sciencecareers.org
Multiply The Power of Science
TWO ASSISTANT PROFESSORSHIPS POSTDOCTORAL POSITIONS

Systems Biology and Texas A&M Institute for Regenerative
Cell & Molecular Biology Medicine at Temple, TX
University of California, Merced
The Texas A&M Institute for Regenerative
The School of Natural Sciences at the Uni- Medicine is seeking POSTDOCTORAL FEL-
versity of California, Merced seeks applicants LOWS for research on adult stem/progenitor
for two tenure-track assistant professor posi- cells. The Institute is dedicated to research both
tions in the areas of Systems Biology and Cell on the basic biology of stem/progenitor cells
& Molecular Biology (CMB). Candidates are and their potentials for therapies of human
expected to have an excellent publication diseases. It occupies newly created laborato-
Science Careers record and to teach biology effectively at both ries and a series of core laboratories in an ex-
undergraduate and graduate levels. Systems panding academic medical center. Postdoctoral
Classified Advertising Biology is used here to mean a research approach appointments will be for one year with the op-
For full advertising details, go to that uses comprehensive datasets and multiple portunity to renew for a second or third year
ScienceCareers.org and click For Employers, types of analysis to relate overall biological func- subject to performance. Please send curricu-
or call one of our representatives. tion to the underlying biochemical or biophys- lum vitae, brief statement of research interests,
ical processes, with an ultimate goal of a predictive and three recommendation letters to the atten-
Tracy Holmes understanding. For the CMB search priority will tion of Darwin J. Prockop, M.D., Ph.D., Di-
Worldwide Associate Director be given to candidates studying the molecular rector, Texas A&M Institute for Regenerative
Science Careers and cellular mechanisms of biological processes Medicine at e-mail: regenerate@medicine.
Phone: +44 (0) 1223 326525 in model organisms. For both positions, appli- tamhsc.edu. The Texas A&M Health Science Center
cations of special relevance to research emphases is an Affirmative Action/Equal Opportunity Employer.
UNITED STATES & CANADA at UC Merced include the mechanisms of cell
fate decisions, complex diseases such as diabetes
E-mail: advertise@sciencecareers.org or inflammatory disorders, and microbial systems
Fax: 202-289-6742 relevant to human disease. For more information,
see website: http://jobs.ucmerced.edu/n/ ASSISTANT OR ASSOCIATE PROFESSOR
Tina Burks
Midwest/West Coast/ academic/listings.jsf?seriesId010. The Division of Musculoskeletal Sciences, Depart-
South Central/Canada Interested applicants should submit online: ment of Orthopaedics and Rehabilitation at The Penn-
Phone: 202-326-6577 curriculum vitae, statements of research and sylvania State University, College of Medicine announces
teaching interests, and the names and addresses a search for an Assistant or Associate Professor (ten-
Elizabeth Early of five references. Applications will be con- ured or tenure-track) in the area of musculoskeletal sci-
East Coast & Industry sidered starting on November 25, 2010. UC ences or engineering. This position includes a highly
Phone: 202-326-6578 Merced is an AA/EOP employer. competitive salary and significant startup funds. The
Marci Gallun successful candidate will be an individual who can ap-
Sales Administrator ply modern biomedical science and engineering con-
Phone: 202-326-6582 cepts to the study of musculoskeletal tissues. This is a
unique opportunity to join a well-established, highly
Online Job Posting Questions interactive research group consisting of engineers, ma-
Phone: 202-326-6577 BIO-COMPUTER SCIENTIST terial, clinical, and basic scientists focusing on muscu-
Wilkes University invites applications for a tenure- loskeletal research. A joint academic appointment will
EUROPE & REST OF WORLD track ASSISTANT PROFESSOR appointment in include a primary appointment in the College of Med-
E-mail: ads@science-int.co.uk the College of Science and Engineering starting fall icine and a secondary appointment in an appropriate
Fax: +44 (0) 1223 326532 2011. This position is in part sponsored by the Howard department at Penn State. The Penn State College of
Hughes Medical Institute to enhance quantitative skills Medicine is located in Hershey, Pennsylvania and of-
Alex Palmer of undergraduates in the life sciences. We seek a can- fers a highly desirable lifestyle and an affordable cost of
Phone: +44 (0) 1223 326527 didate who can teach and conduct research at the living in close proximity to many metropolitan areas
Susanne Kharraz Tavakol interface between biology and computer science in an including Baltimore, Washington, D.C., Philadelphia,
Phone: +44 (0) 1223 326529 undergraduate setting. The applicant must have a Ph.D. and New York City. Applications will be accepted until
in a relevant field; postdoctoral experience is preferred. the position is filled.
Dan Pennington Potential areas of specialty include but are not limited to Send curriculum vitae to: Ananya Das, Faculty
Phone: +44 (0) 1223 326517 mathematical modeling and simulation, bioinformatics, Search Committee, Division of Musculoskeletal Sci-
computational biology, epidemiology, or geospatial ences, Department of Orthopaedics and Rehabili-
Lisa Patterson
statistics. tation, The Pennsylvania State University, College
Phone: +44 (0) 1223 326528
The successful candidate will develop a strong re- of Medicine, 500 University Drive, H089, Hershey,
search program involving undergraduates and will par- PA l7033.
JAPAN Penn State is committed to Affirmative Action, Equal Oppor-
ticipate in efforts to provide a strong interface between
ASCA Corporation Biology and CS. Teaching responsibilities will include tunity, and the diversity of its workforce.
Jie Chin participation in introductory and upper-level under-
Phone: +81-3-6802-4616 graduate courses appropriate to the candidate_s areas
Fax: +81-3-6802-4615 of expertise. A competitive startup package is available. TENURE-TRACK FACULTY POSITIONS in
E-mail: careerads@sciencemag.jp Wilkes University is an independent institution of Cardiovascular Physiology, Metabolism, Stress
To subscribe to Science: higher education with approximately 2,200 under- The Department of Integrative Biology and Phys-
In United States call 866-434-2227 graduates located in Wilkes-Barre, Pennsylvania, a iology at the University of Minnesota Medical School
In the rest of the world call +1 202-326-6417 mid-sized city within two and one-half hours driving seeks outstanding faculty candidates in integrative sys-
distance of New York City and Philadelphia. The new tems biology of the cardiovascular system, metabolism,
All ads submitted for publication must comply hire will join a productive and collaborative cluster of and stress open to all ranks (ASSISTANT, ASSO-
with applicable U.S. and non-U.S. laws. Science computational scientists from mathematics, computer CIATE, FULL PROFESSOR). Successful candidates
reserves the right to refuse any advertisement
at its sole discretion for any reason, including science, biology, and chemistry. will have an innovative research program that em-
without limitation for offensive language or Please send application letter, curriculum vitae in- braces biological complexity from molecular building
inappropriate content, and all advertising is cluding contact information for three references, re- blocks to the living organism with a focus on cardio-
subject to publisher approval. Science encour-
ages our readers to alert us to any ads that search and teaching statements, and recent publications vascular biology, metabolic syndrome, diabetes, obesity,
they feel may be discriminatory or offensive. in PDF format. For full consideration, submit all mate- and stress. A commitment to excellence in teaching is
rials by January 7, 2011, to e-mail: william.terzaghi@ essential. Minimum requirements are a Ph.D., M.D., or
wilkes.edu. Wilkes University is an Equal Opportunity employer M.D.-Ph.D. with two or more years of postdoctoral
committed to a diverse faculty, staff and student body. Applicants training. Applicants should apply online (website:
from diverse backgrounds are strongly encouraged to apply. http://employment.umn.edu; requisition number
For further information about this position, please 169479) and submit curriculum vitae, cover letter,
contact the Chair of the search committee, Dr. William and references. The University of Minnesota is an Equal
Terzaghi at e-mail: william.terzaghi@wilkes.edu. Opportunity Educator and Employer.
1420 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencecareers.org

featured employer online @sciencecareers.org
FACULTY POSITIONS in
INFECTIOUS DISEASES
HARVARD UNIVERSITY Tulane National Primate
Assistant/Associate Professors Research Center
TULANE UNIVERSITY
Department of Genetics
and Complex Diseases The Tulane National Primate
Research Center (TNPRC) wishes to expand its
infectious disease research programs involving
The Department of Genetics and Complex Diseases (GCD) at the Harvard School of Public molecular virology, bacteriology, and parasitology
Health (HSPH) invites applications for tenure-track positions at the level of assistant professor. by reaching into areas covered by the NIH/NIAID
Exceptional associate professor candidates will also be considered. Successful applicants will National Biodefense Program, as well as vaccin-
hold a PhD and/or MD degree and will have a record of outstanding productivity. Individu- ology and vector-borne and emerging diseases.
als are sought particularly in the following areas to complement the existing research and Agent-specific interests for the expansion include
training goals of the department: signal transduction related to energy and nutrient sensing but are not limited to HIV/AIDS; tick-borne bacte-
pathways, regulation of metabolic homeostasis, inflammatory and stress response pathways rial diseases, e.g. Lyme disease; bacterial diseases
that affect the respiratory and nervous systems,
related to chronic metabolic diseases and aging, cancer metabolism, and epigenetic regula-
e.g. tuberculosis and pneumococcal pneumonia;
tion of metabolism. Individuals using systems and/or computational approaches applied at a brucellosis; and malaria.
mechanistic level to problems of metabolic homeostasis, gene-environment interactions and/or
adaptive responses are also encouraged to apply. The candidate should possess the ability Four positions are available at the rank of Assis-
to work collaboratively with other scientists and the scholarly qualities required to mentor tant, Associate, or Full Professor. Designations
doctoral students in the graduate program in the Division of Biological Sciences. Generous may be either in the Tenure-Track or within the
startup packages and state-of-the-art research facilities are available. Research Professor Series, depending on qualifi-
cations. Academic appointments will be in appro-
Please send a letter of application, including a statement of current and future research interests, priate Departments of either the Tulane University
curriculum vitae, sample publications, and the names of four references to the following address. School of Medicine or the Department of Tropical
Applicants should ask their four references to write independently to this address. Medicine of the Tulane School of Public Health
and Tropical Medicine. The research portfolio and
Chair, GCD Search, c/o Holly Johnsen infrastructure of the TNPRC, which is undergo-
Department of Genetics & Complex Diseases ing a vigorous expansion, currently includes
Harvard School of Public Health NIH-funded research programs on AIDS, Lyme
655 Huntington Avenue, Building II, 101 disease, tuberculosis, varicella, malaria, and Cat-
egory A-C Select Agents, using chiefly nonhuman
Boston, MA 02115 primates but also other animal models. The suc-
gcddept@hsph.harvard.edu cessful candidate will be expected to contribute to
The Harvard School of Public Health is committed to increasing the representation of existing research programs and to build or bring
his/her own independent research agenda. All
women and minorities in its faculty, and encourages applications from such candidates.
necessary resources to assure that the candidate
is successful will be provided including ample
laboratory and office space.
The TNPRC has excellent infrastructure to sup-
port collaborative and independent research using
nonhuman primates. In addition to holding one of
THE CHINESE UNIVERSITY OF HONG KONG the largest colonies of nonhuman primates in the
country, the TNPRC is the only National Primate
Applications are invited for:- Research Center that houses a Regional Biosafety
School of Biomedical Sciences Laboratory, to support research under the NIH/
Associate Professors / Assistant Professors (Non-clinical) NIAID National Biodefense Program. Research
(Ref. 1011/037(665)/2) (Closing date: December 28, 2010) resources include extensive BSL2/ABSL2 and
The School of Biomedical Sciences was established under the Faculty of Medicine in June 2009 through BSL3/ABSL3 facilities and highly integrated
re-organization of its preclinical departments. It is the Faculty’s vision to become a world-recognized clinical and laboratory support for infectious
leader in selected fields of biomedical sciences while maintaining excellence in postgraduate and disease studies using nonhuman primates. This
undergraduate educational programmes. Currently, the School has research programmes focusing on includes a full-time staff of clinical veterinarians
neuro-degeneration, cancer biology and inflammation, developmental biology, reproduction and and technicians and core services commonly
endocrinology, vascular and metabolic biology, and stem cell and regeneration. used for infectious disease research including:
The School invites applications for Associate Professorships / Assistant Professorships. Individuals who are (1) Diagnostic Parasitology, (2) Vector-Borne
strongly motivated by the biological importance of their research and who value the opportunity to work in Diseases (maintains ixodid ticks and anoph-
close collaboration with others are welcome to apply. Those with expertise in stem cell biology, eline mosquitoes), (3) DNA Microarray and
developmental genomics, and organogenesis are particularly welcome. The School will provide
Gene Expression, (4) Anatomic Pathology, (5)
state-of-the-art research facilities, appropriate laboratory and office space, and a start-up package.
Applicants should have (i) a PhD, MD, or DVM degree or equivalent in biological sciences or related
Clinical Pathology, (6) Molecular Pathology, (7)
disciplines; (ii) experience in using cutting-edge approaches including but not limited to live cell imaging, Confocal Microscopy and Image Analysis, (8)
genetics, genomics, proteomics, bioinformatics, or transgenic animal models in research; and (iii) a good Flow Cytometry, (9) Cellular Immunology, (10)
track record of research and publication in international peer-reviewed scientific journals in one or more Virus Characterization, Isolation and Production,
related areas. The appointees will (a) teach undergraduate, postgraduate, and general education courses; and (11) Pathogen Detection and Quantification, and
(b) lead a vigorous independent research programme. Appointments will normally be made on contract basis (12) Infectious Disease Aerobiology. More infor-
for up to three years initially commencing May 2011, which, subject to mutual agreement, may lead to mation is available at the following link: http:
longer-term appointment or substantiation later. //www.tnprc.tulane.edu/research_resou.html.
Salary and Fringe Benefits
Salary will be highly competitive, commensurate with qualifications and experience. The University offers a To apply, send a letter indicating your research
comprehensive fringe benefit package, including medical care, plus a contract-end gratuity for appointments interests and experience, curriculum vitae, and the
of two years or longer, and housing benefits for eligible appointees. Further information about the names of three individuals who may be contacted
University and the general terms of service for appointments is available at for references to: Ms. Rita Haynes, Coordinator,
http://www.cuhk.edu.hk/personnel. The terms mentioned herein are for reference only and are subject to TNPRC Search Committee, Tulane National
revision by the University. Primate Research Center, 18703 Three
Application Procedure Rivers Road, Covington, LA 70433; E-mail:
Please send full resume, copies of academic credentials, a publication list and/or abstracts of selected rita@tulane.edu.
published papers together with names, addresses and fax numbers/e-mail addresses of three referees to
whom the applicants’ consent has been given for their providing references (unless otherwise specified), to Tulane University is an Affirmative Action and
the Personnel Office, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong (Fax: (852) 2696 Equal Opportunity Educator and Employer.
1462) by the closing date. The Personal Information Collection Statement will be provided upon request. Women and minorities are strongly encouraged
Please quote the reference number and mark ‘Application - Confidential’ on cover. to apply.
HOWARD HUGHES MEDIC AL INSTITUTE
GRANTS FOR BIOMEDICAL RESEARCH

INTERNATIONAL EARLY CAREER SCIENTISTS
Science is a multinational, cross-cultural The International Early Career Scientist • Have trained at the doctoral,
endeavor that connects researchers Program will select and support highly medical, or postdoctoral level in
across the borders created by discipline qualified scientists working in selected the United States.
countries outside the United States
and continent.The Howard Hughes • Hold a full-time position as an
who are in the critical beginning stages
Medical Institute (HHMI) believes independent researcher at a research-
of their independent careers. HHMI
oriented university, medical school,
it is vital to encourage the careers international early career scientists will
or nonprofit institution in any of the
and scientific creativity of scientists receive five-year grants—$250,000 in
following countries: Argentina, Brazil,
working abroad. HHMI is announcing the first year and $100,000 for each of
Chile, China, Czech Republic, Egypt,
an International Early Career Scientist the following four years.
Hungary, India, Italy, Mexico, Poland,
Program, which will support up to Successful applicants will have trained Portugal, Russia, South Africa, South
35 outstanding scientists working in through the postdoctoral level in a Korea, Spain, Taiwan, Turkey.
vigorous basic research environment.
selected countries outside the • Have made significant contributions
Eligible candidates must have trained
United States who are, or have the in fundamental biomedical research
in the United States at the doctoral,
potential to become, scientific leaders. on basic biological processes or
medical, or postdoctoral level. Appli-
disease mechanisms, and have been
cants are expected to have outstand-
the first or senior author on at least
ing scientific training records and
two peer-reviewed, English-language,
exceptional potential for significant
original research publications.
productivity and originality in their
Five-year basic biomedical independent careers. • Have started their first indepen-
reseach grant dent research position on or after
HHMI recognizes that a supportive
January 1, 2004.
research environment is crucial to
Application deadline: launching a successful research program, • Control their own research direction,
February 23, 2011, so awards will be made only to institu- laboratory space, and funding and
at 2 p.m. ET, U.S. tions that can clearly support the devote most of their professional time
research activities of the grant recipient. to research, mentoring, and teaching.
Application information: Applicants must meet the following
http://www.hhmi.org/research/ HHMI, a nonprofit medical research
eligibility requirements:
competitions organization, plays a powerful role in
• Hold a doctoral degree or medical
advancing biomedical research and
degree and have completed post-
science education. To learn more, visit
doctoral research training.
www.hhmi.org.
Assistant/Associate/Full Professor
Faculty Position in Infectious Diseases Research
Tenure/Tenure Track Faculty, 9-Month Appointment
(80% Research and 20% Teaching) Do you have a passion for cutting-edge
Position Number 103761 science and educating the public about it?
The Virginia-Maryland Regional College of Veterinary Medicine at the University of Maryland-College Five Chief Scientists for the Nature
Park, MD, invites applications from the qualified individuals for a tenured/tenure-track faculty position
Research Center (NRC), North Carolina
in Immunology of Infectious Diseases and Microbial Pathogenesis. This 9-month appointment will be at
Museum of Natural Sciences
the Assistant, Associate or Full Professor level, depending on the qualifications of the selected candidate.
The Department has an excellent core facility, which includes BSL-2 and BSL-3 suites. The North Carolina Museum of Natural Sciences
QUALIFICATIONS: A DVM/PhD or PhD degree with relevant postdoctoral training. in Raleigh, NC will open an 80,000 square-foot
innovative facility in early 2012 to serve as a
RESEARCH: Current focus is on host-pathogen interaction with emphases on virology, immunology, public laboratory for both local and global sci-
microbial pathogenesis, epidemiology, and public health. The position requires the candidate to focus on ence research (www.naturesearch.org). The
host immunity/host-pathogen interactions with importance for human and animal health, zoonosis and NRC seeks five Science Directors to lead its new
public health. The selected candidate will be expected to develop, maintain, and conduct a productive, extra- research laboratories: Genetics/Microbiology;
murally funded research program that will strengthen the current research goals of the College. Applicants Paleontology/GeoSciences; Space Observation;
at the Associate/Full Professor level are expected to have an established, extramurally-funded research Biodiversity/Earth Observation; and Science
program and a strong publication record. The selected candidate will also be expected to develop and Communication. Over 40 additional staff will
maintain active and productive collaborations, both within and outside the College. Excellent opportunities be hired to operate four research-and-education
exist for collaboration with federal agencies (USDA, FDA, NIH) and other University departments. platforms: (1) A three-story theater, Daily Planet,
TEACHING: Active participation in the University’s Graduate Program will be required, to include with the latest technologies for communicating
mentoring of graduate students and serving on student advisory committees. The selected candidate will be science; (2) Investigate Labs for citizen sci-
responsible for the development of one graduate course to be included in the current veterinary medicine ence participation; (3) Exhibits featuring staff
curriculum. This new course will be developed in conjunction with active participation on the college’s research and other critical global issues; and (4)
current teaching objectives and mission goals. Salary will be commensurate with rank and experience. Hands-On Programs of citizen science/distance
learning/mentoring/outreach to diverse audiences
TO APPLY: Applications are accepted through https://jobs.umd.edu (sort for position number 103761). including policy-makers. Directors will execute
Required are (1) cover letter, (2) CV, (3) statement of research interests and plans, (4) statement of teach- cutting-edge research programs under the leader-
ing philosophy and (5) three reference letters. For best consideration, applications should be received by ship of Dr. Meg Lowman, Director of the NRC,
January 31, 2011, or until a suitable candidate is identified. as well as lead a team of lab managers, education
THE UNIVERSITY OF MARYLAI\ID IS AN ATFIRMATTVE ACTION/EQUAL OPPORTUNITY staff, students, and citizen scientists. In addition,
EMPLOYER WOMEN AND MINORITIES ARE ENCORAGED TO APPLY. Directors will have joint appointments at the level
of Associate or Full Professor with one of the 16
campuses of the UNC System, offering additional
research status and some teaching opportuni-
ties. Salary is funded between University and
Museum, with a competitive benefits package.
Applicants who enthusiastically embrace educa-
tion outreach as part of their outstanding research
programs are invited to submit a letter of interest
by December 30, 2010. Suitable candidates must
have a PhD or equivalent in fields relevant to
Full Professors/Group Leaders one of the research labs, 3-5 years postdoctoral
Availableat the State Key Laboratory or research/teaching/ administrative experience,
of Ophthalmology in China and proven expertise with science communica-
tion to diverse audiences. The application process
Established in 1965, Zhongshan Ophthalmic
requires two stages: (1) Interest Period and (2)
Center of Sun Yat-sen University is a leading
Application Period. During the interest period
eye care institution in China. It comprises the
please send a letter of interest, if you choose
Eye Hospital, the Eye Research Institute, the
you can also send, CV; 3 research publications
Department of Preventive Ophthalmology and
(including one oriented to public audiences); 3
the Department of Optometry.
letters of reference plus 3-5 additional contacts;
The China State Key Laboratory of Ophthalmology and a vision statement outlining an unsolved issue
at the Zhongshan Ophthalmic Center seeks full in your scientific arena, and how you could con-
professors or group leaders. Candidates should
• LIFE SCIENCES have a strong academic record in eye and vision
duct research/communicate outreach in the NRC
• CHEMISTRY research. Scientific excellence and an interactive
facilities. Please mail to: Deputy Museum Direc-
• ENVIRONMENTAL SCIENCES personality are the most important criteria
tor, Alvin Braswell, NC Museum of Natural
AND SUSTAINABLE for selection. Non-Chinese scientists are also
Sciences, 11 W Jones St, Raleigh NC 27601;
DEVELOPMENT encouraged to apply. All positions are full-time
or email to: Cindy Bogan, administrative
• HUMANITIES AND SOCIAL and based in Guangzhou, China.
assistant, cindy.bogan@ncdenr.gov (919-733-
SCIENCES 7450 x 262).
• COMPUTER SCIENCE Applicants should submit a curriculum vitae, a
• ENGINEERING letter explaining their research plan in the position Subsequent to submitting the letter of interest,
• MATHEMATICS and an indication of their salary expectation to: interested persons will be notified when to submit
• PHYSICS The Searching Committee a state application form (PD-107) listing the posi-
• NUCLEAR AND PARTICLE Zhongshan Ophthalmic Center tion number and job title, relevant information/
PHYSICS 54 South Xianlie Road work history by 5 PM on the due date listed on the
• EARTH SCIENCES AND Guangzhou, 510060 state website. Please note all relevant knowledge,
ASTRONOMY China skills, abilities, training and experience must be
Fax +8620-87333271 documented on the PD-107 state application form
ZOC@mail.sysu.edu.cn to receive proper credit and consideration (http:
www.gzzoc.com //www.osp.state.nc.us/jobs/gnrlinfo.htm).
The close date is December 31, 2010. NCMNS is an Equal Opportunity/Affirmative
Action Employer.
GROUP LEADER POSITIONS at IMBA, Vienna
We invite applications for two fully funded groupleader positions at IMBA, Vienna. We aim to identify outstanding candidates
who will establish a strong and innovative research program in any area of cell or molecular biology or molecular medicine.
We offer internationally competitive salaries, generous funding IMBA is an internationally recognized research institute that
for up to four positions including all set up and running costs. focuses on stem cell research, RNA biology and molecular
We provide free access to our core service infrastructure includ- medicine and is home to 140 scientists of over 26 nationalities.
ing proteomics, Drosophila facility, FACS sorting, deep sequenc- Together with the neighbouring IMP and the Max Perutz labo-
ing, bio-optics and a 2000m2 mouse house. PhD students are ratories, we provide a dynamic and highly interactive research
recruited through the Vienna Biocenter PhD program, one of environment.
the most competitive graduate programs in Europe. Child care
facilities are available.
Applications including CV, scientific achievements, a five page future research plan and contact details for three referees should be sent
Deadline for
electronically applications: 15th January
to tlsearch@imba.oeaw.ac.at 200931st, 2011. For further information on IMBA see: www.imba.oeaw.ac.at
by January
IMBA
Institute of Molecular Biotechnology
of the Austrian Academy of Sciences
UNIVERSITY PROFESSOR POSITIONS

The Pohang University of Science and Technology (POSTECH) Desired Qualifications:
is Korea’s first research-oriented university and is known for its Nobel laureates, Fields medalists or scholars of equivalent
unique contribution to the advancement of science and caliber
technology in Korea. POSTECH is ranked first among Asia’s Members of world-renowned national academies of
specialized universities in the 2010 Chosun Ilbo - QS Asian sciences/engineering such as the National Academy of
University Rankings. Sciences (USA), the National Academy of Engineering
(USA), and the Royal Society (UK)
POSTECH invites distinguished scholars to join in Editors of the world's top scientific journals or those with
POSTECH’s efforts to become a global leader in science & equivalent experience
technology research. Those who have demonstrated potential to grow to become
a University Professor are also welcome to apply.
Position Announcement:
We are seeking distinguished scholars in all fields of science and Interested individuals should send a CV, a statement of
engineering. Appointments will be made for multiple positions research plans, and a teaching statement to e-mail:
at the rank of University Professor. POSTECH will provide recruit@postech.ac.kr or mailing address: Faculty Search
startup funds of up to 7 million US dollars for each Committee, Office of Academic Affairs, POSTECH, San
distinguished scholar. Salary will be competitive and 31, Hyoja-dong, Nam-gu, Pohang 790-784, Republic of
commensurate with candidate’s qualifications and experience. Korea.
The exact amount of financial support may vary depending on
the field.
Office of the Science and

Technology Adviser to the
Secretary of State
Jefferson Science Fellowship Promega is

The National Academies is pleased to announce a call for applica-
tions for the 2011 Jefferson Science Fellows program. This pro-
Hiring Due to Growth in
gram is a model for engaging the American academic science, Research & Applied Markets
technology and engineering communities in the formulation and With strong corporate growth and double digit investment in R&D,
implementation of U.S. foreign policy. Promega continues to expand its scientific staff to develop new
technological capabilities for the life sciences.
Jefferson Science Fellows spend one year at the U.S.
Department of State or the U.S. Agency for International Develop- Promega is seeking additional scientific expertise across multiple areas,
ment in Washington, DC and may periodically travel to U.S. for- including analytical methods in cellular biology and proteomics,
nucleic acid isolation and analysis, high throughput assays, laboratory
eign embassies and/or missions.
automation, toxicology, and human identity.
Jefferson Science Fellow awards are open to tenured academic Requirements include:
scientists, technologists and engineers from U.S. institutions of • Desire to innovate new tools for advancing basic research, drug
higher learning. Applicants must be U.S. citizens and will be re- discovery, human identity, and molecular diagnostics
quired to obtain a security clearance.Detailed information on the • Willingness to work in a dynamic team environment engaged in
Jefferson Science Fellows program is available online: diverse disciplines.
www.nas.edu/jsf • Interest in sharing scientific achievements and collaborating with
innovators from other companies, universities, and institutes.
The deadline for applications for the 2011 program year is Promega is located in Madison, WI, a community of international
January 14, 2011. scientists, beautiful natural settings and an award winning quality of life.
The Jefferson Science Fellows program is sponsored by the U.S. Established in 1978, Promega is a privately held company
Department of State and U.S. Agency for International Development. supplying 2000+ products to over 100 countries.
Visit www.promega.com/RDjobs
Promega Corporation is an equal opportunity/
affirmative action employer.
Chair, Department of Biological Sciences

Pocatello, Idaho
Department: Biological Sciences

TheMaxPlanckInstituteofMolecularCellBiologyandGenetics(MPI-CBG)in Primary Purpose: Serve as a faculty member and Chair of Biological
Dresden is seeking outstanding candidates for
Sciences.
Research Group Leader Minimum Qualifications: Doctoral Degree and credentials to qualify for
tenure and appointment at the level of full professor.
in Membrane Biology Preferred Qualifications: The successful applicant will have a Doctorate
in biological sciences or related field, a commitment to undergraduate
Research at the MPI-CBG focuses on the molecular mechanisms underlying and graduate education, an excellent record of publication and extramu-
the structure and organization of cells and tissues (see http://www.mpi-
ral support, and strong leadership qualities; candidate should have a
cbg.de).Inthissearch,weareparticularlylookingforapplicantsinterestedin
membrane biochemistry, though all excellent candidates will be considered. proven track record in research.
Research Group Leader positions are initially for 5 years at the EG15 level ac- Salary/Pay Information: Commensurate with qualifications and experi-
cording to theTVÖD scale with the possibility of extension for up to an addi- ence. Competitive benefits package.
tional 4 years. Funds are available for a postdoctoral fellow, a PhD student, a
technician, as well as for consumables and equipment. Please send your CV, Term of Employment: 12 Month /Full-time
publication list and a short description of research accomplishments and
future plans to Prof. Marino Zerial (see address below) by 7 January 2011. Ple- Location: Pocatello
ase also arrange for three letters of reference to be sent separately before the
deadline. We especially encourage women to apply. Application Process: Please visit ISU at
https://isujobs.net/applicants/jsp/shared/Welcome_css.jsp
Prof. Marino Zerial Special Instructions to Applicants: Please include in your
Max Planck Institute cover letter a statement of leadership philosophy.
of Molecular Cell Biology and Genetics
Pfotenhauerstr. 108 Closing Date: Open Until Filled
01307 Dresden, Germany
Please send your applications to: 2010-RGL-1080@mpi-cbg.de ISU is an equal opportunity/affirmative action employer. We have an
institution-wide commitment to inclusion and diversity and encourage all
qualified individuals to apply. Veterans' preference.
TheMaxPlanckSocietyiscommittedtoemploymorehandicappedindividu- Upon request, reasonable accommodations in the application process will be
als and actively seek their applications. provided to individuals with disabilities.
Assistant Professor (Tenure Track) of
Cartilage Engineering and Regeneration
Faculty of Science
The future Department of Health Sciences and Techno-

logy at ETH Zurich invites applications for the above men-
The Faculty of Science invites applications for an tioned position. The new professor is expected to bridge
the gap between the fundamental biosciences and clini-
cal applications by working out an outstanding research
Assistant Professorship program, developing innovative methods that promote
cartilage regeneration. Key components of this research
in Physical Chemistry program could center around the development of new
insights into the molecular structure of cartilage matrix
proteins, cartilage signaling, mechanobiological aspects,
in the area of the structure and dynamics of molecular
mechanisms regulating cartilage metabolism, as well
systems in the condensed phase (experimental or theoretical). as repair and regeneration mechanisms. These insights
The new professor is expected to pursue cutting-edge would be utilized to define novel tissue engineering strat-
egies to replace or regenerate cartilage (both articular
research in an area that is complementary to that of existing
and septal) and joint tissues (i.e. meniscus and synovium)
groups at the University of Zurich. He or she is furthermore that interact with cartilage. The primary criterion is either
expected to teach special courses in Chemistry at the Master demonstrated or potential excellence in research and
technology developments, rather than on one particular
or PhD level in either German or English.
scientific aspect. The professorship comes with generous
resources, including start-up funds and annual alloca-
The Institute of Physical Chemistry is located on the Irchel tions, to establish a significant research program. Close
campus, where most of natural science Institutes of the collaboration with researchers at ETH Zurich, the FIFA
Medical Assessment Research Center/Schulthess Clinic
University are located. The Faculty of Science provides for
and University Hospital Zurich are envisioned, thereby
a very stimulating and attractive environment for inter- complementing already existing efforts.
disciplinary research, which is further strengthened by the
The candidate will contribute to the interdisciplinary
nearby science campus of the ETH Zurich, the Empa and the
education programs offered at ETH Zurich; this includes
Paul Scherrer Institute. the ETH Health Sciences and Technology Bachelor and
Master Program, and the ETH Master Program in Bio-
medical Engineering. The new professor will be expected
The position will be filled at the Assistant Professor level
to teach undergraduate level courses (German or Eng-
(non-tenure track, for six years). Candidates are invited to
lish) and graduate level courses (English). The candidate’s
submit an application package by January 31, 2011, including educational program will support a vital link between
curriculum vitae, publication list, outline of current and future biological and physical scientists and engineers and clini-
cal professionals.
research interests, teaching philosophy and names and
addresses of three potential referees. Documents should be Assistant professorships have been established to promote
addressed to Prof. Dr. Michael Hengartner, Dean of the the careers of younger scientists. The initial appointment
is for four years with the possibility of renewal for an
Faculty of Science, University of Zurich, and submitted as a
additional two-year period and promotion to a permanent
single PDF file to jobs@mnf.uzh.ch. For further information, position.
please contact Prof. Dr. P. Hamm at phamm@pci.uzh.ch
Please submit your application together with a curricu-
lum vitae, a list of publications and a brief statement of
The University of Zurich is an equal opportunity employer.
present and future research interests to the President
Applications from women are particularly encouraged. of ETH Zurich, Prof. Dr. Ralph Eichler, ETH Zurich, Rae-
mistrasse 101, 8092 Zurich, Switzerland (or via e-mail as
one single PDF to faculty-recruiting@sl.ethz.ch), no later
than January 31, 2011. With a view towards increasing
the number of female professors, ETH Zurich specifically
encourages qualified female candidates to apply.
Department of Environmental Health Sciences,

Professor and Chair
The Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) and the Max
The Johns Hopkins Bloomberg School of Public Health invites applications for Planck Institute for the Physics of Complex Systems (MPI-PKS), both located in Dresden,
chair of the Department of Environmental Health Sciences. The successful applicant are seeking outstanding candidates for
will have an outstanding record of academic and research accomplishment, and
demonstrated leadership and administrative abilities. Research Group Leaders in Systems Biology
The Department integrates diverse disciplines in its quest to increase understand- The successful applicants could be theoretical, experimental or both, and are expected
ing of the impact of environmental factors on individuals and human populations. to develop an independent research program. Research at the MPI-CBG focuses on the
It has over 60 faculty, 80 Doctoral and Master’s students, and a diverse research structure and dynamic organization of cells and tissues in several model organisms (see
http://www.mpi-cbg.de). This experimental research is greatly strengthened by theory,
program. The Bloomberg School of Public Health has ten departments and more contributed through close collaborations with physicists at the MPI-PKS (http://www.
than 500 full-time faculty and 2000 graduate-level students. The School is located mpipks-dresden.mpg.de).
on the Johns Hopkins University East Baltimore medical campus, a collaborative
InDresden,wedefinesystemsbiologybroadly.Inadditiontomoretraditionalactivitiesin
and highly interactive environment with a superb research infrastructure. systemsbiologysuchastheanalysisofgeneticandproteinsignalingnetworks,wehavea
The Johns Hopkins University is committed to recruiting, supporting and fostering stronginterestinthequantitativeunderstandingofmorphogenesisatseverallevels:how
do molecules dynamically organize into organelles and cells, and how do interactions
a diverse community of outstanding faculty, staff and students. All applicants who between cells give rise to tissues and organs? Answering these questions requires expe-
share this goal are encouraged to apply. Women and underrepresented minority rimental and theoretical approaches, in which there is considerable strength in Dresden,
candidates are particularly encouraged to apply. The Johns Hopkins University is but also advances in the processing and synthesis of biological data, especially images.
an Equal Opportunity Employer and does not discriminate on the basis of race, We therefore seek applicants not just from biology, bioinformatics and physics, but also
color, gender, religion, age, sexual orientation, national or ethnic origin, disability, from computer science and engineering.
marital status, veteran status, or any other occupationally irrelevant criteria. The MPI Research Group Leader positions are initially for 5 years at the EG15 level according to
University promotes affirmative action for minorities, women, disabled persons, theTVÖDscalewiththepossibilityofanextension.Fundsareavailableforpersonnel,con-
sumables and equipment according to the procedures at the two Institutes. Please send
and veterans. your CV, publication list and a short description of research accomplishments and future
Applications should include a curriculum vitae and statement of research inter- planstoProf.MarinoZerialorProf.RoderichMoessner(seeaddressesbelow),mentioning
est and vision of leadership. Please submit electronic applications as PDF or doc Systems Biology search, by 7 January 2011. Please also arrange for three letters of reference
to be sent before the deadline. We especially encourage women to apply.
files. Review of candidates will begin in January 2011. Applications should be
submitted to: Prof. Marino Zerial
Max Planck Institute of Molecular Cell Biology and Genetics
Chair, Search Committee for Pfotenhauerstr. 108, 01307 Dresden, Germany
Chair of Environmental Health Sciences
Prof. Roderich Moessner
c/o Ms. Susan Williams, Special Assistant Max Planck Institute for the Physics of Complex Systems
Office of the Dean Noethnitzer Str. 38, 01187 Dresden, Germany
Johns Hopkins Bloomberg School of Public Health
Please send your applications to: 2010-RGL-1080@mpi-cbg.de
615 N. Wolfe St., Room W1041
Baltimore, MD 21205 The Max Planck Society is committed to employ more handicapped individuals and ac-
tively seek their applications.
suwillia@jhsph.edu
Positions in Regenerative Biology

Faculty Position Opportunity
and Tissue Engineering
Division of Cancer Medicine, Department of
Lymphoma/Myeloma The Marine Biological Laboratory (MBL) and the Harvard University
Department of Stem Cell and Regenerative Biology announce a joint
The University of Texas MD Anderson Cancer Center seeks an M.D. or Ph.D. search for outstanding, highly innovative candidates at Assistant,
scientist to join its full-time faculty. This opportunity provides dedicated Associate, and/or Senior levels in the recently established Eugene Bell
research effort to actively engage in cutting-edge translational research.
Multiple opportunities for collaboration with scientists within the institution Center for Regenerative Biology and Tissue Engineering at the MBL
and faculty mentorship. The selected applicant should have expertise in in Woods Hole, Massachusetts. Successful candidates are expected to
laboratory research. Faculty rank is Assistant Professor, tenure track. A develop independent research programs in the areas of stem cell and
generous start-up package is available. regenerative biology or tissue engineering, focusing in particular on
MD Anderson, ranked as the top cancer center by U.S. News & World Report, marine, aquatic or lower vertebrate models.
is the world’s largest treatment facility for oncological diseases. Our location
in Houston provides access to a world-renowned medical community and the
Areas of emphasis include, but are not limited to, gene regulatory
splendid cultural and recreational diversity of a sophisticated, metropolitan
area that is the country’s fourth largest city. networks and systems analysis, genomics, comparative evolutionary
biology, bioelectric currents, advanced imagining, and computational
Interested applicants should send a copy of their curriculum vitae to:
approaches. Exploration of collaborative opportunities and synergies
Larry W. Kwak, M.D., Ph.D.
with other research programs at both the MBL and Harvard is
Professor and Chairman
Justin Distinguished Chair in Leukemia Research
encouraged. Opportunities for teaching in undergraduate and graduate
Department of Lymphoma/Myeloma programs at Harvard University are provided through an affiliated
The University of Texas MD Anderson Cancer Center faculty appointment in the Department of Stem Cell and Regenerative
1515 Holcombe Blvd., Unit 429 Biology.
Houston, TX 77030
Dr. Gary Borisy is the chair of the search committee.
ADVISORY COMMITTEE: Doug Melton, Chair, Harvard University;

Alejandro Sanchez Alvarado, University of Utah; Eric Davidson,
California Institute of Technology; Linda Griffith, Massachusetts
Institute of Technology; Michael Levin, Tufts University; Elly Tanaka,
Max Planck Institute, Dresden.
MD Anderson Cancer Center is an equal opportunity employer and does not discriminate on
the basis of race, color, national origin, gender, sexual orientation, age, religion, disability
or veteran status except where such distinction is required by law. All positions at The For applications and more information please visit:
University of Texas MD Anderson Cancer Center are security sensitive and subject to
examination of criminal history record information. Smoke-free and drug-free environment. https://mbl.simplehire.com
The MBL is an Equal Opportunity/Affirmative Action workplace.
Improving human health requires…
PO Box 2600
Pretoria 0001
South Africa
Tel: (012) 481 4000
Fax: (012) 349 1179
Int. Code: +27 12
info@nrf.ac.za
www.nrf.ac.za
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Cancerologists Epidemiologists Immunologists
DIRECTOR: SOUTH AFRICAN ASTRONOMICAL OBSERVATORY (SAAO)
The SAAO is a National Facility operated by the National Research Foundation of
South Africa and is the primary facility for optical/infrared astronomy in Africa.
It has a staff of about 120, divided between the headquarters in Cape Town,
where the Director will be based, and the observing facilities at Sutherland in
the Northern Cape.
The Southern African Large Telescope (SALT), the single largest optical telescope
in the southern hemisphere, is operated by SAAO, under contract to the Geneticians Neurobiologists Biochemists
12-member international SALT consortium. SAAO has a divisional structure
tenure positions are offered
122
that consists of (a) astronomy research, (b) SALT astronomy and technical
operations, (c) small telescope operations, (d) instrumentation (which has been Inserm to researchers m/f dedicated
responsible for major components of two of the SALT primary instruments), (e) is recruiting: to biomedical research
IT (recently upgraded through its location to a modern, dedicated building), (f)
education and public outreach (through the SALT collateral benefits program, Candidates to Research Associates and Research Directors positions
and recently enhanced through the IAU award to SAAO of its Office of Astronomy must have a PhD (or equivalent degree). There is no nationality restriction.
for Development), (g) administration.
Inserm is the only French public research body entirely dedicated to
We are seeking a dynamic astronomer with excellent leadership skills and human health. Its researchers are committed to studying all diseases,
diplomatic qualities, an established track record in optical/infrared astronomy, whether common or rare.
proven experience and skills in research management and a reputation for
high quality research at an international level. Experience with large telescope Through its diversity of approaches, Inserm provides a unique environment
for researchers.
science and/or instrumentation would be an advantage. 13 000 researchers, engineers and technicians work in the 318 Inserm
Further information about SAAO, SALT and the NRF may be obtained through laboratories housed in hospitals, universities and research campuses, all
http://www.saao.ac.za, together with a more detailed job description. over France.
Applicants should submit a letter of application, motivating their candidacy for Application modalities: visit our website : http//www.eva2.inserm.fr
the position, together with a detailed CV, which includes the names, addresses Application deadline: January 6th, 2011 - 4.00.pm (GMT+1)
and contact numbers of at least three referees, before 30 January 2011 to:
Mr Patrick Thompson
Group Executive: HR and Stakeholder Relations
Fax: +27 (0)12 481 4006 / E-mail: Patrick@nrf.ac.za
The NRF is committed to employment equity and redress.
Download your
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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
Brought to you by the AAAS/Science Business Office
VCU
TENURE-TRACK/TENURED FACULTY POSITIONS
DEPARTMENT OF BIOCHEMISTRY AND
TheMaxPlanckInstituteofMolecularCellBiologyandGenetics(MPI-CBG)in
Dresden is seeking outstanding candidates for
MOLECULAR BIOLOGY
Virginia Commonwealth University School of Medicine
Research Group Leader
We are seeking exceptional investigators pursuing innovative questions in Electron Microscopy
relevant to the department’s multi-disciplinary research strengths in cel-
lular and molecular biology, signaling, metabolism, inflammation, and Research at the MPI-CBG focuses on the molecular mechanisms underly-
cancer. Candidates should have a PhD or MD/PhD with an outstanding ing the structure and organization of cells and tissues (see http://www.
research record and will be considered at all ranks based upon qualifica- mpi-cbg.de). In this search, we are particularly looking for applicants using
electron microscopy, including cryotomography, to approach these issues,
tions and experience. A strong record of research accomplishments, an
though all excellent candidates will be considered.
independent externally funded research program, and experience working
in and fostering a diverse faculty, staff, and student environment or com- Research Group Leader positions are initially for 5 years at the EG15 level ac-
mitment to do so as a faculty member at VCU are required. A leadership cording to theTVÖD scale with the possibility of extension for up to an addi-
position is available for an outstanding senior individual. Substantial tional 4 years. Funds are available for a postdoctoral fellow, a PhD student, a
startup and salary packages are available. The Department belongs to a technician, as well as for consumables and equipment. Please send your CV,
very active community of investigators and is committed to providing publication list and a short description of research accomplishments and
an outstanding research environment. future plans to Prof. Marino Zerial (see address below) by 7 January 2011. Ple-
ase also arrange for three letters of reference to be sent separately before the
Interested candidates should send curriculum vitae, letter of interest deadline. We especially encourage women to apply.
including research outline and the name, address, telephone number,
fax number, and e-mail address of three references. Electronic submis- Prof. Marino Zerial
Max Planck Institute
sions preferred to: mwmaceyka@vcu.edu. Mailing address: Dr. Sarah of Molecular Cell Biology and Genetics
Spiegel, Chair, Virginia Commonwealth University, Department of Pfotenhauerstr. 108
Biochemistry and Molecular Biology, Box 980614, Richmond VA 01307 Dresden, Germany
23298-0614.
Please send your applications to: 2010-RGL-1080@mpi-cbg.de
Virginia Commonwealth University is an Equal Opportunity/
Affirmative Action Employer. Women, minorities and persons TheMaxPlanckSocietyiscommittedtoemploymorehandicappedindividu-
with disabilities are encouraged to apply. als and actively seek their applications.
Department of Health and Human Services
National Institutes of Health
Clinical Director
National Institute of Arthritis and Musculoskeletal and Skin Diseases
The Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases
(NIAMS) is seeking a physician-scientist to serve as Clinical Director. This individual will direct the NIAMS Pro-
gram in Translational Research, which includes training and clinical care branches as well as multiple investigative
laboratories and branches. Investigators in the Clinical Program conduct studies in natural history and treatment
as well as basic investigations into the etiology and/or pathophysiology of disease. The candidate should have the
ability to manage this diverse clinical research enterprise and to provide the leadership to maintain the outstand-
ing track record of the NIAMS Clinical Program. The ideal candidate for this position is an M.D. or M.D.-Ph.D.
who is board-certified or board-eligible in either Pediatrics, Internal Medicine and Rheumatology or Allergy/
Immunology. Potential areas of concentration would include rheumatoid arthritis, systemic lupus erythematosus,
ankylosing spondylitis, vasculitis, scleroderma, myositis, osteoarthritis, or other inflammatory/rheumatic diseases.
The candidate should have experience in conducting clinical or translational research and in immunology, cell
biology, genetics, or other areas of research relevant to rheumatic or autoimmune disease. The candidate will also
be provided generous independent resources to develop his/her own clinical or translational research program as
a tenured investigator within NIAMS.
The NIAMS Clinical Program is part of the NIH Intramural Research Program, located in Bethesda, Maryland.
Investigators in this program have full privileges to admit their research subjects, free of charge to the patient, to
the new Mark O. Hatfield Clinical Research Center, a state-of-the-art 230-bed hospital fully devoted to clinical
and translational studies. Ample resources will be provided to the successful applicant to establish a first-rate
Clinical Program and their own research program in his or her area of concentration. The NIAMS Intramural
Research Program, headed by its Scientific Director, Dr. John O’Shea, comprises several outstanding programs
in cytokine biology, signaling, genetics, and structural biology, and the broader NIH Bethesda campus provides
a rich and highly interactive environment within a wide range of basic and translational research disciplines that
relate directly to rheumatology.
Salary will be commensurate with experience. A full package of benefits, including retirement, health, life, and
long-term care insurance, and a Thrift Savings Plan, is available. Qualified international applicants who have
passed the ECFMG or USMLE examinations or have comparable qualifications are welcome to apply.
Interested applicants should send a curriculum vitae, a one-page summary of research interests, and the names of
three referees to Ms. Linda Peterson, 31 Center Drive, MSC 2350, Building 31, Room 4C12, Bethesda, MD
20892-2350, e-mail petersonl@arb.niams.nih.gov. If you need additional information, please call Dr. John
O’Shea at 301-496-2612. Review of applications will begin on or about January 3, 2011, but applications will
be accepted until the position is filled.
NIH and DHHS are equal opportunity employers.

FACULTY POSITIONS
Department of Bioengineering
Bourns College of Engineering
The Department of Bioengineering at

the University of California, Riverside
invites applications for junior level
tenure-track faculty positions in the areas
of translational technologies, including
biomaterials, medical devices, diagnos-
tic instrumentation, drug design and
delivery, and/or the area of healthcare
informatics. Exceptional senior candi-
dates may be considered. Applicants
should have a doctoral degree in the
relevant engineering discipline or a
related field.
Details and application materials

can be found at www.engr.ucr.edu/
facultysearch. The search committee
will review applications beginning on
January 25, 2011 and will continue to
receive applications until the positions
are filled.
EEO/AA Employer.
Nontraditional
Careers:
CELL/MOLECULAR BIOLOGIST
Assistant Professor Opportunities
Department of Biology Away From
The Biology Department at the University of New Mexico is seeking
applications for a probationary appointment leading to a tenure deci-
the Bench
sion Assistant Professor in the area of Cell/Molecular Biology. We seek
a colleague who will establish and maintain a vigorous, independent
research program, is committed to excellence in teaching at the under-
graduate through graduate levels, and is interested in joining a broadly
Webinar
based Biology Department. Applicants must have a Ph.D. in biology or a
related discipline and at least two years relevant postdoctoral experience.
For complete job requirements see Assistant Professor, Cell Molecular
Biologist at https://unmjobs.unm.edu.
Now Available
Applicants must submit a cover letter, curriculum vitae, three
representative reprints, and statements of current and future research On Demand
and teaching interests. All application material must be uploaded www.sciencecareers.org/webinar
and submitted through UNMjobs, https://unmjobs.unm.edu.
Applicants must also arrange for at least three letters of reference to
be sent directly to Cellbios@unm.edu. Application materials must
be received by December 15, 2010, for best consideration. Questions
related to this posting may be directed to Dr. Mary Anne Nelson, Produced by the
manelson@unm.edu. Science/AAAS Business Office.
The University of New Mexico is an Equal Opportunity/Affirmative

Action Employer and Educator. Women and underrepresented
minorities are encouraged to apply.
UAB Stem Cell Institute
Department of Biochemistry and Molecular Genetics
University of Alabama at Birmingham
Schools of Medicine and Dentistry
Tenure track junior faculty positions and tenured senior faculty positions
Neurobiology Faculty Position are available for investigators focused on stem cell biology, biochemistry,
The Department of Anatomy and Neurobiology (http://neurobiology. epigenetics and transplantation biology. Areas of special emphasis include,
umaryland.edu) is recruiting for tenured/tenure-track faculty positions but are not limited to, mechanistic studies of stem cell self-renewal and
in Neuroscience. We are particularly interested in candidates who will lineage specification and mechanisms of somatic cell reprogramming to
strongly complement existing strengths in the Department: chemical pluripotency. Structural biology of stem cell proteins by X-ray crystal-
senses, peptidergic circuits, sensorimotor functions, neurodegeneration lography and high-field NMR is an additional area of interest. State of
and neural circuits subserving higher order cortical functions. Candidates the art X-ray crystallography instrumentation and a new 800MHz NMR
should have a strong history of scholarly activity and preference will be system with a cryoprobe are available for Departmental faculty and Insti-
given to those with an independent funded research program and whose tute members. Nationally competitive salaries, startup packages and space
presence will catalyze multi-PI initiatives within the department. allocations will be offered to successful candidates. UAB is a highly inter-
active environment with strong basic and clinical sciences. Birmingham
We offer an outstanding intellectual and collaborative environment with is a beautiful and affordable city with many cultural attractions.
highly competitive salary and recruitment packages. All department
faculty are members of the Graduate Program in Life Sciences and Applicants should send a C.V., a summary of research interests and the
the interdisciplinary Program in Neuroscience (http://neuroscience. names of three references before January 31, 2011 to:
umaryland.edu). Dr. Tim Townes
Director, UAB Stem Cell Institute
Candidates should submit the following as one single PDF file to Chairman, Department of Biochemistry and Molecular Genetics
facsearch@umaryland.edu: detailed curriculum vitae, a brief state- University of Alabama at Birmingham
ment of research interests and goals, and names/contact information for Kaul Genetics Building, Room 502
three references. For best consideration candidates should submit their 720 20th Street South
application by February 1, 2011 and should be addressed to the attention Birmingham, AL 35294
of: Dr. Joseph Cheer, Chair of Faculty Search Committee. E-mail: ttownes@uab.edu
University of Maryland, Baltimore is an Equal Opportunity, Affirmative
Action Employer. Minorities, women, veterans, and individuals with UAB is an Equal Opportunity Employer committed to
disabilities are encouraged to apply. building a culturally diverse environment.
Guide the next

generation of
scientists.
Tator-Campeau Endowed Chair in
Brain and Spinal Cord Injury Research
The Toronto Western Research Institute of the University Health
Dean, Graduate School of Biomedical Sciences
Network invites applicants to apply for a Scientist position. The ideal Reporting directly to the Executive Vice President for Academic and Clinical Affairs, you
candidate will hold a PhD and/or MD degree (or equivalent) and have an will be a key member of our leadership team. As chief academic and administrative officer
established record of achievement in areas that have relevance to spinal of the Graduate School of Biomedical Sciences (GSBS) your primary responsibility will
cord injury or neurotrauma, e.g. neuroprotection, neuroregeneration and be to oversee the planning, development, and conduct of the academic programs of
related disciplines such as neural stem biology, axon regeneration, neural the school. Your interpersonal skills will serve you well as you represent GSBS in dealing
plasticity or restorative neural engineering. The successful candidate will with other schools within UMDNJ and with external academic, civic, governmental, and
professional organizations. You will be proficient in fundraising and an innovative leader
be appointed to an appropriate department at the University of Toronto
who can advance graduate training to meet the future needs of the biomedical and health
at a level commensurate with their experience (from Assistant to Full
sciences workforce.
Professor).
The UHN has a strong interdisciplinary research program in spinal cord The inspired candidate we seek must have a Ph.D., M.D. or M.D./PhD. degree, along
with professional accomplishments that qualify for a Professorship with tenure. Previous
injury and regenerative neuroscience. The successful candidate will have
experience as a Dean, Vice-Dean, Senior Associate Dean or Department Chair in an
the ability to establish an independent, well funded program of international
academic health sciences/research institution is preferred, as is a strong record of NIH
prominence and to collaborate with other members of our research and funded research. A successful history of graduate student mentoring and program
clinical staff in the development of treatments for injuries and disorders development, together with the skills to engender collaboration among groups of diverse
of the central nervous system. backgrounds, are also essential.
Applications (CV, statement of interest, referees) must be e-mailed
For consideration, please send your letter of interest and curriculum vitae to: UMDNJ-
by January 30, 2011 to: Dr. C.J. Paige, Vice President, Research, GSBS Dean Search Committee, c/o P.R. Falk, Office of Academic Affairs, University
University Health Network, E-mail: TCSearch@uhnresearch.ca. of Medicine and Dentistry of New Jersey, 65 Bergen Street, Suite 1441, Newark, NJ
We wish to thank all applicants for their interest, however, only those 07107. Priority will be given to letters of interest and curricula vitae received by February
selected for an interview will be contacted. The Toronto Western 1, 2011. If you have any questions, please contact: Dr. Andrew Thomas, Chair, Search
Research Institute of the Toronto Western Hospital, along with the Committee, 973-972-1396; thomasap@umdnj.edu. Equal Opportunity Employer.
Princess Margaret Hospital and the Toronto General Hospital, is
a member of the University Health Network, an equal opportunity
Employer. In accordance with Canadian Immigration requirements,
this advertisement is initially directed to Canadian citizens and
permanent residents.
POSITIONS OPEN POSITIONS OPEN POSITIONS OPEN
CONSERVATION BIOLOGIST OR FACULTY POSITION THREE FULL-TIME TENURE-TRACK

RESTORATION ECOLOGIST ASSISTANT OR ASSOCIATE PROFESSOR in ASSISTANT PROFESSOR POSITIONS in
The Biological Sciences Department at California Genetics/Genomics Discipline-Based Science Education Research
State Polytechnic University (Cal Poly Pomona), invites San Diego State University_s Department of Biology The University of Maine invites applications for
applications for a tenure-track, ASSISTANT PRO- announces a tenure-track position in Genetics/Genomics three full-time tenure-track faculty positions at the
FESSOR position in Conservation Biology or Res- at the associate or assistant professor level with the ap- Assistant Professor level with expertise in discipline-
toration Ecology, beginning September 2011. This pointment beginning fall 2011. We seek a productive based education research. The positions will be in
position is intended to integrate with and contribute scientist who is addressing fundamental questions in ge- the fields of (1) Physics, Chemistry, or Mathematics;
to our growing Environmental Biology Program. A netics and/or genomics. We are especially interested in (2) Biology, Earth Sciences, or Marine Sciences; and
Ph.D. in Biology or a related field, a demonstrated candidates who creatively bridge traditional boundaries (3) Science Education. These positions were created
record of publication, and some teaching experience between evolution and cell and molecular biology using through the Maine Physical Sciences Partnership (PSP)
at the college level are required; postdoctoral expe- state-of-the-art experimental approaches. A Ph.D. and Project, under a grant to the Maine Center for Research
rience is preferred. The area of expertise is open, but postdoctoral experience in genetics, genomics, evolu- in STEM Education to target the teaching and learning
candidates with a strong field-based research program tionary biology, or a related field are required. Can- of science in grades 6–9 as well as the preparation of
in conservation/restoration biology are particularly en- didates at all levels should have a strong record of science teachers at the University of Maine. By the
couraged to apply. The successful candidate will be research accomplishments and funding, an active research time of appointment, the candidate must have an
expected to teach courses in general ecology, conser- program, current extramural (NSF, NIH, DOE, or com- earned Doctorate in the discipline, in discipline-based
vation biology, and other upper division and/or grad- parable) funding, a strong publication record, and a education research, or in a closely related field; strong
uate level courses related to his or her area of expertise. commitment to undergraduate and graduate teaching. experience in discipline-based education research; and
Teaching duties may also include introductory biology Send curriculum vitae, separate statements of research demonstrated excellence in teaching. The new faculty
or a general education course. The successful candidate and teaching interests, three representative publications, member will be expected to (1) engage in research,
must be strongly committed to teaching, mentoring and arrange for three letters of reference to be sent to: writing, and other scholarly activities that contribute
of undergraduate and graduate (M.S.) students, and Genetics/Genomics Search Committee, Department to discipline-based education research; (2) teach
developing an externally funded research program. Cal of Biology, SDSU, San Diego, CA 92182-4614. undergraduate- and graduate-level courses; (3) super-
Poly Pomona is a comprehensive Master_s level uni- Review of applications will begin on 15 January and will vise graduate and undergraduate research; (4) develop
versity with a diverse student body. The successful can- continue until position is filled. For more information, an externally-funded research program; (5) serve as a
didate will have demonstrated ability to be responsive see website: http://www.bio.sdsu.edu. student advisor; and (6) participate in leadership for
to the educational equity goals of the university and its SDSU is an Equal Opportunity Employer and does not dis- collaborative efforts in STEM-education with col-
increasing ethnic diversity and international character. criminate against persons on the basis of race, religion, national origin, leagues in the home department, the Maine RiSE
Applicants should mail: (1) curriculum vitae, (2) state- sexual orientation, gender, gender identity and expression, marital Center, the University, and other professional com-
ment of teaching philosophy, (3) proposed plan of status, age, disability, pregnancy, medical condition, or covered vet- munities. The positions are academic year appointment
research, (4) reprints of up to three publications, and eran status. with preferred starting date no later than September 1,
(5) the names and contact information of five refer- 2011. See website: http://www.umaine.edu/
ences to: Chair, Conservation Biologist/Restoration center/positions/PSPfaculty for full position an-
Ecologist Search Committee, Biological Sciences nouncements and application instructions. Review of
Department, California State Polytechnic University, applications will begin January 3, 2011, and continue
3801 West Temple Avenue, Pomona, CA 91768- ASSISTANT OR ASSOCIATE PROFESSOR in until the positions are filled. Incomplete applications
4132. Review of applications will begin on January 5, Cell Biology cannot be considered. Appropriate background checks
2011. Official transcripts and three letters of refer- University of California at San Francisco will be required.
ence will be required of all finalists. For full position The Department of Cell and Tissue Biology, School The University of Maine is an Equal Employment Opportunity/
description, please visit the Department website at of Dentistry, invites applications for a tenure-track fac- Affirmative Action Employer and is committed to excellence through
website: http://www.csupomona.edu/Èbiology. ulty position at the Assistant or Associate Professor diversity in its faculty, staff and students. We strongly encourage
California State Polytechnic University, Pomona is an Equal level. Candidates should hold a Ph.D. degree and are all qualified individuals to apply.
Opportunity/Affirmative Action Employer. expected to establish a dynamic research program in
eukaryotic cell biology, and to contribute to teaching
and training. Areas of interest include but are not lim-
PLANT CELL PHYSIOLOGIST ited to cell biological mechanisms relevant to cancer, ANIMAL PHYSIOLOGIST
TENURE-TRACK stem cells, or host-pathogen interactions, which are TENURE-TRACK
The Department of Biology at California State focus areas of our recently established department
The Department of Biology at California State
University San Bernardino invites applications for a (website: http://ctb.ucsf.edu/). The successful can-
University San Bernardino invites applications for a
tenure-track position at the rank of ASSISTANT didate will become a member of the UCSF Biomedical
tenure-track position at the rank of ASSISTANT
PROFESSOR in the area of cellular plant physiology. Sciences and the Oral and Craniofacial Sciences grad-
PROFESSOR in the area of animal physiology. The
The successful applicant will develop an independent uate programs.
successful applicant will develop an independent
research program and is expected to excel in teaching Applications received by February 1, 2011, will
research program, and is expected to excel in teach-
core courses related to cell and plant biology at the receive preferential consideration, but the position
ing core courses related to organismal and human
undergraduate and M.S. levels. Candidates must have will remain open until filled. A single PDF file in-
physiology at the undergraduate and M.S. levels.
a record of published research and show potential for cluding curriculum vitae, a brief description of research
Candidates must have a record of published research
developing and sustaining an independent, externally accomplishments, future research plans, and teaching
and show potential for developing and sustaining an
funded research program involving both undergraduate interests should be submitted electronically to e-mail:
independent, externally funded research program in-
and M.S. students. Candidates must have a Ph.D. in ctb_search@ucsf.edu. Three to five reference letters
volving both undergraduate and M.S. students. Can-
the biological sciences; postdoctoral experience is desir- should be sent directly to the same e-mail address.
didates must have a Ph.D. in the biological sciences;
able. Application deadline is January 3, 2011, or until UCSF seeks candidates whose experience, teaching, research, or
postdoctoral experience is desirable. Application dead-
position is filled. Submit curriculum vitae, statement of community service has prepared them to contribute to our commit-
line is January 3, 2011, or until position is filled. Submit
research accomplishments and goals, statement of teaching ment to diversity and excellence. UCSF is an Equal Opportunity/
curriculum vitae, statement of research accomplishments
philosophy, and three letters of reference to: Dr. David Affirmative Action Employer. All qualified applicants are encouraged
and goals, statement of teaching philosophy, and three
Polcyn, Chair, Department of Biology, Attn: Plant to apply, including minorities and women.
letters of reference to: Dr. David Polcyn, Chair,
Cellular Physiology Search. California State Uni-
Department of Biology, Attn: Animal Physiology
versity, 5500 University Parkway San Bernardino,
Search. California State University, 5500 Univer-
CA 92407.
sity Parkway San Bernardino, CA 92407.
ASSISTANT PROFESSOR OF BIOLOGY
ASSISTANT/ASSOCIATE PROFESSOR Clarkson University
Harvard Medical School Clarkson University is seeking candidates for As-
Beth Israel Deaconess Medical Center sistant Professor of Biology. The successful candidate PRECEPTOR IN THE LIFE SCIENCES
Department of Obstetrics and Gynecology. The can- will develop a strong, externally funded research pro- Harvard University
didate should be a Ph.D. committed to research in one gram that complements our strengths in biotechnol- Harvard University is seeking a preceptor for a large
of the followings areas: Reproductive Biology, Repro- ogy and teach undergraduates and graduate students. introductory undergraduate course that integrates
ductive Endocrinology, or Gynecologic Oncology. The position requires expertise in physiology, cellular genetics, genomics, and evolution. The preceptor is
Attractive opportunity for the candidate to establish a and molecular biology, and an interest in developing responsible for designing the laboratory and discussion
new program in an outstanding and vibrant environ- collaborations with faculty in biochemistry, physics, or components, preparing course assessments, and super-
ment. Send inquiries and curriculum vitae to: John rehabilitation engineering. For additional information vising teaching fellows. The applicant must have a
Yeh, M.D., Professor and Chairman, Obstetrics about this position and to apply please visit website: Ph.D. in a Life Sciences field with proven experience
and Gynecology, Beth Israel Deaconess Medical http://www.clarkson.edu/hr and click BCareer Op- teaching undergraduates. For more information, see
Center, Harvard Medical School, Boston, MA; portunities.[ Clarkson University is an Equal Opportunity/ website: http://www.mcb.harvard.edu/Jobs/
e-mail: tlcole@bidmc.harvard.edu. Affirmative Action Employer. TeachingFellows.php.

PROGRAMS
Research Opportunities in Luxembourg.
See what’s behind.
PEARL ATTRACT
LUXEMBOURG’S RESEARCH PROGRAMME FOR LUXEMBOURG’S RESEARCH PROGRAMME FOR OUTSTANDING
INTERNATIONALLY RECOGNISED SENIOR RESEARCHERS YOUNG RESEARCHERS FROM ALL OVER THE WORLD
If you are an internationally recognised senior researcher, If you are an outstanding young researcher, our research
our research programme PEARL gives you the opportunity programme ATTRACT helps you to set up an independent
to transfer your research programme to a public-sector research team within a public-sector research institution
research institution in Luxembourg and thus to accelerate in Luxembourg. The innovation, dynamism and creativity
the development of and to strengthen Luxembourg’s of your project as well as its high scientific quality should
research priorities. 3-5M€ are offered to Luxembourg’s enhance Luxembourg’s position in the international world
public-sector research institutions through this programme of R&D. Projects selected under ATTRACT have a lifespan of
to compete for the best candidates. The FNR foresees to five years and the financial contribution will be up to 1.5M€.
grant 1 to 2 PEARL awards per year. The 5th ATTRACT Call will be launched in December 2010.
The call is open all year.
meetings and announcements

More information about ATTRACT and PEARL as well as the other funding
opportunities offered by the National Research Fund Luxembourg can be found
on the FNR’s website. Go and see what’s behind on www.fnr.lu INVESTIGATING FUTURE CHALLENGES
PRIZES
FERNANDO GIL INTERNATIONAL PRIZE

FOR THE PHILOSOPHY OF SCIENCE 2011
The creation of the FERNANDO GIL des Hautes Études en Sciences Sociales, Paris
INTERNATIONAL PRIZE for the Philosophy of Donald Gillies University College London
Science was announced by the Portuguese Giulio Giorello Università degli Studi di
government at the time of his death, to Milano Eberhard Knobloch Institut für
honor his memory and work (1937-2006). Philosophie, Technische Universität Berlin
The prize is launched as a joint initiative of Maria Filomena Molder Departamento de
the Portuguese government, represented by Filosofia, Universidade Nova de Lisboa
FCT, the Portuguese Foundation for Science Frédéric Nef École des Hautes Études
FERNANDOGIL
and Technology (Ministry of Science, en Sciences Sociales, Paris Jean Petitot École
Technology and Higher Education), and FCG, des Hautes Études en Sciences Sociales, Paris
the Calouste Gulbenkian Foundation. Bertrand Saint–Sernin Institut de France
The Fernando Gil International Prize will be Manuel Silvério Marques Centro de Filosofia,
awarded every year in Lisbon. The 2010 prize Universidade de Lisboa.
was awarded to Ladislav Kvasz, a Slovak
Nominations to the Fernando Gil
Mathematician and Philosopher.
International Prize 2011 should be sent to
The Prize intends to award a work of
premio-filosofia@fernando-gil.org.pt
particular excellence in the domain of the
until February 28, 2011 at 12h00 UTC, and
Philosophy of Science, whether regarding
include the following elements:
general epistemological problems or
• The complete publication data of the
particular scientific areas.
nominated work;
For the prize, it will be solely considered
original and recent works of a single author • A critical review of the work being
from any nationality or professional proposed for the prize emphasizing the
affiliation, published during the three elements considered to be more relevant for
previous years (2008, 2009 and 2010). the awarding of the prize (2000 characters);
The recipient of the Prize is requested • A brief comment on the author biography
to deliver an original public lecture that will (2000 characters).
be published by the Calouste Gulbenkian
Simultaneously, a paperback copy of the
Foundation and to conduct a specialized
nominated publication should be sent to the
seminar for students and researchers in
Foundation for Science and Technology, for
Lisbon on the occasion of the Prize ceremony.
the address indicated bellow:
The amount to be paid to the laureate
will be 125,000 Euros. Foundation for Science and Technology
c/o Andreia Rosa
The jury of the Fernando Gil International Prize Av. D. Carlos I, 126,
2010 includes the following members: 1249-074 Lisboa - Portugal
Henri Atlan Hadassah University Hospital,
B2 Design
Jerusalem & École des Hautes Études en Nominations from the nominees
themselves cannot be accepted.
Sciences Sociales, Paris Per Aage Brandt Case
Western Reserve University Marcelo Dascal For more information:
Tel Aviv University Vincent Descombes École www.fernando-gil.org.pt
POSITIONS OPEN POSITIONS OPEN
FACULTY POSITION in TENURE-TRACK ASSISTANT PROFESSOR in

Ecological or Evolutionary Genomics Human Physiology
Saint Louis University, a Catholic, Jesuit institution The Department of Biology, University of Wisconsin-
dedicated to student learning, research, health, and ser- Stevens Point, offers a tenure-track, nine-month faculty
vice, is seeking applicants for a tenure-track faculty posi-
tion in Ecological or Evolutionary Genetics/Genomics
in the Department of Biology. Competitive applicants
position in Human Physiology (Assistant Professor),
beginning August 2011. Teaching assignment includes
human physiology, introductory biology, senior seminar,
Do what
will have a Ph.D., postdoctoral experience, a record of
research productivity, and a commitment to undergrad-
uate and graduate student training. The successful can-
didate will be expected to establish an independent,
and, eventually, an advanced course in specialty. Re-
search involving undergraduates, department service,
and student advising are expected. Ph.D. with emphasis
in mammalian physiology (for tenure), and teaching and
you love.
extramurally funded research program that applies research experience are required. Coursework and/or
genetic and/or genomic approaches to fundamental research in neurobiology is desirable. Experience may
questions in ecology or evolutionary biology. The fac-
ulty member will contribute to an undergraduate course
in Genetics, a graduate course in the candidate_s area of
include grants, publications, evidence of teaching excel-
lence, and postdoctoral work. The faculty seeks appli-
cants from underrepresented groups.
Love what
expertise, and/or a general biology course.
All applications must be made online at website:
http://jobs.slu.edu (Req ID 20100990) and include
a cover letter, curriculum vitae, a research statement,
Include curriculum vitae, statements of teaching
philosophy and research interests, three letters of rec-
ommendation, and undergraduate and graduate tran-
scripts. Send application materials to: Dr. C. Yahnke,
you do.
and a statement of teaching experience and philosophy. Chair; Biology Department, University of Wisconsin-
In addition, please have three letters of reference sent Stevens Point; Stevens Point, WI 54481. Review
to: Dr. Robert Wood, Department of Biology, Saint begins 1 February 2011, and continues until filled. For
Louis University, 3507 Laclede Avenue, St. Louis, more information, telephone: 715-346-2455; fax:
MO, 63103. Review of applications will begin on 15
December 2010, and continue until the position is
715-346-3624; e-mail: cyahnke@uwsp.edu.
Affirmative Action/Equal Opportunity Employer.
www.sciencecareers.org
filled. Additional information on the Department of
Biology can be found at website: http://www. POSTDOCTORAL POSITIONS in
slu.edu/x14762.xml. Cancer Biology and Therapeutics
Saint Louis University is an Affirmative Action/Equal Oppor- Several postdoctoral positions are available in the
tunity Employer (AA/EOE), and encourages nominations of and area of antibody engineering and cancer, including
applications from women and minorities. the use of advanced fluorescence microscopy and murine
cancer models to develop improved therapeutics. Strong
applicants with a Ph.D. in biological or physical sciences,
including bioengineering, will be considered. Please send
curriculum vitae as well as names and contact infor-
mation of three references to: Professor E. Sally Ward
VISITING ASSISTANT PROFESSOR OF
(e-mail: wardlab@utsouthwestern.edu). UT Southwestern
BIOLOGY
is an Equal Opportunity/Affirmative Action Employer.
Kenyon College is seeking applications for an ap-
pointment as a two-year Visiting Assistant Professor
of Biology. The teaching commitment is five courses
per year; laboratories count as separate courses. The
successful candidate will be expected to teach intro-
ductory and advanced courses in animal physiology
or ecology, including lecture and laboratory courses.
There are opportunities for undergraduate research
mentoring and support for professional development.
To apply, candidates should visit the online ap-
Find your
A Career future here.
↓
plication site found at website: https://employment.
kenyon.edu. A complete application will comprise:
(1) a cover letter discussing the applicant_s research
and scholarship undertaken, its relevance to the field
or discipline, and prior teaching experience; (2) a
statement of the applicant_s teaching philosophy; (3)
in science
curriculum vitae; (4) unofficial graduate and under-
graduate transcripts; and (5) letters of reference from
three (3) recommenders. All application materials must
be submitted electronically through Kenyon_s employ-
is more
ment website.
Review of applications will begin January 15, 2011,
and will continue until the position is filled. Com-
than just www.ScienceCareers.org
pleted applications received by the January 15 dead-
line will be guaranteed full consideration. A Ph.D in
Biology or related field is required.
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APOPTOSIS
UNDERSTANDING
“Mechanism is where
APOPTOSIS it’s at these days.”
Apoptosis is a simple concept with complex underpinnings—damaged
cells are prompted to self-destruct through a series of molecular events,
like dominos toppling over one by one. Unraveling the mechanisms underlying d l i apopto-
pto-
sis is akin to determining the role each individual domino plays in a falling cascade—one one
strategically placed tile can trigger a cascade of several lines, and one misplaced tile can
an
mean a dead end in the chain reaction. Techniques that pick apart this intricate process ss
have been rapidly advancing, now offering more than the ability to look at a single eventt
with high throughput, multiplexing capabilities, and in vivo imaging. Future technolo-
gies aim to help scientists visualize human cellular processes in vivo to aid research-
ers in tailoring and monitoring treatment for a variety of diseases, especially cancer.
By Paul Smaglik
A
poptosis differs from necrosis, although the end results tiating apoptosis from necrosis. However, some scientists bridge
are the same—cell death. Whereas apoptosis mirrors the two approaches when studying a specific disease, especially if
the actions implied by the word’s Greek origins for “drop- they are investigating whether a particular compound can inhibit or
ping off,” akin to flowers losing their petals one by one, promote apoptosis, says Mike Earley, market segment manager at
necrosis causes a cell to abruptly explode and spill its potentially Sigma-Aldrich.
toxic contents into the surrounding environment. Scientists became
fascinated with the orderly apoptotic process, or programmed cell ONE AT A TIME
death, because of its key role in a host of diseases—most notably Although the most basic approaches for detecting apoptosis have
in cancer, which results from rogue cells proliferating instead of self- been around for a while, “the technology hasn’t changed that much,”
destructing when apoptotic signals go awry. Earley explains. However, these simple assays and tools play a valu-
The “dropping off” process often begins with an environmental able role for researchers who simply need to confirm whether apop-
stress, like heat, radiation, or viral infection. These stressors can initi- tosis is happening. Several stages in the apoptotic cascade have dis-
ate a molecular signaling cascade, either by moving proteins around tinct events that can be easily detected, such as protein rearrange-
from inside the cell to the outside or by activating proteins inside ments, caspase activity, and DNA alterations.
the cell, such as caspases that tell the cell to start dismantling itself. For scientists who want a simple way to determine whether apop-
The final stages of this systematic self-destruction leave signs of tosis happened, without looking at individual components of the
the wreckage behind, for instance DNA fragments that resemble complex pathway, Annexin V-specific stains can detect the calcium-
broken ladders. Each of these steps can be isolated for detection dependent protein, which moves from the inner surface of the cy-
through a variety of methods. toplasm to outside the cell during apoptotic activity. This approach’s
Researchers started identifying the numerous apoptotic markers speed and ease-of-use has made it one of the most popular simple
and mechanisms in the early ‘90’s. The first kits developed to aid assays. “The Annexin V assay is a one-step procedure requiring just
their search simply detected individual steps within the entire pro- 10 minutes to perform,” says Suvarna Gandlur, product manager of
cess—whether an individual domino tipped. However, subsequent Clontech in Mountain View, Colorado. “The assay is nonenzymat-
generations of technology have become more sophisticated with
advances that detect apoptotic markers at different stages of the
cascade and identify the underlying mechanisms at play. UPCOMING FEATURES
CREDIT: C. BICKEL/SCIENCE
Scientists usually have one of two goals when studying apop-

tosis, says Terri Borree, EMD Millipore’s product manager. Some Proteomics
P i Meets
M Glycomics—January
Gl i J 7
seek general knowledge about apoptosis—whether it’s happening,
Forensic Technologies—March 4
when it’s triggered, and what is either causing or inhibiting it. Others
want to reveal more about the process itself—perhaps by finding Genomics: Synthetic Genomics—March 25
missing links in the domino chain of chemical events or differen-
1416 www.sciencemag.org/products
ic, does not require fixation, and is suitable for both adherent and
APOPTOSIS (
suspension cells.” Besides Clontech, many other companies offer Scientists became fascinated
Annexin V assays, including Life Technologies, Cambrex, and
with the orderly apoptotic
BioVision.
However, Annexin V assays do not tease apart the individual stages process, or programmed cell
of the signaling cascade; they only detect whether or not it is under- death, because of its key role
way. Caspase assays, on the other hand, can reveal more about the
early stages of the process. For instance, caspase 8 and caspase 9
in a host of diseases—most
are involved in the induction of apoptosis, and caspase 3 plays a key notably in cancer, which results
role in the execution of the process. Promega in Madison, Wiscon-
from rogue cells proliferating
sin, focused its R&D efforts on developing more sensitive and easily
automated bioluminescent probes to detect activation of several of instead of self-destructing when
these key caspases, explains Pam Guthmiller, strategic marketing apoptotic signals go awry.
manager of cellular analysis. “Mechanism is where it’s at these days,”
says Guthmiller.
Scientists seeking markers of the later stages of the apoptotic son Whalley, flow cytometry manager at EMD Millipore. Flow cy-
cascade can look for signs of DNA fragmentation. This programmed tometry also provides increased speed with the ability to “read”
destruction of the genome can be detected using a terminal deoxy- for multiple apoptotic markers in single cells, rather than in popula-
nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) tions of cells, and provides a time-lapse view of what’s happening
assay. The technique allows researchers to look for breaks in the in these individual cells. This technique enables researchers to see
DNA strands, which can result from apoptosis-induced nuclease when multiple events occur, says Terri Borree, an EMD Millipore
activity, by labeling the exposed 3’ hydroxyl ends. However, these product manager. Given the multiplicity of samples that need to be
assays were originally difficult to perform and could not differentiate screened and the variety of treatment conditions that need to be
between necrotic and apoptotic induced DNA damage. evaluated in apoptosis studies, benchtop flow cytometers can ease
Many academic researchers wanted clearer evidence of this end- researchers’ workloads.
stage, which shows that the apoptotic process reached its conclu- EMD Millipore’s latest addition has been flow cytometry assays
sion, says Sallie Cassel, director of product management, at EMD that detect mitochondrial dysfunction, which is often an early indica-
Millipore. In response, companies have refined these assays. For tor of cellular stress and can trigger apoptosis or other cell death
example, EMD Millipore’s ApopTag line is able to differentiate the pathways. EMD Millipore’s MitoDamage Kit provides simultaneous
characteristic blunt ends of DNA found only in apoptosis from the information on mitochondrial perturbations, apoptosis, and cell death,
DNA overhangs created during both apoptosis and necrosis. while the MitoStress kit allows researchers to study the relationship
between mitochondrial oxidative stress and apoptosis.
MULTITASKING, MULTIPLEXING Life Technologies has also worked to make flow cytometry easier
Though assays have become more sensitive and accurate over the to use, and more effective for apoptosis research. “Given that no
years, researchers also require enhanced speed, higher throughput, single parameter fully defines apoptosis in all systems, we have fo-
and increased multiplexing capabilities. As a result, companies have cused on developing and supplying products that focus on a multi-
been adapting their technologies to multiple approaches, from anti- parametric approach that enable researchers to not only determine
body-based assays to flow cytometry, and expanding the number of the relative stage of apoptosis but to also distinguish between live,
samples that can be read at once using microwell plate readers. apoptotic, and necrotic cells in their experimental system”, says Kath-
The ability to detect multiple apoptotic markers simultaneously leen Free, senior manager of product development at Life Technolo-
allows researchers to gain a better overview of the process, since gies (formerly Invitrogen). These products include kits designed to
apoptosis encompasses so many events. Promega, for example, go along with detection systems that have the most recent advanc-
has optimized methods that use both fluorescent and luminescent es, such as flow cytometers with a 405 nm laser—which enhances
probes in one sample well. Last year, they released the ApoTox- these machines’ multiplexing capabilities—as well as high-content
Glo Triplex Assay, which measures viability, membrane integrity imaging platforms.
CREDIT: © ISTOCKPHOTO.COM/ERAXION
as a marker of necrosis, and caspase-3/7 activity as a marker of Life Technologies also has a novel way to label DNA, RNA, and
apoptosis, all in the same sample well. “The assay not only allows proteins that further enhances the sensitivity of these detection
you to obtain more biologically relevant information from the technologies. The company provides small molecule labeling and
same cell sample well without manipulation, but also minimizes detection reagents that are significantly smaller than conventional
cell culture expenses by streamlining three assays into one plate,” antibodies, biotin, or fluorescent tags. The Click-iT TUNEL assay, for
says Guthmiller. example, uses an alkyne-modified dUTP to label fragmented DNA
Using flow cytometry, researchers can obtain multiplexed an- ends. This molecule is more easily incorporated into DNA strand
swers on a large number of samples simultaneously, explains Ja- breaks than the more traditional nucleotide modifications, such
APOPTOSIS
The next frontier for probing apoptosis in complex systems is real-

FEATURED PARTICIPANTS time, in vivo imaging. This effort is already underway for animal re-
search, and companies are racing to deliver techniques that will also
Aposense work in human clinical studies.
www.aposense.com Promega has developed a bioluminescent caspase-3/7 probe for in
BioVision vivo animal imaging of apoptosis. However, this requires the use of
www.biovision.com the luciferase gene, either by using genetically modified mice that
express the gene or by surgically implanting tumor cells containing
Cambrex the lucfierase gene, which is commonly used for screening potential
www.cambrex.com
cancer drug candidates.
Clontech Aposense, an Israeli-based company, has generated a new class
www.clontech.com of rationally designed, small molecular probes that detect cell mem-
brane alterations that are indicative of apoptosis, including loss of
EMD Millipore
www.millipore.com membrane potential, exposure of phosphatidylserine (PS), and phos-
pholipid scrambling. On such molecule, Apo-TRACE, can be used for
ImmunoChemistry Technologies pre-clinical in vivo imaging studies. Aposense has also developed an
www.immunochemistry.com agent for PET (positron emission tomography) imaging in patients.
Life Technologies The [18F]-ML-10 imaging agent, which is labeled with the radioiso-
www.lifetechnologies.com tope fluorine-18 (18F), is currently in clinical trials at multiple leading
cancer centers in the United States. This cutting-edge technology
Promega may enable clinicians to see in real-time whether chemotherapy
www.promega.com
treatments that target apoptotic pathways work, and possibly aid in
Sigma-Aldrich the individualization of patient treatment plans. Personalized cancer
www.sigmaaldrich.com treatment is one the major unmet needs in oncology today, says
Yoram Ashery, CEO of Aposense.
Providing in vivo imaging for the apoptosis pathway will be chal-
as biotin or fluorescein, because of its smaller size. To detect the lenging because it will require a small molecule, nontoxic approach,
molecule, an azide-coupled fluorophore is then fused to the alkyne unlike the in vitro techniques, says Gary Johnson, president of
through a copper-catalyzed click chemistry reaction. As a result, ImmunoChemistry Technologies (ICT) in Bloomington, Minneapo-
the Click-iT assay can detect breaks in DNA strands that other lis. ICT is also trying to meet this challenge with tracers designed
approaches—using larger-sized tags—might miss. to detect the intracellular process of apoptosis, explains Johnson.
One such molecule, labeled with the 18F radioactive positron emitter,
COMPLEX SYSTEMS is undergoing testing for use with PET imaging in animals, and is
Looking at what happens throughout an entire cell or organism can anticipated to move into clinical trials by 2011. ICT is also develop-
give researchers a more complete and accurate picture of apoptosis ing tracers labeled with iodine-123 (123I) for single positron emission
than a single assay, or even a time-lapse approach. computational tomography (SPECT) imaging, which emits gamma
Already offering reagents for flow cytometry and developing mul- rays, rather than positrons, to produce a 3-D picture.
tiplexing assays, Sigma-Aldrich is refocusing its efforts towards cre- The potential is great, says Johnson, because physicians will be
ating genetically altered cell and animal models that can be used able to determine if therapeutic treatments are working in less time
to analyze the many components of the apoptosis pathway. These than is currently possible, and without having to perform biopsies.
models are created using their CompoZr zinc finger nuclease tech- Practitioners may also be able to use the technique to immediately
nology, which act as highly specific scissors to create double-strand- assess the impact of neurological injury, and even degenerative
ed DNA breaks for targeted gene deletion or insertion. diseases.
“It’s less about specific reagent kits and more about developing If Aposense’s and ImmunoChemistry Technologies’ approaches
the right model systems to study the genetic basis of apoptosis,” make it through clinical trials, researchers can expect another wave
says Helge Bastian, vice president of Global Marketing for Research of apoptosis detection technologies, potentially including real-time
Biotech at Sigma-Aldrich. Bastian says this approach came about be- “movies” that could help clinicians personalize treatments for pa-
cause the company was not content to just keep improving the ease tients, and watch whether and how these treatments work. This
of sample preparation and making technical workflow improvements would move apoptosis detection off the bench and into the clinic—
via speeding processing through ever-larger multiplexing approach- and greatly expand utility of tracking this complex, but key, process.
es. Now, they are geared more towards providing products that are
designed to specifically analyze genes and proteins, or the pathways Paul Smaglik is a freelance writer living in Milwaukee, Wisconsin.
in which they function. DOI: 10.1126/science.opms.p1000050
1418 www.sciencemag.org/products
MICROPLATE READER
NEW PRODUCTS: APOPTOSIS (

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(TRF), TR-FRET, and BRET assays with ultrahigh sensitivity. Synergy H1 includes the Gen5 Data Analy-
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CELL HEALTH ANALYSIS MICROPATTERNED SUBSTRATES

FlowCellect kits are designed for cell health analysis using flow cytom- Patterns of extracellular matrix proteins can control important cellular
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derstanding the mechanism of diseases. The six new kits include the actions. The new BioWrite line and grid protein micropatterns on glass
MitoDamage kit, which provides simultaneous information on mitochon- coverslips are designed for cell-based applications. The fibronectin pat-
drial perturbations, apoptosis, and cell death. The MitoStress kit allows terns have feature sizes ranging from 15 µm to 100 µm, which restrict
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apoptosis. For researchers who want to further understand mechanis- define the location, size, and morphology of cultured cells; control cel-
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CONTENTS Volume 330 Issue 6009

EDITORIAL LETTERS PERSPECTIVES

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1277

>> Perspective p. 1326 After Brain Damage

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1279

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1281

written words, rather than being able to rely An Upside of Asymmetry

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of Plant Caspases Shake It to Wake It Further analysis of more complex, tetrapodal

Sunny and Cool online 11 November; see the Perspective by

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1283

Better Brain Maps

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www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010

Bruce Alberts is Editor-

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science-based industrial establishment.

*http://americasclimatechoices.org. T. E. Bowman et al., Science 330, 1044 (2010).

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1287

Good for You, Good for Me, Too

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the tubulin/FtsZ-like protein, TubZ, fate and transport

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1289

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network perturbations in biological systems

SENIOR EDITORIAL BOARD F. Fleming Crim, Univ. of Wisconsin

1292 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

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www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1297

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1298 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

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control arms of trials of vaccines or other

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1299

New HIV Infections Drop, But Treatment Demands Rise

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and I certainly hope they will take the deci-

Q: Do you think we’re at a point where we

www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1301

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1302 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

From Science’s Online Daily News Site

Meet the Squidworm

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www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1303

U.S. RESEARCH UNIVERSITIES

Panel Explores New Funding Pact With Washington

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1304 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

U.S. ENERGY POLICY

With Money Tight, White House Panel From the Science

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www.sciencemag.org SCIENCE VOL 330 3 DECEMBER 2010 1305

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CREDITS (TOP TO BOTTOM): DANNY LEHMAN/CORBIS; A. REGALADO/SCIENCE

Proud moment. Neuroscientist Miguel Nicolelis

1306 3 DECEMBER 2010 VOL 330 SCIENCE www.sciencemag.org

Science hub. Brazil’s richest city, São Paulo, and

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