Science Apr 06 07

Tools for Cell Biology Research
R&D Systems Surveyor™ IC Immunoassays

The ease of ELISA applied to intracellular factors.
Surveyor IC Analytes: R&D Systems Surveyor IC ELISA kits simplify the detec-
tion and quantification of intracellular factors impor-
Phospho-Akt tant for cell signaling. These kits provide all of the
(S473) Pan Specific components necessary to run a successful sandwich
ELISA with a clear, easy-to-follow protocol.
Phospho-ERK1/
ERK2 ASSAY FEATURES
Phospho-ERK2 High specificity
(T185/Y187) Quantitative
Total HIF-1 alpha Rapid
Colorimetric detection
Total HSP70 Validated for a variety of cell lysates
Phospho-p38 Superb alternative to Western blot
alpha (T180/Y182)
Total Survivin For more information visit our website at www.RnDSystems.com/go/SurveyorIC
Phospho-TOR
(S2448) 25
uninduced
LY294002
ERK2
IGF-1
20
p-Akt
ng of p-Akt per 106 cells
15 Akt MCF-7 cells were incubated with or

without IGF-1 and the PI3 Kinase
inhibitor LY294002. Cells were lysed
and phosphorylated Akt was quant-
10 ified with phospho-Akt Surveyor IC
U.S. & Canada
R&D Systems, Inc.
(Catalog # SUV887). The same lysates
Tel: (800) 343-7475
were also immunoblotted (inset) with
[email protected] either anti-phospho-Akt (S473) (p-Akt)
5
or anti-Akt antibodies.The Surveyor IC
Europe Immunoassay results correlate well
R&D Systems Europe Ltd. with the amounts of phosphorylated
Tel: +44 (0)1235 529449
[email protected] 0 Akt detected by Western blot.
uninduced IGF-1 IGF-1 + LY294002
R&D Systems is a registered
trademark of TECHNE Corporation. For research use only. Not for use in diagnostic procedures.
Selection expanding weekly—visit www.RnDSystems.com

to sign up for weekly new product updates. ®
6 April 2007 | $10
Inducible Expression Systems
Tet-On and Tet-Off Advanced
The Benchmark System Just Got Better

• Highly Sensitive and Controllable Visit our website today
• Proven for Both Inducible RNAi and cDNA www.clontech.com/tetsys
and plan for tomorrow!
• High Maximal Expression Levels
Products for your research needs

Fluorescent & Luminescent Reporters
Gene Expression & Delivery
Gene Expression Profiling
HEK 293Tet-On Advanced cells transfected with pTRE-Tight-BI-AcGFP1, grown in the absence (left) and
presence (right) of doxycycline (Dox), and observed by fluorescence microscopy (40x magnification). Non-coding RNA Research & RNAi
Tet-On Advanced Tet-Off Advanced

Nucleic Acid Purification
Dox Dox Oligo Modification
– + – +
PCR & RT-PCR Products
Luciferase
Protein Arrays
Protein Expression & Purification
ß-actin
Protein-Protein Interaction Systems
Luciferase expression in HEK 293Tet-On Advanced and HEK 293Tet-Off Advanced Cells transfected RNA
with pTRE-Tight-Luc grown in the presence and absence of Dox.
Clontech Laboratories, Inc.

A Takara Bio Company
www.clontech.com
United States/Canada: +1.800.662.2566 • Europe: +33.(0)1.3904.6880 • Asia Pacific: +1.650.919.7300 • Japan: +81.(0)77.543.6116
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech, Clontech logo, and all other trademarks
are the property of Clontech unless noted otherwise. ©2007 Clontech Laboratories, Inc.
AD732279
GE Healthcare
Bringing protein analysis

to life with Ettan DIGE and
Amersham ECL
When it comes to life sciences, GE Healthcare is setting the
standard. Tens of thousands of scientists worldwide rely on our
products and proven expertise in protein analysis and detection
every day. But we’re never content to stand still. We’re constantly
striving for innovations that boost accuracy and deliver
quantitative data.
The result is new fluorescence platforms like the Ettan™ DIGE Imager
and Amersham ECL Plex™ for quantitative protein analysis.
By continually developing technology that can turn your scientific
ideas into reality, we’re bringing science to life and helping to
transform drug discovery and healthcare.
We call it Protein Analysis Re-imagined.
Discover the full power of Ettan DIGE and the Amersham ECL™
family of Western blotting systems.
www.gehealthcare.com/protein_analysis
GE Healthcare Bio-Sciences AB, a General Electric Company.

Björkgatan 30, 751 84 Uppsala, Sweden.
© 2007 General Electric Company - All rights reserved.
GE08-06
CONTENTS
Volume 316, Issue 5821
COVER DEPARTMENTS
A Chihuahua walking with a Great Dane 11 Science Online
more than 50 times its mass. The extreme 13 This Week in Science
diversity in body size among purebred dogs 19 Editors’ Choice
is greater than that of any other mammalian 24 Contact Science
species. Researchers have identified a gene 27 Random Samples
that helps explain this size diversity. 29 Newsmakers
See page 112. 129 Science Careers
Photo: Deanne Fitzmaurice
EDITORIAL
17 A Two-Pronged Climate Strategy
by Rosina M. Bierbaum and
Peter H. Raven
NEWS OF THE WEEK LETTERS

Turnover at the Top, but Problems Persist at the 30 Making Articles Available for Flu Planning M. Berger 49
Smithsonian Pneumococcal Vaccines and Flu Preparedness
Design Flaw Could Delay Collider 31 K. P. Klugman and S. A. Madhi
Timing of a Back-Migration into Africa
Attosecond Laser Pulses Illuminate Fleeting Dance 33
P. Forster and V. Romano
of Electrons
Response A. Olivieri et al.
SCIENCESCOPE 33 Speeding Up the EPA Review Process M. Peacock
Hobbit’s Status as a New Species Gets a Hand Up 34
Chemistry Reports Warn of Eroding American 35 BOOKS ET AL.
Research Lead Charles Darwin and Victorian Visual Culture 54
Indonesia to Share Flu Samples Under New Terms 37 J. Smith, reviewed by H. Ritvo
Appointee ‘Reshaped’ Science, Says Report 37 Dr. Golem How to Think About Medicine 55
H. Collins and T. Pinch, reviewed by R. A. Ankeny
NEWS FOCUS
An Asian Tiger’s Bold Experiment 38
POLICY FORUM
Hard Data on Hard Drugs, Grabbed From 42 Framing Science 56
the Environment M. C. Nisbet and C. Mooney
The World Through a Chimp’s Eyes 44
PERSPECTIVES
American Physical Society Meeting 46
Experimenters Agree: You Can Cross Off Flowing Crystals
Rapid Consolidation 57
Ultrashort Laser Pulses See Inside the Body
L. R. Squire
Pulling Strings to Untangle Catastrophe
>> Research Article p. 76
55
Processive Motor Movement 58
D. D. Hackney
>> Report p. 120
Roots of Biosynthetic Diversity 60
D. W. Christianson
>> Research Article p. 73
High Bond Orders in Metal-Metal Bonding 61
F. Weinhold and C. R. Landis
So Small Yet Still Giant 63
I. V. Lerner
>> Report p. 99
Searching for a Solid-State Terahertz Technology 64
M. Lee and M. C. Wanke
38
CONTENTS continued >>
www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 5

Sample technologies by QIAGEN
Prepare for results
I Protein, RNA, and DNA purification

I Whole genome amplification
I Epigenetics
I Sample collection, stabilization, and preparation
I Plug and play automation with QIAcube
SampleTech0407S1WW
Contact QIAGEN today or visit www.qiagen.com/goto/SampleTech .
Sample & Assay Technologies

CONTENTS
SCIENCE EXPRESS
www.sciencexpress.org
PHYSICS
CELL BIOLOGY Functional Quantum Nodes for Entanglement Distribution over Scalable
Genes Required for Mitotic Spindle Assembly in Drosophila S2 Cells Quantum Networks
G. Goshima et al. C.-W. Chou et al.
A whole-genome screen identifies the 204 genes involved in assembling the mitotic Entanglement between atomic gas clouds 3 meters apart forms a quantum repeater,
spindle in flies and how they might contribute to cancer and other abnormalities. an essential tool for passing information in long-distance quantum communication.
10.1126/science.1141314 10.1126/science.1140300
CLIMATE CHANGE CELL BIOLOGY

Model Projections of an Imminent Transition to a More Arid Climate Positive Regulation of Itk PH Domain Function by Soluble IP4
in Southwestern North America Y. H. Huang et al.
R. Seager et al. A kinase phosphorylates the inositol pyrophosphate IP3 to generate IP4 and is
A collection of 19 climate models predicts that southwest North America will dry necessary for cell signaling during positive selection of immune cells.
significantly in the coming century, a transition that may already be under way. >> Reports pp. 106 and 109
10.1126/science.1139601 10.1126/science.1138684
TECHNICAL COMMENT ABSTRACTS BREVIA

ECOLOGY ECOLOGY
Comment on “Divergent Induced Responses to an 53 Rapid and Recent Changes in Fungal Fruiting Patterns 71
Invasive Predator in Marine Mussel Populations” A. C. Gange, E. G. Gange, T. H. Sparks, L. Boddy
P. D. Rawson, P. O. Yund, S. M. Lindsay The length of the autumn fruiting season for fungi in forest soil has
full text at www.sciencemag.org/cgi/content/full/316/5821/53b increased for the past five decades, in parallel with temperature and
Response to Comment on “Divergent Induced Responses rainfall increases in the United Kingdom.
to an Invasive Predator in Marine Mussel Populations”
A. S. Freeman and J. E. Byers RESEARCH ARTICLES
full text at www.sciencemag.org/cgi/content/full/316/5821/53c BIOCHEMISTRY
Chimeras of Two Isoprenoid Synthases Catalyze All 73
REVIEW Four Coupling Reactions in Isoprenoid Biosynthesis
H. V. Thulasiram, H. K. Erickson, C. D. Poulter
OCEAN SCIENCE
A synthetic protein made from the four enzymes that synthesize
Atlantic Meridional Overturning Circulation During 66
isoprenoids can effectively catalyze all four reactions, suggesting
the Last Glacial Maximum
their origin from a common ancestor. >> Perspective p. 60
J. Lynch-Stieglitz et al.
NEUROSCIENCE
Schemas and Memory Consolidation 76
D. Tse et al.
Rats learn to associate a place with a taste much more rapidly if they
have already been given a chance to learn the spatial context of the
new location. >> Perspective p. 57
REPORTS
MATERIALS SCIENCE
Nonstoichiometric Dislocation Cores in α-Alumina 82
N. Shibata et al.
Electron microscopy reveals that in aluminum oxide,
nonstoichiometric dislocations form on adjacent planes
and slip together during high-temperature deformation.
CHEMISTRY
Acid Catalysis in Basic Solution: A Supramolecular 85
Host Promotes Orthoformate Hydrolysis
M. D. Pluth, R. G. Bergman, K. N. Raymond
102 The electrostatic environment within the cavity of a synthetic
metal-ligand cluster enables acid catalysis in a basic solution.

CONTENTS
REPORTS CONTINUED...
CLIMATE CHANGE GENETICS
The Deep Ocean During the Last Interglacial Period 89 A Single IGF1 Allele Is a Major Determinant of 112
J. C. Duplessy, D. M. Roche, M. Kageyama Small Size in Dogs
North Atlantic Deep Water was warmer during the last interglacial N. B. Sutter et al.
than it is today and probably warmed Antarctic waters, accelerating Small dogs are small because they carry a particular allele of the
ice loss and raising sea levels. gene encoding insulin-like growth factor 1.
PLANETARY SCIENCE BIOCHEMISTRY
Subsurface Radar Sounding of the South Polar 92 Binding of the Human Prp31 Nop Domain to a 115
Layered Deposits of Mars Composite RNA-Protein Platform in U4 snRNP
J. J. Plaut et al. S. Liu et al.
Radar mapping of layered deposits at Mars’ south pole shows that A protein within the particle that assembles mature mRNAs has both
they are pure water ice, sitting on cratered terrain, with a volume RNA and protein binding surfaces, and it achieves binding specificity
equivalent to a global water layer 11 meters thick. by acting as a molecular ruler.
APPLIED PHYSICS BIOCHEMISTRY
Synchronized Oscillation in Coupled 95 An ATP Gate Controls Tubulin Binding by the 120
Nanomechanical Oscillators Tethered Head of Kinesin-1
S.-B. Shim, M. Imboden, P. Mohanty M. C. Alonso et al.
A rich dynamic response, including synchronization and entrainment, The two-headed motor kinesin is gated by ATP independently of
is seen when two coupled nanomechanical beams are driven over a the microtubule along which it moves, contrary to current models
range of oscillating frequencies. of kinesin motion.
>> Perspective p. 58
PHYSICS
Giant Fluctuations of Coulomb Drag in a 99
Bilayer System
A. S. Price et al.
Electrons flowing in one thin layer drag electrons in an underlying
layer more than expected, implying that local electron properties
are important in momentum exchange. >> Perspective p. 63
APPLIED PHYSICS
Direct-Current Nanogenerator Driven by 102
Ultrasonic Waves
X. Wang, J. Song, J. Liu, Z. L. Wang
Through their variable bending, which separates charge, a series of
zinc oxide nanowires can convert sound waves to continuous electrical
current to power nanoscale devices.
CELL BIOLOGY
A Conserved Family of Enzymes That Phosphorylate 106
CREDIT: M. C. WAHL/MAX PLANCK INSTITUTE FOR BIOPHYSICAL CHEMISTRY, GÖTTINGEN, GERMANY
Inositol Hexakisphosphate
S. Mulugu et al.
A yeast enzyme is regulated by pH and can both synthesize and
metabolize the inositol pyrophosphate IP6.
>> Science Express Report by Y. H. Huang et al.
CELL BIOLOGY
Regulation of a Cyclin-CDK-CDK Inhibitor Complex 109
by Inositol Pyrophosphates
Y.-S. Lee, S. Mulugu, J. D. York, E. K. O’Shea 115
When yeast are starved for the nutrient phosphate, the inositol
pyrophosphate IP7 activates gene expression and a metabolic
network for nutrient homeostasis.
>> Science Express Report by Y. H. Huang et al.
SCIENCE (ISSN 0036-8075) is published weekly on Friday, except the last week in December, by the American Association
for the Advancement of Science, 1200 New York Avenue, NW, Washington, DC 20005. Periodicals Mail postage (publication No.
484460) paid at Washington, DC, and additional mailing offices. Copyright © 2007 by the American Association for the Advancement
of Science. The title SCIENCE is a registered trademark of the AAAS. Domestic individual membership and subscription (51 issues): $142
($74 allocated to subscription). Domestic institutional subscription (51 issues): $710; Foreign postage extra: Mexico, Caribbean (surface
mail) $55; other countries (air assist delivery) $85. First class, airmail, student, and emeritus rates on request. Canadian rates with GST
available upon request, GST #1254 88122. Publications Mail Agreement Number 1069624. Printed in the U.S.A.
Change of address: Allow 4 weeks, giving old and new addresses and 8-digit account number. Postmaster: Send change of address to AAAS, P.O. Box 96178, Washington, DC 20090–6178. Single-copy sales:
$10.00 current issue, $15.00 back issue prepaid includes surface postage; bulk rates on request. Authorization to photocopy material for internal or personal use under circumstances not falling within the
fair use provisions of the Copyright Act is granted by AAAS to libraries and other users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that $18.00 per article is
paid directly to CCC, 222 Rosewood Drive, Danvers, MA 01923. The identification code for Science is 0036-8075. Science is indexed in the Reader’s Guide to Periodical Literature and in several specialized indexes.

RNAi Solutions
New
siLentMer™
siRNA!
Check out our
new validated and
predesigned 27-mer
siRNA duplexes
Bio-Rad and RNAi.Come have a look.

From design to detection, Bio-Rad supports your RNAi research.
With a broad range of proven delivery technologies, award-winning

detection systems, and a suite of high-quality support products,
it’s clear that Bio-Rad has a vision for RNAi.
I High-performing potent siRNA for ≥85% knockdown
with as low as 5 nM siRNA
I Greatest choice of delivery technologies
I RNA and protein purification products
I Automated microfluidic RNA analysis
I Sensitive, optimized cDNA synthesis kits
MCF-7 cells transfected using siLentFect™
I Systems for protein and mRNA detection reagent. RNA purified and analyzed using
the Aurum™ total RNA kit and Experion™
For a close look at Bio-Rad’s tools for RNAi, visit us system. Real-time PCR detection performed
on the Web at www.bio-rad.com/rnai/ using iScript™ cDNA synthesis kit.
Practice of the polymerase chain reaction (PCR) may require a license.
Visit us on the Web at discover.bio-rad.com

Call toll free at 1-800-4BIORAD (1-800-424-6723);
outside the US, contact your local sales office.
ONLINE
www.sciencemag.org
SCIENCENOW
www.sciencenow.org DAILY NEWS COVERAGE
Interspecies Tryst While Out of Africa?
Other researchers question interpretation of oldest
human skeleton found in China.
Not Your Type? Don’t Sweat It
Enzymes convert type A and type B blood to type O.
Tatooine’s Twin Suns Not So Farfetched
Planets may abound around double star systems.
Scientific writing
and publishing.
SCIENCE CAREERS
www.sciencecareers.org CAREER RESOURCES FOR SCIENTISTS
GLOBAL: Special Feature—Getting Published in

Scientific Journals
E. Pain
CREDITS (TOP TO BOTTOM): CREATIVE COMMONS/ATTRIBUTION; IMAGE ADAPTED FROM TERESE WINSLOW, NATIONAL CANCER INSTITUTE
Publications can make or break your career, but how can you
Cancer progression. improve your chance of success?
US: Tips for Publishing in Scientific Journals
SCIENCE’S STKE K. Kelner
www.stke.org SIGNAL TRANSDUCTION KNOWLEDGE ENVIRONMENT Science Deputy Editor Katrina Kelner offers advice on how to get
your research published.
EDITORIAL GUIDE: Focus Issue—Exploring New Avenues for
Cancer Treatment US: The Story’s the Thing
E. M. Adler and N. R. Gough R. Ness
Recent research on cancer cell pathophysiology provides reasons to One key to scientific writing is spinning a good (nonfiction) yarn.
be hopeful about development of novel therapies. EUROPE: Publishing for Non-Native Writers
MEETING REPORT: Tumor Biology—How Signaling Processes E. Pain
Translate to Therapy When writing up their research for Western journals, non-native
K. Friedrich, O. Janssen, R. Hass English speakers face extra challenges.
The most recent meeting of the Signal Transduction Society US: From the Archives—How to Write a Winning Résumé
highlighted the translation of signaling research into advances P. Fiske
in the cancer clinic (in Perspectives). If your résumé isn’t well honed, you may lose out to an inferior—
PERSPECTIVE: Metabolic Targeting as an Anticancer but more polished—candidate.
Strategy—Dawn of a New Era? GRANTSNET: April 2007 Funding News
J. G. Pan and T. W. Mak GrantsNet Staff
Could targeted therapies directed against aerobic metabolism Learn about the latest in research funding, scholarships, fellowships,
represent a viable approach to treating cancer? and internships.
REVIEW: Sequestration and Segregation of Receptor Kinases
in Epithelial Cells—Implications for ErbB2 Oncogenesis
C. A. C. Carraway and K. L. Carraway
Can oncogenesis occur by co-opting normal physiological responses
to epithelial damage?
Separate individual or institutional subscriptions to these products may be required for full-text access.

NEW
ENGLAND
BIOLABS
in a word, trust.
T4 DNA Ligase from New England Biolabs

WHEN YOU NEED LIGASE, TURN TO THE INDUSTRY STANDARD
New England Biolabs is dedicated to providing our customers with guaranteed enzyme performance.
Our recombinant T4 DNA Ligase is the most extensively used ligase for cloning experiments. It is
available at exceptional value, and an even greater value when purchased in large quantities for high
throughput technologies. For cohesive, blunt, simple or complex reactions, make T4 DNA Ligase from
NEB your first choice.
I T4 DNA Ligase* r Advantages:

Regular Concentration M0202S/L I Quality – Highly pure enzyme with no lot-to-lot variation
For standard cloning reactions I Convenience – Choose original T4 DNA Ligase or the
High Concentration M0202T/M Quick Ligation Kit to meet the demands of a variety of 0 0.1 0.2
T4 DNA Ligase (μl)
1.0 0 10 20 30
Time (min)
60
For large or difficult constructs reaction conditions

Ligation of blunt-ended Ligation of HindIII fragments
I Flexibility – Active at room temperature or 16°C; reaction HaeIII fragments of Lambda (4-base overhang) of Lambda
I Quick Ligation™ Kit* r M2200S/L
times run from 5 minutes to overnight DNA using various amounts DNA using 1 cohesive end
For ligation of cohesive or blunt-end DNA of T4 DNA Ligase (400,000 unit (1μl of 1:400 dilution) of
fragments in 5 minutes at room temperature. I Robustness – Active in a variety of reaction buffers
cohesive end units/ml) in T4 DNA Ligase. Reactions
See our website for more details. a 20 μl reaction volume. were incubated at 25°C.
Reactions were incubated
r = Recombinant
for 30 minutes at 16°C.
*NEB ligase products are BSA-free
For more information and our international distribution network, please visit www.neb.com
New England Biolabs, Inc. is an ISO 9001 certified company
New England Biolabs Inc. 240 County Road, Ipswich, MA 01938 USA 1-800-NEB-LABS Tel. (978) 927-5054 Fax (978) 921-1350 [email protected]
Canada Tel. (800) 387-1095 [email protected] • China Tel. 010-82378266 [email protected] • Germany Tel. 0800/246 5227 [email protected]
Japan Tel. +81 (0)3 5669 6191 [email protected] • UK Tel. (0800) 318486 [email protected] the leader in enzyme technology
EDITED BY STELLA HURTLEY AND PHIL SZUROMI
Glaciers on Mars
Most of the water ice on the surface of Mars is locked up in
the polar caps. The Mars Express orbiter has used its radar
to penetrate to the base of the layered deposits on the
north pole. Now Plaut et al. (p. 92, published online 15
March) have mapped the south polar–layered deposits.
The radar penetrates 3.7 kilometers with little attenua-
tion, which suggests that these deposits are almost pure
water ice. The base of the deposits shows a set of buried
depressions that may be past impact craters. The deposits
themselves total 1.6 × 106 cubic kilometers, equivalent to
a global water layer approximately 11 meters thick.
Ocean Conditions known about the structure of dislocations, which

are defects in the crystalline ordering, and the
tually simple system that nonetheless shows rich
dynamic behavior. The beams are driven at a
Past and Present role they may play. Shibata et al. (p. 82) use wide range of frequencies. Frequency-locking or
The formation of cold, dense water in the North high-resolution electron microscopy to study dis- entrainment occurred in a number of regions in
Atlantic Ocean today helps drive meridional locations in aluminum oxide. Two nonstoichio- which the two beams synchronize to a single res-
overturning circulation, in which warm water metric partial defects form close to each other, onance. These resonators may be of use in signal
flows north over cold water flowing south, but and at high temperatures, the motion of the par- processing and communication.
conditions may have differed during the Last tial defects occurs with those on adjacent planes.
Glacial Maximum (LGM) 21,000 years ago.
Lynch-Stieglitz et al. (p. 66) review our under- Electrons Feel the Drag
standing of this problem. The pace of deep Acid Buried in Base When current flows in one layer of a bilayer sys-
Atlantic circulation during the LGM was nearly as Chemists often tailor reaction conditions tem, electron-electron interactions can drag cur-
vigorous as it is now, but patterns of sea surface by manipulating the temperature or rent in the other layer. Measurements of this
temperatures and the distribution of water acidity of the medium. In contrast, Coulomb drag effect are important for under-
masses were different, indicating that different enzymes cannot grossly alter standing coupled electronic and correlated
mechanisms drove circulation then. Further- their surroundings, and rely electron systems. Price et al. (p. 99; see
more, during the last interglacial, around instead on internal cavi- the Perspective by Lerner) report on the
125,000 years ago, land and sea surface tem- ties that tune the molec- observation of giant fluctuations, four
peratures at high latitudes were higher than they ular environment of an orders of magnitude greater than
are today, and sea level was 4 to 6 meters individual docked substrate. that expected, of the Coulomb drag
higher. Did deep ocean conditions contribute to Pluth et al. (p. 85) mimic resistance, which result in an alternating pos-
CREDITS (TOP TO BOTTOM): NASA/JPL/ASI/UNIVERSITY OF ROME; PLUTH ET AL.
melting of the Greenland and Antarctic ice this strategy using a synthetic itive and negative frictional force on electrons.
sheets? Duplessy et al. (p. 89) analyzed cores cage-like cluster that self-assembles The authors propose a model in which electrons in
from the Atlantic and Southern oceans and show from ligands and metal ions in solution. the two layers interact in the ballistic regime,
that North Atlantic Deep Water was warmer dur- The electrostatic environment inside the cluster characterized by large momentum transfers,
ing the last interglacial than it is today. Using stabilizes cations, and so favors protonation of where the local electron properties become
two models, they infer that extra heat would guest molecules. The cage can function as an important.
have been transferred to Circumpolar Deep acidic enclave in a basic solution and be used to
Water in the Southern Hemisphere, which would perform acid-catalyzed orthoformate hydrolysis
have melted more of the Antarctic Ice Sheet. in a surrounding basic medium. Engineering Isoprenoid
Builders
Dissecting Oxide In Sync Several Times Isoprenoids, a diverse family of natural products,
An organism or cell can synchronize its oscilla- are built from five-carbon building blocks using
Dislocations tory behavior with that of its neighbors, as in the four coupling reactions. Enzymes that catalyze
Imbalances in stoichiometry in layered oxides, blinking of fireflies or the beating of cardiac chain elongation and cyclopropanation have
such as at grain boundaries, can affect their cells. Shim et al. (p. 95) studied the behavior of been identified, however, enzymes that catalyze
electrical and mechanical properties. Less is two coupled nanomechanical beams, a concep- Continued on page 15

This Week in Science
Continued from page 13
branching and cyclobutanation have not. Thulasiram et al. (p. 73; see the Perspective by Christian-
son) show that all four reactions can be catalyzed by engineered enzymes that are chimeras of a chain
elongation enzyme and a cyclopropanation enzyme. The products have the same stereochemistry as
the natural products, suggesting that enzymes catalyzing the four reactions evolved from a common
ancestor.
The More You Know, the More You Learn

The ability to remember complex new information often depends on prior knowledge of the topic.
This is because we have already formed a relevant mental schema as a framework. Tse et al. (p. 76;
see the Perspective by Squire) used rats to study the effects of prior learning of schemas on the abil-
ity to acquire new episodic associations. These associations were acquired faster when the animals
were first trained on a consistent set of associations than when they occurred in the context of a novel
set of associations. The acquisition of novel associations was dependent on the hippocampus. How-
ever, within 48 hours the associations were independent of the hippocampus, which is substantially
faster than typical memory consolidation. Thus, animals—like people—can bring activated mental
schemas to bear during learning.
Motor Mechanics
Kinesin-1 is a two-headed molecular motor that takes 8-nm
steps along microtubules. At each step, one molecule of
adenosine triphosphate (ATP) is hydrolyzed and between
steps kinesin pauses until another molecule of ATP binds.
Now Alonso et al. (p. 120; see the Perspective by
Hackney) show that kinesin-1 interacts with free tubulin
heterodimers in solution and that this system too is gated
by ATP. The observed behavior would not be predicted by
current models for the motor mechanism that include a
role for the microtubule lattice in the gating mechanism.
Understanding Inositol Pyrophosphates

Inositol pyrophosphates are relatively poorly understood, highly phosphorylated members of the
inositol polyphosphate family. Two studies describe related advances in signaling involving inositol
pyrophosphates. Mulugu et al. (p. 106) purified an inositol pyrophosphate synthase from yeast that
has two catalytic domains. The enzyme, called Vip1, appears to act as a switch, with its catalytic activ-
ity determined by the local pH. Lee et al. (p. 109) purified a molecule that regulates the yeast
Pho80-Pho85-Pho81 complex, a protein complex containing a cyclin, a cyclin-dependent kinase
(CDK), and a CDK inhibitor. The active molecule turned out to be myo-D-inositol heptakisphosphate
(IP7), which is synthesized through the kinase activity of Vip1.
Sizing Up Man’s Best Friend

In contrast to most mammalian species, Canis familiaris (the domestic dog) shows extreme diver-
sity in body size. Sutter et al. (p. 112, cover) show that a single allele of the gene encoding
insulin-like growth factor–1 (IGF-1) is shared by all small dog breeds but is nearly absent from
giant dog breeds, implying that sequence variation in the IGF-1 gene plays a causal role in dog
size. Discovery of the IGF-1 gene was facilitated by its localization within a genomic signature, or
haplotype block, that probably arose as a result of centuries of dog breeding by humans.
Spliceosome Assembly
Human Prp31 is a protein in the spliceosome that is essential for pre-mRNA splicing. It is assembled
onto the spliceosome after 15.5K protein binds to an RNA component, U4 small nuclear (sn)RNA.
CREDIT: ALONSO ET AL.
Liu et al. (p. 115) present structural and biochemical data of hPrp31-15.5K-U4 snRNA complexes
that give insight into this hierarchical assembly. hPrp31 presents both RNA and protein binding
surfaces, making it a true ribnucleoprotein (RNP) binding protein. Binding occurs through the
nucleolar protein (Nop) domain, which may act as a molecular ruler that measures the length of an
RNA stem to achieve RNP binding specificity.

The National Institutes of Health
invites applications for the inaugural
Announced in early 2007, the Open to New Investigators Who

NIH Director’s New Innovator Have not yet obtained an NIH R01 or
Award will support research by similar grant
new investigators who propose Hold an independent research position
highly innovative projects with at an institution in the United States
the potential for exceptionally Received a doctoral degree or completed
great impact on biomedical medical internship and residency in 1997
or behavioral science. or later
Propose research in any scientific area
NIH expects to make at least
relevant to the NIH mission
14 awards in September
2007. Each grant will be for
Apply Electronically
5 years and up to a total of
Get instructions at http://grants.nih.gov/
$1.5 million in direct costs
grants/guide/rfa-files/RFA-RM-07-009.html
plus applicable facilities
Prepare the short application; preliminary
and administrative costs.
data allowed, but not required
Women and members of groups Submit the application through Grants.gov
that are underrepresented between April 25 and May 22, 2007
in biomedical or behavioral
research are especially More Information
encouraged to apply. See http://grants.nih.gov/grants/
new_investigators/innovator_award/
E-mail questions to [email protected]
or call 301-594-4469
EDITORIAL
A Two-Pronged Climate Strategy

A SENSIBLE STRATEGY TO MINIMIZE THE DAMAGES FROM ANTHROPOGENIC CLIMATE
change has two objectives: mitigate the pace and ultimate magnitude of the changes that occur and
Rosina M. Bierbaum
adapt to the changes that cannot be avoided. To underline this two-pronged approach, the recent
is co-chair (with Peter
report Confronting Climate Change, prepared for the United Nations (UN) by an international
H. Raven) of the SEG
panel we co-chaired, was subtitled Avoiding the Unmanageable and Managing the Unavoidable
on Climate Change and
Global Development,
(www.unfoundation.org/SEG/). On 27 February 2007, we presented UN Secretary-General
and professor and dean
Ban Ki-moon with this urgent call for new levels of commitment and coordination by the UN and
of the School of Natural its member states to avoid the worst climate-change dangers while there is still time.
Resources and The Scientific Expert Group (SEG) on Climate Change and Sustainable Development was
Environment, University organized by the scientific research society Sigma Xi and the UN Foundation at the request of
of Michigan. the UN Department on Economic and Social Affairs. Our 18 expert
[email protected] members come from 11 countries and a wide range of disciplines and
institutions. Unlike the Intergovernmental Panel on Climate Change
(IPCC), which may not make recommendations for action, the SEG was
invited to tell the UN what it should do to address the climate-change
challenge, and after over 2 years of work, it did.
The group’s unanimous recommendations focus equally on mitigation
and adaptation. The SEG concludes that unmanageable changes in the
future are avoidable only if the world community acts now. Global carbon
dioxide emissions must level off by 2015 or 2020 at little more than their
current level and then decline to no more than a third of that level by 2100.
Emissions of methane and black soot must also be controlled. This can be
done with a mix of existing and new technologies, with many subsidiary
benefits. We recommend several urgent goals: improved efficiency in the
transportation sector and in the energy efficiency of buildings; expanded
Peter H. Raven is use of biofuels; and, very important, the design and deployment of coal-fired power plants
co-chair (with Rosina capable of environmentally sound retrofits for carbon capture.
CREDITS (TOP TO BOTTOM): STEVE KUZMA PHOTOGRAPHY; MICHAEL JACOB/MISSOURI BOTANICAL GARDEN; UN FOUNDATION
M. Bierbaum) of the SEG Equally important is our capacity to adapt to unavoidable change by improving preparedness
on Climate Change and and response strategies to meet the needs of the world’s poor, who will bear the heaviest burden of
Global Development, climate change. The summary of the new IPCC report on Impacts, Adaptation and Vulnerability,
and president of the expected this week, makes it clear that the future will be very different from the past and that
Missouri Botanical Garden. changes under way challenge our land management regimes and our ability to maintain ecosystem
[email protected] services. Climate-resilient energy-efficient cities must become the norm, and institutions must be
strengthened to cope with weather-related disasters and climate-change refugees, whose numbers
may reach tens of millions in the future. Building on land less than 3 feet above sea level is certainly
not sustainable. Preserving a major proportion of the poorly known biological diversity of the
world requires curbing the rates of climate change but also needs enhanced and innovative efforts
to save surviving species.
Global climate has already changed noticeably, with more than half of the increase in
temperature since preindustrial times occurring since 1970. Heat waves; ice melt; shifting
ranges of plants and animals; sea-level rise; and droughts, floods, and wildfires are increasing,
as expected. Even if emissions were completely halted today, the total temperature increase from
greenhouse gases already in the atmosphere would be approximately 1.5°C globally. Unless we
can keep global average temperature from exceeding 2° to 2.5°C above preindustrial levels, we
may reach tipping points that could produce intolerable human impacts. Business as usual could
have us 3° to 5°C above preindustrial temperatures by 2100—a temperature jump equaling that
from the height of the last ice age to the present warm period. Unless the world acts now, we will
fail miserably to meet the UN Millennium Development Goals, fail to improve the fate of the
poor, and fail to achieve global sustainability. The human race, now numbering 6.5 billion
people, has never faced a greater challenge, and there is no time for further delay.
– Rosina M. Bierbaum and Peter H. Raven
10.1126/science.1143220

www.roche-applied-science.com
Introducing Our Second Generation Genome Sequencing System
Genome Sequencer FLX

Be First to the Finish with...
I Increased read lengths averaging 200 to 300 bases per read
I Throughput of more than 400,000 reads per run
I High single-read accuracy greater than 99.5% over 200 bases
I Consensus-read accuracy greater than 99.99%
...more Flexibility and more Applications.

Visit www.genome-sequencing.com to learn about the
expanding number of peer-reviewed publications appearing weekly.
Roche Diagnostics GmbH

Roche Applied Science
454 and GENOME SEQUENCER are trademarks of
454 Life Sciences Corporation, Branford, CT, USA. 68298 Mannheim
© 2007 Roche Diagnostics GmbH. All rights reserved. Germany
EDITORS’CHOICE
EDITED BY GILBERT CHIN AND JAKE YESTON
ECOLOGY/EVOLUTION
Perennial Infection
Although grazing and fire have been proposed as explanations for the
remarkable success of exotic annual grasses in California, where they
have established themselves among the native perennials over wide
swathes of the landscape despite being inferior competitors for
resources, active management based on these factors has failed to stem
the invasions. It is known that disease can alter the competitive balance
between species in ecological communities, and Borer et al. have devel-
oped a model showing quantitatively how invasion has been mediated
by viral disease (barley and cereal yellow dwarf viruses, which are a
major pathogen in crops, including wheat, barley, and oats). They find
that the key to the success of the annual grasses is that virus is horizon-
tally transmitted by aphids, rather than vertically via seeds; hence, seed
survival is unaffected, and each generation suffers infection anew.
Central California
In contrast, perennial grasses serve as long-term reservoirs for the virus
grasslands.
and experience deleterious effects on survival and on lifetime seed pro-
duction, thus facilitating the invasion by annuals. — AMS
Proc. Natl. Acad. Sci. U.S.A. 104, 5473 (2007).
PHYSICS has, in some environments, credentialed bioin- substrates and products differ, which may
formatic analysts and relegated experimental reflect an evolutionary adaptation to changes in
Probing Quantum Memories metabolite concentrations. — GJC
work to the back-benchers. Nevertheless, the
Can’t quite place a name to the face, or associate significance of mutations can be hard Biochemistry 46, 10.1021/bi6024879 (2007).
a singer with a song? You know, or at least hope, to predict without actually mak-
that the information lies intact somewhere in your ing the proteins and assessing CHEMISTRY
head, needing only the correct memory trick or their behavior.
stimulus to retrieve it. For quantum communica-
A Less Radical Pathway
Kona et al. have taken this
tions, where information is transmitted along approach in trying to understand Enediyne molecules, in which two doubly
quantum channels and stored in quantum memo- the role of a Cd2+-binding cys- bonded carbons tether two sets of triply
ries, it is necessary that the stored information be teine in the Escherichia coli bonded carbons, have been known for over
robust and retrievable. However, quantum memo- enzyme KDO8P synthase in compar- 30 years to cyclize to the intriguing para-
CREDITS (TOP TO BOTTOM): ELIZABETH BORER; KONA ET AL., BIOCHEMISTRY 46, 10.1021/BI6024879 (2007)
ries are known to decay because of decoherence, ison to an asparagine in the Aquifex benzyne biradical. This species has been
and physicists therefore have to develop their own aeolicus version of the same enzyme. observed in many cases to behave as a
set of tricks to probe and measure how reliable The reaction they catalyze is an aldol benzene ring with two diametrically
these memories are. Staudt et al. look at quantum condensation of phospho- opposed trivalent carbons, which each
information stored in an optical memory, where enolpyruvate and arabinose 5- react rapidly with hydrogen or halogen
the information is encoded in the coherent trans- phosphate. This enzymatic atom sources.
fer of the phase and amplitudes of light pulses step is a critical one in the Perrin et al. have observed a sur-
onto a suitable solid-state medium. They use a bacterial biosynthetic pathway prisingly different mode of reactiv-
photon-echo technique whereby a sequence of leading to lipopolysaccha- ity, which is more consistent with nucle-
pulses initializes the memory cell, encodes the rides and hence is a poten- KDO8P synthase ophilic attack at one of the unsaturated
data onto it, and uses a read pulse to generate a tial drug target. A compari- intermediate. carbons than with radical atom abstraction.
stimulated echo pulse which replicates the stored son of the structures Their studies show that slight heating of an
information. The advantage of this scheme is that, enabled them to make a series of mutations enediyne in the presence of lithium halide salts
though memories may be lost, if they are recalled bridging the metallo- and nonmetallo-KDO8P and acid results in a halide and proton adding
they remain undistorted. — ISO synthases; follow-up kinetic and structural to opposite ends of the resultant benzene ring.
Phys. Rev. Lett. 98, 113601 (2007). analyses yielded several insights. The cysteine- Isotopic labeling reveals that even as weak an
coordinated metal fulfills the same function as acid as dimethylsulfoxide can serve as the pro-
BIOCHEMISTRY the asparagine carboxamide in binding and ori- ton donor, implicating a highly basic phenyl
enting a water molecule for attack on the si side anion intermediate formed after halide attack.
Step by Step
at C2. Even though the metallo- and non- The reaction is high-yielding for chloride, bro-
Recent exponential growth in databases as a metallo-KDO8P synthases produce the same mide, and iodide salts, and shows kinetics con-
consequence of big-science projects such as chemical intermediate, probably via the same sistent with p-benzyne formation as the rate-
genome sequencing and structural genomics reaction pathway, the binding constants of the Continued on page 21

EDITORS’CHOICE The Art of Global
Continued from page 19
Discovery Chemistry
limiting step. These findings offer a compelling intense when the field cut across the cube’s cor-
rationale for the puzzling isolation from marine ners; more rounded truncated cubes showed lit-
sources of monochlorinated cyanosporaside iso- tle variation with polarization direction. Similar
mers whose structures were inconsistent with effects were seen in simulations of the local
established radical or electrophilic chlorination fields for these particles. — PDS
pathways. — JSY Nano Lett. 7, 10.1021/nl070157q (2007).
J. Am. Chem. Soc. 129, 10.1021/ja070023e
(2007). MOLECULAR BIOLOGY
CHEMISTRY
The Evolution of Origins
Prokaryote genomes are generally organized as
SERS from Sharp Silver
a single circular chromosome with a single ori-
Surface-enhanced Raman scattering (SERS) is gin of DNA replication; most eukaryotes, on the
observed on a variety of silver and gold surfaces other hand, have multiple chromosomes, each
where nanoscale roughness creates high local with multiple replication origins. This latter fea-
fields, and giant enhancements have been ture has recently been found in a number of
observed in “hot spots” created between two archaea, including Sulfolobus species, which
nanoparticles. However, even single nanoparti- have several origins on a single chromosome.
cles can create fields large enough to enable Might these have arisen simply by duplication?
single-molecule detection. Robinson and Bell show that origins that are
To better understand the origin of this effect, conserved across Sulfolobus species share the
McLellan et al. have deposited silver nanoparti- gene copG, encoding a plasmid copy-number
cles of various shapes on silicon substrates that control protein, as well as a number of stress
have registration marks. Scanning electron response genes. Furthermore, one of the two
microscopy was used to determine the orienta- origins in the archaeal Aeropyrum pernix bears
a striking resemblance to two origins
found in a distantly related Sul-
folobus species; several genes and
evidence of a putative prokaryotic
viral integration site are conserved.
Among the genes is a protein that is
similar to RepA, a bacterial plasmid
initiator protein, as well as the yeast
replication initiation protein Cdt1.
Altogether, this evidence points to a
Field amplitudes around a silver cube. captured extrachromosomal element, possibly a
virus/plasmid hybrid, as the source of the
tion of the particles so that the effect of laser supernumerary origins. A hybrid phage/eukary-
polarization on SERS spectra could be studied. otic replication initiation site on the yeast 2μ
For nanocubes, the SERS intensity of adsorbed plasmid hints at a similar genesis for the multi-
1,4-benzenedithiol varied greatly with the direc- ple origins on eukaryotic chromosomes. — GR
tion of polarization, and the spectra were more Proc. Natl. Acad. Sci. U.S.A. 104, 5806 (2007).
<< Better Bones Without Bax

CREDIT: MCLELLAN ET AL., NANO LETT. 7, 10.1021/NL070157Q (2007)
At about age 50, the depletion of ovarian follicles through apoptosis CHEMBRIDGE CORPORATION IS THE
leads to the loss of cyclic ovarian function in women. Although aging WORLD’S LARGEST GLOBAL DISCOVERY
female mice do not undergo menopause, they do suffer a depletion of
CHEMISTRY CRO AND PREMIER PROVIDER
www.stke.org ovarian follicles and health complications similar to those of post-
menopausal women. After their earlier finding that oocyte loss was mit- OF ADVANCED SCREENING LIBRARIES
igated in mice lacking the proapoptotic protein Bax, Perez et al. investigated aging Bax-deficient
FOR SMALL MOLECULE DRUG DISCOVERY.
female mice and found them to be leaner and more active than their wild-type counterparts. They
retained more of their hair, developed fewer cataracts, experienced less wrinkling of the skin, and PLEASE VISIT WWW.CHEMBRIDGE.COM
had stronger bones. Although older Bax knockout mice failed to become pregnant, they did ovu-
late in response to gonadotropin, and when their ovarian tissue was grafted into young wild-type
females, the oocytes produced viable pups. Finally, behavioral analyses indicated that the knock-
outs were less anxious and more attentive than wild-type mice. — EMA
www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007

Does your next career step
need direction?
I have a great new research idea.
Where can I find more grant options?
For a career in science,

I turn to Science
You know, ScienceCareers.org

is part of the non-profit AAAS
That means they’re putting

something back into science
I got the offer I've been
dreaming of.
With thousands of job postings,

it’s a lot easier to track down a
career that suits me
Now what?
I want a career,
not just a job
There’s only one place to go for career advice if you value the
expertise of Science and the long experience of AAAS in supporting
career advancement - ScienceCareers.org. The pages of Science
and our website ScienceCareers.org offer:
• Thousands of job postings • Funding information

• Career advice articles and tools • Networking opportunities
www.sciencecareers.org
www.sciencemag.org EDITOR-IN-CHIEF Donald Kennedy EXECUTIVE PUBLISHER Alan I. Leshner
EXECUTIVE EDITOR Monica M. Bradford PUBLISHER Beth Rosner
DEPUTY EDITORS NEWS EDITOR
R. Brooks Hanson, Barbara R. Jasny, Colin Norman FULFILLMENT & MEMBERSHIP SERVICES ([email protected]) DIRECTOR
Katrina L. Kelner Marlene Zendell; MANAGER Waylon Butler; SYSTEMS SPECIALIST Andrew
Vargo; CUSTOMER SERVICE SUPERVISOR Pat Butler; SPECIALISTS Laurie Baker,
EDITORIAL SUPERVISORY SENIOR EDITOR Phillip D. Szuromi; SENIOR EDITOR/
Tamara Alfson, Karena Smith, Vicki Linton, Latoya Casteel; CIRCULATION
PERSPECTIVES Lisa D. Chong; SENIOR EDITORS Gilbert J. Chin, Pamela J. Hines,
ASSOCIATE Christopher Refice; DATA ENTRY SUPERVISOR Cynthia Johnson;
1200 New York Avenue, NW Paula A. Kiberstis (Boston), Marc S. Lavine (Toronto), Beverly A. Purnell, SPECIALISTS Tomeka Diggs, Tarrika Hill, Erin Layne
Washington, DC 20005 L. Bryan Ray, Guy Riddihough, H. Jesse Smith, Valda Vinson, David Voss;
Editorial: 202-326-6550, FAX 202-289-7562 ASSOCIATE EDITORS Jake S. Yeston, Laura M. Zahn; ONLINE EDITOR Stewart BUSINESS OPERATIONS AND ADMINISTRATION DIRECTOR Deborah Rivera-
News: 202-326-6500, FAX 202-371-9227 Wills; ASSOCIATE ONLINE EDITOR Tara S. Marathe; BOOK REVIEW EDITOR Sherman Wienhold; BUSINESS MANAGER Randy Yi; SENIOR BUSINESS ANALYST Lisa
J. Suter; ASSOCIATE LETTERS EDITOR Etta Kavanagh; EDITORIAL MANAGER Cara Donovan; BUSINESS ANALYST Jessica Tierney; FINANCIAL ANALYSTS Michael
Tate; SENIOR COPY EDITORS Jeffrey E. Cook, Cynthia Howe, Harry Jach, LoBue, Farida Yeasmin; RIGHTS AND PERMISSIONS: ADMINISTRATOR Emilie
Bateman House, 82-88 Hills Road Barbara P. Ordway, Jennifer Sills, Trista Wagoner; COPY EDITORS Lauren David; ASSOCIATE Elizabeth Sandler; MARKETING: DIRECTOR John Meyers;
Cambridge, UK CB2 1LQ Kmec, Peter Mooreside; EDITORIAL COORDINATORS Carolyn Kyle, Beverly MARKETING MANAGERS Darryl Walter, Allison Pritchard; MARKETING
+44 (0) 1223 326500, FAX +44 (0) 1223 326501 Shields; PUBLICATIONS ASSISTANTS Ramatoulaye Diop, Chris Filiatreau, Joi S. ASSOCIATES Julianne Wielga, Mary Ellen Crowley, Catherine Featherston,
Granger, Jeffrey Hearn, Lisa Johnson, Scott Miller, Jerry Richardson, Brian Alison Chandler, Lauren Lamoureux; INTERNATIONAL MARKETING MANAGER
SUBSCRIPTION SERVICES For change of address, missing issues, new White, Anita Wynn; EDITORIAL ASSISTANTS Maris M. Bish, Emily Guise, Wendy Sturley; MARKETING EXECUTIVE Jennifer Reeves; MARKETING/MEMBER
orders and renewals, and payment questions: 866-434-AAAS (2227) Patricia M. Moore, Jennifer A. Seibert; EXECUTIVE ASSISTANT Sylvia S. Kihara; SERVICES EXECUTIVE Linda Rusk; JAPAN SALES Jason Hannaford; SITE LICENSE
or 202-326-6417, FAX 202-842-1065. Mailing addresses: AAAS, P.O. ADMINISTRATIVE SUPPORT Maryrose Police SALES: DIRECTOR Tom Ryan; SALES AND CUSTOMER SERVICE Mehan Dossani,
Box 96178, Washington, DC 20090-6178 or AAAS Member Services, NEWS SENIOR CORRESPONDENT Jean Marx; DEPUTY NEWS EDITORS Robert Kiki Forsythe, Catherine Holland, Wendy Wise; ELECTRONIC MEDIA: MAN-
1200 New York Avenue, NW, Washington, DC 20005 Coontz, Eliot Marshall, Jeffrey Mervis, Leslie Roberts; CONTRIBUTING AGER Lizabeth Harman; PROJECT MANAGER Trista Snyder; ASSISTANT
EDITORS Elizabeth Culotta, Polly Shulman; NEWS WRITERS Yudhijit MANAGER Lisa Stanford PRODUCTION ASSOCIATES Nichele Johnston,
I NSTITUTIONAL S ITE L ICENSES please call 202-326-6755 for any
Bhattacharjee, Adrian Cho, Jennifer Couzin, David Grimm, Constance Kimberly Oster
questions or information
Holden, Jocelyn Kaiser, Richard A. Kerr, Eli Kintisch, Andrew Lawler (New
REPRINTS: Author Inquiries 800-635-7181 England), Greg Miller, Elizabeth Pennisi, Robert F. Service (Pacific NW), ADVERTISING DIRECTOR WORLDWIDE AD SALES Bill Moran
Commercial Inquiries 803-359-4578 Erik Stokstad; John Simpson (intern); CONTRIBUTING CORRESPONDENTS Barry PRODUCT ([email protected]); MIDWEST Rick Bongiovanni:
Corrections 202-326-6501 A. Cipra, Jon Cohen (San Diego, CA), Daniel Ferber, Ann Gibbons, 330-405-7080, FAX 330-405-7081 • WEST COAST/W. CANADA Teola Young:
PERMISSIONS 202-326-7074, FAX 202-682-0816 Robert Irion, Mitch Leslie, Charles C. Mann, Evelyn Strauss, Gary Taubes, 650-964-2266 EAST COAST/E. CANADA Christopher Breslin: 443-512-
MEMBER BENEFITS Bookstore: AAAS/BarnesandNoble.com bookstore Ingrid Wickelgren; COPY EDITORS Linda B. Felaco, Rachel Curran, Sean 0330, FAX 443-512-0331 • UK/EUROPE/ASIA Julie Skeet: +44 (0) 1223-
www.aaas.org/bn; Car purchase discount: Subaru VIP Program Richardson; ADMINISTRATIVE SUPPORT Scherraine Mack, Fannie Groom; 326-524, FAX +44 (0) 1223-325-532 JAPAN Mashy Yoshikawa: +81 (0)
202-326-6417; Credit Card: MBNA 800-847-7378; Car Rentals: BUREAUS Berkeley, CA: 510-652-0302, FAX 510-652-1867, New 33235 5961, FAX +81 (0) 33235 5852 TRAFFIC MANAGER Carol Maddox;
Hertz 800-654-2200 CDP#343457, Dollar 800-800-4000 #AA1115; England: 207-549-7755, San Diego, CA: 760-942-3252, FAX 760- SALES COORDINATOR Deiandra Simms
AAAS Travels: Betchart Expeditions 800-252-4910; Life Insurance: 942-4979, Pacific Northwest: 503-963-1940 COMMERCIAL EDITOR Sean Sanders: 202-326-6430
Seabury & Smith 800-424-9883; Other Benefits: AAAS Member Services PRODUCTION DIRECTOR James Landry; SENIOR MANAGER Wendy K. Shank;
ASSISTANT MANAGER Rebecca Doshi; SENIOR SPECIALISTS Jay Covert, Chris
CLASSIFIED ([email protected]); U.S.: RECRUITMENT SALES MAN-
202-326-6417 or www.aaasmember.org. AGER Ian King: 202-326-6528, FAX 202-289-6742; U.S./INDUSTRY:
Redwood; SPECIALIST Steve Forrester; PREFLIGHT DIRECTOR David M.
[email protected] (for general editorial queries) Tompkins; MANAGER Marcus Spiegler; SPECIALIST Jessie Mudjitaba Darrell Bryant: 202-326-6533; MIDWEST/CANADA: Daryl Anderson: 202-
[email protected] (for queries about letters) 326-6543; NORTHEAST: Allison Millar: 202-326-6572; SOUTHEAST:
ART DIRECTOR Kelly Buckheit Krause; ASSOCIATE ART DIRECTOR Aaron
[email protected] (for returning manuscript reviews) Fernando Junco: 202-326-6740; WEST: Katie Putney: 202-326-6577;
Morales; ILLUSTRATORS Chris Bickel, Katharine Sutliff; SENIOR ART ASSOCIATES
SALES COORDINATORS Erika Bryant, Rohan Edmonson, Shirley Young;
[email protected] (for book review queries) Holly Bishop, Laura Creveling, Preston Huey; ASSOCIATES Nayomi
INTERNATIONAL: SALES MANAGER Tracy Holmes: +44 (0) 1223 326525, FAX
Published by the American Association for the Advancement of Science Kevitiyagala, Jessica Newfield; PHOTO EDITOR Leslie Blizard
+44 (0) 1223 326532; SALES Christina Harrison, Svitlana Barnes; SALES
(AAAS), Science serves its readers as a forum for the presentation and S CIENCE I NTERNATIONAL ASSISTANT Louise Moore; JAPAN: Jason Hannaford: +81 (0) 52 757 5360,
discussion of important issues related to the advancement of science, EUROPE ([email protected]) EDITORIAL: INTERNATIONAL MANAGING FAX +81 (0) 52 757 5361; ADVERTISING PRODUCTION OPERATIONS MANAGER
including the presentation of minority or conflicting points of view, EDITOR Andrew M. Sugden; SENIOR EDITOR/PERSPECTIVES Julia Fahrenkamp- Deborah Tompkins; ASSOCIATES Christine Hall, Amy Hardcastle; PUBLICA-
rather than by publishing only material on which a consensus has been Uppenbrink; SENIOR EDITORS Caroline Ash (Geneva: +41 (0) 222 346 TIONS ASSISTANTS Robert Buck, Mary Lagnaoui
reached. Accordingly, all articles published in Science—including 3106), Stella M. Hurtley, Ian S. Osborne, Stephen J. Simpson, Peter
editorials, news and comment, and book reviews—are signed and reflect AAAS BOARD OF DIRECTORS RETIRING PRESIDENT, CHAIR John P. Holdren;
Stern; ASSOCIATE EDITOR Joanne Baker EDITORIAL SUPPORT Alice Whaley; PRESIDENT David Baltimore; PRESIDENT-ELECT James J. McCarthy; TREASURER
the individual views of the authors and not official points of view adopted Deborah Dennison ADMINISTRATIVE SUPPORT Janet Clements, Jill White;
by the AAAS or the institutions with which the authors are affiliated. David E. Shaw; CHIEF EXECUTIVE OFFICER Alan I. Leshner; BOARD John E.
NEWS: EUROPE NEWS EDITOR John Travis; DEPUTY NEWS EDITOR Daniel Clery;
Dowling, Lynn W. Enquist, Susan M. Fitzpatrick, Alice Gast, Linda P. B.
AAAS was founded in 1848 and incorporated in 1874. Its mission is to CORRESPONDENT Gretchen Vogel (Berlin: +49 (0) 30 2809 3902, FAX
advance science and innovation throughout the world for the benefit Katehi, Cherry A. Murray, Thomas D. Pollard, Kathryn D. Sullivan
+49 (0) 30 2809 8365); CONTRIBUTING CORRESPONDENTS Michael Balter
of all people. The goals of the association are to: foster communication (Paris), Martin Enserink (Amsterdam and Paris), John Bohannon
among scientists, engineers and the public; enhance international (Vienna); INTERN Krista Zala
cooperation in science and its applications; promote the responsible ASIA Japan Office: Asca Corporation, Eiko Ishioka, Fusako Tamura, 1-8-
conduct and use of science and technology; foster education in science
and technology for everyone; enhance the science and technology 13, Hirano-cho, Chuo-ku, Osaka-shi, Osaka, 541-0046 Japan; +81 (0) 6
workforce and infrastructure; increase public understanding and 6202 6272, FAX +81 (0) 6 6202 6271; [email protected]; ASIA NEWS
appreciation of science and technology; and strengthen support for EDITOR Richard Stone +66 2 662 5818 ([email protected]); JAPAN NEWS
the science and technology enterprise. BUREAU Dennis Normile (contributing correspondent, +81 (0) 3 3391
0630, FAX 81 (0) 3 5936 3531; [email protected]); CHINA REP-
RESENTATIVE Hao Xin, + 86 (0) 10 6307 4439 or 6307 3676, FAX +86 (0)
I NFORMATION FOR A UTHORS 10 6307 4358; [email protected]; SOUTH ASIA Pallava Bagla (con-
See pages 120 and 121 of the 5 January 2007 issue or access tributing correspondent +91 (0) 11 2271 2896; [email protected])
www.sciencemag.org/feature/contribinfo/home.shtml AFRICA Robert Koenig (contributing correspondent, [email protected])
S ENIOR E DITORIAL B OARD J. M. Claverie, CNRS, Marseille Steven Jacobsen, Univ. of California, Los Angeles J. Roy Sambles, Univ. of Exeter
Jonathan D. Cohen, Princeton Univ. Peter Jonas, Universität Freiburg Jürgen Sandkühler, Medical Univ. of Vienna
John I. Brauman, Chair, Stanford Univ. Stephen M. Cohen, EMBL Daniel Kahne, Harvard Univ. David S. Schimel, National Center for Atmospheric Research
Richard Losick, Harvard Univ. Robert H. Crabtree, Yale Univ. Bernhard Keimer, Max Planck Inst., Stuttgart Georg Schulz, Albert-Ludwigs-Universität
Robert May, Univ. of Oxford F. Fleming Crim, Univ. of Wisconsin Elizabeth A. Kellog, Univ. of Missouri, St. Louis Paul Schulze-Lefert, Max Planck Inst., Cologne
Marcia McNutt, Monterey Bay Aquarium Research Inst. William Cumberland, UCLA Alan B. Krueger, Princeton Univ. Terrence J. Sejnowski, The Salk Institute
Linda Partridge, Univ. College London George Q. Daley, Children’s Hospital, Boston Lee Kump, Penn State David Sibley, Washington Univ.
Vera C. Rubin, Carnegie Institution of Washington Edward DeLong, MIT Mitchell A. Lazar, Univ. of Pennsylvania Montgomery Slatkin, Univ. of California, Berkeley
Christopher R. Somerville, Carnegie Institution Emmanouil T. Dermitzakis, Wellcome Trust Sanger Inst. Virginia Lee, Univ. of Pennsylvania George Somero, Stanford Univ.
George M. Whitesides, Harvard University Robert Desimone, MIT Anthony J. Leggett, Univ. of Illinois, Urbana-Champaign Joan Steitz, Yale Univ.
Dennis Discher, Univ. of Pennsylvania Michael J. Lenardo, NIAID, NIH Elsbeth Stern, ETH Zürich
W. Ford Doolittle, Dalhousie Univ. Norman L. Letvin, Beth Israel Deaconess Medical Center Thomas Stocker, Univ. of Bern
B OARD OF R EVIEWING E DITORS Jennifer A. Doudna, Univ. of California, Berkeley Olle Lindvall, Univ. Hospital, Lund Jerome Strauss, Virginia Commonwealth Univ.
Julian Downward, Cancer Research UK Richard Losick, Harvard Univ. Marc Tatar, Brown Univ.
Joanna Aizenberg, Bell Labs/Lucent Denis Duboule, Univ. of Geneva Ke Lu, Chinese Acad. of Sciences Glenn Telling, Univ. of Kentucky
R. McNeill Alexander, Leeds Univ. Christopher Dye, WHO Andrew P. MacKenzie, Univ. of St. Andrews Marc Tessier-Lavigne, Genentech
David Altshuler, Broad Institute Richard Ellis, Cal Tech Raul Madariaga, École Normale Supérieure, Paris Michiel van der Klis, Astronomical Inst. of Amsterdam
Arturo Alvarez-Buylla, Univ. of California, San Francisco Gerhard Ertl, Fritz-Haber-Institut, Berlin Anne Magurran, Univ. of St. Andrews Derek van der Kooy, Univ. of Toronto
Richard Amasino, Univ. of Wisconsin, Madison Douglas H. Erwin, Smithsonian Institution Michael Malim, King’s College, London
Meinrat O. Andreae, Max Planck Inst., Mainz Virginia Miller, Washington Univ. Bert Vogelstein, Johns Hopkins
Barry Everitt, Univ. of Cambridge Christopher A. Walsh, Harvard Medical School
Kristi S. Anseth, Univ. of Colorado Paul G. Falkowski, Rutgers Univ. Yasushi Miyashita, Univ. of Tokyo
John A. Bargh, Yale Univ. Ernst Fehr, Univ. of Zurich Richard Morris, Univ. of Edinburgh Graham Warren, Yale Univ. School of Med.
Cornelia I. Bargmann, Rockefeller Univ. Tom Fenchel, Univ. of Copenhagen Edvard Moser, Norwegian Univ. of Science and Technology Colin Watts, Univ. of Dundee
Brenda Bass, Univ. of Utah Alain Fischer, INSERM Andrew Murray, Harvard Univ. Julia R. Weertman, Northwestern Univ.
Marisa Bartolomei, Univ. of Penn. School of Med. Jeffrey S. Flier, Harvard Medical School Naoto Nagaosa, Univ. of Tokyo Jonathan Weissman, Univ. of California, San Francisco
Ray H. Baughman, Univ. of Texas, Dallas Chris D. Frith, Univ. College London James Nelson, Stanford Univ. School of Med. Ellen D. Williams, Univ. of Maryland
Stephen J. Benkovic, Pennsylvania St. Univ. John Gearhart, Johns Hopkins Univ. Roeland Nolte, Univ. of Nijmegen R. Sanders Williams, Duke University
Michael J. Bevan, Univ. of Washington Wulfram Gerstner, Swiss Fed. Inst. of Technology Helga Nowotny, European Research Advisory Board Ian A. Wilson, The Scripps Res. Inst.
Ton Bisseling, Wageningen Univ. Charles Godfray, Univ. of Oxford Eric N. Olson, Univ. of Texas, SW Jerry Workman, Stowers Inst. for Medical Research
Mina Bissell, Lawrence Berkeley National Lab Jennifer M. Graves, Australian National Univ. Erin O’Shea, Harvard Univ. John R. Yates III, The Scripps Res. Inst.
Peer Bork, EMBL Christian Haass, Ludwig Maximilians Univ. Elinor Ostrom, Indiana Univ.
Dianna Bowles, Univ. of York Dennis L. Hartmann, Univ. of Washington Jonathan T. Overpeck, Univ. of Arizona Martin Zatz, NIMH, NIH
Robert W. Boyd, Univ. of Rochester Chris Hawkesworth, Univ. of Bristol John Pendry, Imperial College Huda Zoghbi, Baylor College of Medicine
Dennis Bray, Univ. of Cambridge Martin Heimann, Max Planck Inst., Jena Philippe Poulin, CNRS Maria Zuber, MIT
Stephen Buratowski, Harvard Medical School James A. Hendler, Univ. of Maryland Mary Power, Univ. of California, Berkeley
Jillian M. Buriak, Univ. of Alberta Ray Hilborn, Univ. of Washington Molly Przeworski, Univ. of Chicago B OOK R EVIEW B OARD
Joseph A. Burns, Cornell Univ. Ove Hoegh-Guldberg, Univ. of Queensland David J. Read, Univ. of Sheffield
William P. Butz, Population Reference Bureau Ary A. Hoffmann, La Trobe Univ. Les Real, Emory Univ. John Aldrich, Duke Univ.
Peter Carmeliet, Univ. of Leuven, VIB Ronald R. Hoy, Cornell Univ. Colin Renfrew, Univ. of Cambridge David Bloom, Harvard Univ.
Gerbrand Ceder, MIT Evelyn L. Hu, Univ. of California, SB Trevor Robbins, Univ. of Cambridge Angela Creager, Princeton Univ.
Mildred Cho, Stanford Univ. Olli Ikkala, Helsinki Univ. of Technology Barbara A. Romanowicz, Univ. of California, Berkeley Richard Shweder, Univ. of Chicago
David Clapham, Children’s Hospital, Boston Meyer B. Jackson, Univ. of Wisconsin Med. School Nancy Ross, Virginia Tech Ed Wasserman, DuPont
David Clary, Oxford University Stephen Jackson, Univ. of Cambridge Edward M. Rubin, Lawrence Berkeley National Lab Lewis Wolpert, Univ. College, London
24 6 APRIL 2007 VOL 316 SCIENCE www.sciencemag.org

New! BigDye®
XTerminatorTM Kit.
Game over blobs.
Find out more @
appliedbiosystems.com/
gameoverblobs
25 years of delivering next-generation systems
Accelerate your time to publish

Human disease researchers bridge diverse research areas to advance the medical field, moving forward in small
but steady strides.Your road requires perseverance and demands commitment. Over the past 25 years, Applied
Biosystems has helped to pave the way with cutting-edge technology for genetic analysis. AB offers reagents,
instruments, and software for applications including DNA sequencing, fragment analysis, and genotyping—
accelerating scientific research.
To find out more, visit: info.appliedbiosystems.com/hdr
For Research Use Only. Not for use in diagnostic procedures. This instrument is authorized for use in DNA sequencing and fragment analysis under process claims of U.S. Patent Nos. 5,821,058 and 5,332,666 and under all process
claims for DNA sequence and fragment analysis of U.S. and foreign counterpart patents owned or licensable by Applied Biosystems. The Applied Biosystems 3130/3130xl Genetic Analyzers include patented technology licensed from
Hitachi, Ltd., as part of a strategic partnership between Applied Biosystems and Hitachi. Ltd., as well as patented technology of Applied Biosystems. Applied Biosystems and AB (Design) are registered trademarks and Applera is a
trademark of the Applera Corporation or its subsidiaries in the US and/or certain other countries. ©2007 Applied Biosystems. All rights reserved.
t!
ru w
en
st Ne
More Flexible LCM

m
in
Molecular Devices introduces ArcturusXT™, the only open, modular microdissection instrument to
combine Laser Capture Microdissection (LCM) with UV laser cutting. ArcturusXT is the ideal solution
for researchers who require more flexibility and system ease of use.
Unique IR-enabled LCM—never lose contact with the sample!

Nikon® TE2000U base—research-grade microscope with high-quality optics
Fully upgradable and extendable platform
Optional UV Laser Cutting and Epi-Fluorescence
Phase contrast and DIC options—ideal for live-cell applications
Ergonomic touch-screen monitor and trackball-controlled stage
Full range of objective offerings—2x to 100x
Compatible with glass or membrane slides
By combining gentle LCM and rapid UV laser cutting, you have more choices in the isolation of pure
cell populations to fit any research application. The new ArcturusXT system, along with our Arcturus®
microgenomics reagents and GenePix® microarray scanners and software, provide a more complete
solution for microarray research. Visit www.moleculardevices.com/LCM for more information.
Expect more. We’ll do our very best to exceed your expectations.
See us in Montréal at the Society for Biomolecular Sciences 13th Annual Conference & Exhibition,
April 15–19, Booth #401 and in Los Angeles at the American Association for Cancer Research Annual
Meeting, April 14–18, Booth #1856.
tel. +1-800-635-5577 | www.moleculardevices.com
LIFE SCIENCE TECHNOLOGIES

CELL SIGNALING
IN THIS ISSUE:
P
hosphorylation reactions catalyzed
by cellular kinases are ubiquitous in
signaling cascades, making them important
players in cell function and dysfunction. Read about
new technologies for detecting, characterizing, and
quantitating kinase activity in the Cell Signaling
feature on page 125 of this issue.
UPCOMING FEATURES:
April 20— Stem Cells
June 1— RNAi
June 22— Cell Signaling 2
Also available at www.sciencemag.org/products/articles.dtl

RANDOMSAMPLES
E D I T E D B Y C O N S TA N C E H O L D E N
Going Under
Two-thirds of all cities with populations exceeding 5 million are “especially vulnera-
ble to risks resulting from climate change,” according to a study from Columbia
University and the International Institute for Environment and Development in
London. A team of geographers defined danger zones as areas within 10 meters
above sea level, the places most vulnerable to weather oscillations combined with
the 25- to 60-cm sea-level rise forecast by 2100. China (see map) is in the lead, with
144 million people, or 11% of its population, at or below the 10-meter level. The
world’s poor are the most imperiled, with some 247 million at risk in least developed
nations. Numbers will climb with continued urbanization, note the authors, who say
Persons per km2
nations should develop policies to encourage inland growth.
Ins and Outs of Carbon the puzzle. In a virtual reconstruction, he fol- Bromage told a meeting of the International
lowed a rule that he says applies to all pri- Association for Dental Research last week in
Some parts of the world pump mates: The angle created by drawing a line New Orleans, Louisiana.
more carbon dioxide into the from the eye socket to the ear and then to the Holloway says he’s sticking to his own esti-
NET atmosphere than they remove, top back molar is mate. “I sincerely doubt that
WATCH whereas other regions are net always 45°. Shifting these fragments can
absorbers. A new site for chart- the skull bones to be so radically recon-
ing the ups and downs of the conform to the rule structed,” he says.
greenhouse gas is CarbonTracker from the pushes out the lower “Maybe with a com-
U.S. National Oceanic and Atmospheric Admin- face and leads to a much puter, but not by a
CREDITS (TOP TO BOTTOM): CENTER FOR INFORMATION EARTH SYSTEMS NETWORK; T. BROMAGE; DENNIS KUNKEL/VISUALS UNLIMITED
istration in Boulder, Colorado. smaller brain: about 575 cubic Before and after. trained anatomist’s
CarbonTracker incorporates CO2 measure- centimeters instead of the 752 found hand.” But paleo-
ments from some 60 locations around the by Columbia University anthropologist neurologist Dean Falk of Florida State University
world to provide a broad picture of carbon Ralph Holloway. That downsizing along in Tallahassee says she thinks Bromage’s method
uptake and release for North America, the with the newly prognathous profile just about for hafting faces onto crania is “really exciting. …
globe, and the oceans between 2000 and edge the skull out of the Homo ballpark, We’re exploring applying it ourselves.”
2005. Visitors can also check out the “carbon
weather” to see how storms alter levels of the
gas. The researchers hope other labs will con-
tribute data that could help make CarbonTracker
A Discriminating Parasite >>
an objective tool for gauging whether carbon Toxoplasma gondii is a parasite that requires two hosts. It’s born in a cat’s intestines, devel-
emission targets are being met. >> ops in another animal—such as a rat—and must return to a cat to reproduce. To boost its
www.esrl.noaa.gov/gmd/ccgg/carbontracker chances of making that return trip, researchers at Oxford University have shown, Toxoplasma
makes rodents less afraid of cats. Now a Stanford University team led by
Ajai Vyas has found that rats carrying the parasite don’t mellow out
New Face for Kenya across the board; they just lose their fear of the smell of cats.
In the lab, infected rats showed much less aversion than normal ones
Hominid? to bobcat urine. But they reacted normally when the researchers probed
Homo rudolfensis, a 1.9-million-year-old skull other types of fear responses. That means Toxoplasma has a “remarkably
from Kenya, may not be a Homo after all, says a specific” behavioral effect, says co-author Robert Sapolsky. He says most
scientist who has done a computer reconstruc- parasites control behavior in much cruder ways—for example, by
tion of the skull. destroying muscle metabolism so an organism can’t evade a predator. In
The skull fragments—found in 1972 near the 2 April online Proceedings of the National Academy of Sciences, the
Lake Turkana and put together by Richard scientists report that Toxoplasma cysts form preferentially on the rat
Leakey—have sparked much debate, because amygdala, which Sapolsky calls “ground zero” for fear in the brain.
their owner seemed to have had a much larger “I always found it incredible that the parasite would be able to alter a response, cat aver-
brain than other hominids of similar age. sion, that is so ingrained in the rat’s psyche,” says Oxford veterinary scientist Manuel Berdoy,
Now Timothy Bromage, a paleoanthropolo- an author of the earlier work. He says the new research shows that the parasite may have the
gist and expert on facial bone development at “astonishing” ability to zero in on the neural pathways for processing cat odors.
New York University, claims to have sorted out

Complimentary Pre-Conference Workshop on: WilBio's Featuring Presentations from:
Conferences are Alois Jungbauer, Ph.D. -
Raising Money for consistently rated Austrian Center of Biopharmaceutical Technology
the best for: Joy Adiletta - Amgen
Antibody Development Oscar Salas-Solano, Ph.D. - Genentech, Inc.
Funding Each Stage of the Scale-Up Process Content
Robert DuBridge, Ph.D. - PDL BioPharma, Inc.
Networking
Chaired by Patrick Huddie, Ph.D. Format Jonida Basha - Merck & Co., Inc.
from Evergreen Capital, LLC Cost Dorothee Ambrosius -
Boehringer-Ingelheim Pharma GmbH & Co.
Sunday, April 22 from 4:00-6:30 PM Food
Entertainment Jeanne Novak, Ph.D. - CBR International Corp.
Accommodations Gerald Carson - Abbott Laboratories
The 12th Annual
Stephen Notarnicola, Ph.D. - Biogen Idec MA, Inc.
Waterside Conference Richard Root, M.Sc. - Roche Diagnostics Corp.
Scale-Up and Production of Tongtong Wang, Ph.D. - Eli Lilly & Company
Recombinant and Monoclonal Antibodies Shujun Sun - Wyeth Biotech
The Most Trusted
Missag Parseghian, Ph.D. - Peregrine Pharmaceuticals
April 23-25, 2007 • San Juan, Puerto Rico Source of BioProcessing
Technology NingNing Ma, Ph.D. - Pfizer, Inc.
Topics Include: P.O. Box 1229 Shue-Yuan Wang - Abbott Laboratories
Virginia Beach, VA 23451 USA
• Purification • Cell Culture Scale-Up 757.423.8823 Peter Gagnon - Validated Biosystems, Inc.
Fax 757.423.2065
• Assay Development • Product -Single Characterization Xing Wang, Ph.D. - Pfizer, Inc.
www.wilbio.com
• Single-Use Components • Regulatory Issues [email protected] Cori Gorman, Ph.D. - Selexis USA
NEWSMAKERS
EDITED BY YUDHIJIT BHATTACHARJEE
IT’S NOT GARBAGE. Removing waste from waste-

AWARDS water is great; milking it for energy is even better.
This month, the University of Southern California
awarded its Tyler Prize to Gatze Lettinga, a professor emeritus
of environmental technology at Wageningen University in the
Netherlands, for combining those two good ideas into a technology
called the upflow anaerobic sludge blanket.
The process uses microbes to digest pollutants in domestic
sewage and industrial effluents and turn them into fuel. With help
from the Dutch government, Lettinga built reactors in Colombia,
India, Brazil, and other countries. By waiving any patent rights, he
paved the way for refinements such as an expanded granular
sludge bed. The award comes with a $200,000 prize.
MOVERS 14 June 2002, p. 1957). Fraser-Liggett is them with the wherewithal to really build
PARTING WAYS. Claire Fraser-Liggett is leav- now weighing an appointment at a major something here.”
ing the DNA research institute founded by her academic medical center that will link her lab The new, 10-year Polaris Investigator Awards
former husband, J. Craig Venter, after running research more directly to clinical work, and are for a total of three faculty spots at the uni-
it for nearly a decade. the speculation is that she and several TIGR versities of Alberta, Calgary, and Lethbridge,
Fraser-Liggett’s decision to step down as staffers are being recruited by the University which are sharing the cost of the awards in a
president of The Institute for Genomic of Maryland. quest to make a bigger mark on the biomedical
Research (TIGR) in research frontier. The recipient “creates critical
Rockville, Maryland, M O N E Y M ATT E R S mass,” says University of Alberta President
comes 5 months after HELP WANTED. The province of Alberta, Indira Samarasekera, “and becomes a global
a board chaired by Canada, is offering $17 million packages for magnet for talent in that particular field.”
Venter stripped TIGR a few rising stars in biomedical research who The awards are an offshoot of an economic
of its independent like the idea of making their name on the boom in the province fueled by spiraling oil
status and made it a Canadian plains. “We’re looking to attract prices. And the foundation has sweetened the
division of the J. Craig people who could be really big players 5 to deal by excusing the winners from any admin-
Venter Institute (JCVI), 10 years down the road,” says Kevin Keough, istrative duties for the first 5 years of their
also located in president of the Alberta Heritage Foundation contracts. Applicants should contact one of
Rockville. A JCVI for Medical Research. “The idea is to provide the universities. The West is calling.
spokesperson says,
“We will be making
some announcements in the very near future
about additional changes.” Three Q’s >>
Fraser-Liggett has led a team of pioneer-
ing microbial DNA scientists at TIGR since Thailand’s new science minister, Yongyuth Yuthavong, brings a deep
Venter launched it in 1992 with proceeds understanding of science to a post traditionally headed by bureaucrats.
from a DNA-sequencing deal (Science, A biochemist who along with colleagues deciphered the structure of a
key enzyme of the malaria parasite, Yongyuth, 62, is lobbying the
CREDITS (TOP TO BOTTOM): USC; CLAIRE FRASER-LIGGETT; MUTSUMI STONE
National Assembly to approve a several-fold increase in R&D spend-
They Said It ing over the next 3 years. Science caught up with him recently.
Q: What is the motivation for your draft science law?

“None of us is running for We want a system for science policy development, with the sci-
president, so I think we can get ence minister as the chief scientific adviser to the government.
away with plagiarism.” The science ministry has always been a “grade C” ministry. I want
—Representative Vernon Ehlers (R–MI), at a it to be “grade A.”
28 March meeting of the House Science and
Technology Committee, after committee chair Q: How will the legislation help rank-and-file scientists?
Bart Gordon (D–TN) confessed to “plagiariz- The law designates a level of support for R&D—not less than 3% of the budget. Even if
ing” the recommendations of a 2005 National
Academies report in a bill to improve math
I get 2%, it will be three or four times the present level. [But] we’ll have to lobby.
and science education (H.R. 362) that the
committee then passed unanimously. Q: What are your chances of success?
My first name means “keep on fighting,” and my surname means “fighting family.” And
[he laughs] I have friends on the assembly’s science and technology committee.
Got a tip for this page? E-mail [email protected]

NEWS>>
THIS WEEK Ultrafast getaway
captured
Hobbit’s
archaic wrist
33 34
MUSEUM MANAGEMENT The U.S. Congress foots about 70% of the
Smithsonian’s bills, but increases in this
Turnover at the Top, but Problems federal allocation have not kept up with costs,
in particular the demand for finishing new
museums and repairing old ones. Small
Persist at the Smithsonian helped bring in a lot of private money—about
$1 billion during his term—for this quasi-
When Lawrence Small abruptly federal institution. But most of it was not for
resigned as secretary of the WHO’S IN WHO’S OUT science. Scientists have looked elsewhere for
Smithsonian Institution last SMITHSONIAN SECRETARY research support, with mixed success.
week, you could almost hear the The harsh reality is that money is still
staff ’s collective sigh of relief. tight, and the Smithsonian is groaning under
Although Small shored up the the weight of its obligations. “Our biggest
Smithsonian’s sagging finances need is still facilities,” says Roger Sant,
during his 7-year term, his depar- chair of the board of the Summit Foundation
ture signaled an end to the inter- in Washington, D.C., and a member of the
nal audits, the harsh press cover- Smithsonian’s Board of Regents. “When
age, and congressional outrage your backlog [of obligations] is $2.3 billion,
over high executive salaries and it’s hard to say anything is going to get a
exorbitant personal expenses— greater amount of attention.”
such as first-class tickets for a UNDERSECRETARY The bricks-and-mortar problem dates
Hawaiian vacation. back to the 1980s when then–Smithsonian
The turnover also hinted at Secretary Sidney Dillon Ripley built eight
better times for the Smithson- museums and set up seven new research pro-
ian’s 500 researchers in locations grams, few of which were fully funded by
from Panama to Massachusetts. Congress. When Small came on board, the
Many think science didn’t fully Smithsonian’s finances were in a shambles,
benefit from Small’s fundrais- and construction projects were underfunded.
ing, which focused on “bricks “The place really did need fixing,” says Sant.
and mortar” improvements. They In addition to raising money, Small, a well-
are encouraged that scientists connected banker, got the National Museum
have been made interim leaders. Yellow ties take charge. At the Smithsonian, Christián of the American Indian and a new branch of
The Board of Regents, which Samper (top, left) has replaced Secretary Lawrence Small the National Air and Space Museum up and
oversees the Smithsonian’s activi- (top, right); Ira Rubinoff (lower, left) has stepped in for running; he also began repairing bad heating
ties, picked the 41-year-old direc- David Evans as undersecretary for science. systems and solving other infrastructure
tor of the Smithsonian National problems. But “he seemed to lose sight of the
Museum of Natural History, biol- important research role of the institution,”
ogist Christián Samper, as acting secretary. lot of attention,” Samper said in an interview. says Peter Raven, head of the Missouri
This move is fueling hopes that Samper, or “I want to strengthen the programmatic Botanical Garden in St. Louis.
someone with a research background, might side—the scholarship and science.” Rubinoff Belt-tightening measures did away with an
take charge long term. In another big change, says his goal is to get “more balance” among internal grants program and research fellow- CREDITS: PHOTOS COURTESY OF THE SMITHSONIAN INSTITUTION
David Evans, who oversaw Smithsonian sci- the institution’s priorities, suggesting a closer ships. An ever-larger percentage of congres-
ence for 4 years under Small, also resigned look at research objectives and not a single- sional funding—which remained flat—had to
last week. Ira Rubinoff, director of the Smith- minded emphasis on refurbishing museums. cover mandatory expenses, such as salaries
sonian Tropical Research Institute in Among the staff, “there were a lot of smiling and shortfalls in the infrastructure budget. At
Panama, has stepped in as his temporary faces this week,” says William Fitzhugh, a the Smithsonian Astrophysical Observatory
replacement. Paul Risser, a botanist and chair Smithsonian archaeobiologist. (SAO), based in Cambridge, Massachusetts,
of the University of Oklahoma Research 2-year delays affected both a new spectro-
Cabinet, will be the new acting director of the Unfinished business graph and a new infrared camera for the
natural history museum. Although the new leaders may be more in Multiple Mirror Telescope—both deemed key
Although it’s too soon to tell what this will tune with research, it will be difficult for improvements by the scientific community.
mean for the institution’s programs, the new them—or anyone—to launch programs while Early in his tenure, Small angered sci-
leaders are speaking in a way that’s bound to maintaining the Smithsonian’s sprawling con- entists when he called for a reorganization
please scientists. In the past, “the whole issue glomeration of 19 museums and galleries, the that would have separated the exhibits
of infrastructure and facilities has received a National Zoo, and nine research facilities. from the research programs and closed a
▲

FOCUS Singapore’s
rapid rise
Tracking cocaine
through cash
38 42
conservation research center in Front Administration and other agencies. But these Smithsonian paleontologist Douglas Erwin:
Royal, Virginia, and a materials research sources could dry up. It’s becoming increas- “There are some things you can [easily] raise
lab in Suitland, Maryland (Science, 13 July ingly difficult to maintain the long-term stud- money for … exhibits and flashy research, but
2001, p. 194). The fuss prompted the ies so crucial to distinguishing climate change not for the preservative in jars of fish.” He and
Board of Regents to appoint an 18-member from normal variation in the environment, others think a scientist, or at least a scholar of
commission that in 2003 presented Small notes SERC Director Anson Hines. some sort, needs to be in charge.
and Evans with almost 100 recommenda- Researchers say what the Smithsonian But Board of Regents members are wary.
tions for changes. Since then, “the Smith- really needs is a spokesperson who will lobby “In the best of all worlds, you want a great
sonian has made a huge amount of Congress and the White House more strongly. scholar,” says philanthropist Eli Broad. “But
progress,” says Jeremy Sabloff, the Uni- “We must articulate very well why our science you want someone also [who] can rally the
versity of Pennsylvania anthropologist is important,” says Samper. It’s not enough to troops and can get the resources over and
who chaired that commission. win backing for individual projects; the above what the government provides. It’s a
The threatened research centers survived research enterprise needs a champion, says tough job.” –ELIZABETH PENNISI
and appear to be on firm ground. There is
now money for fellowships and new blood
in charge at the zoo, the natural history HIGH-ENERGY PHYSICS
museum, the Smithsonian Environmental
Research Center (SERC) in Edgewater,
Maryland, and the SAO.
Design Flaw Could Delay Collider
Samper has turned the natural history A magnet for the Large Hadron Collider the tunnel without having to bring [the mag-
museum around since he took over in 2003, (LHC) failed during a key test at the Euro- nets] up to the surface,” says CERN’s Lyndon
hiring young curators to replace about a pean particle physics laboratory CERN last Evans, who leads the construction of the
dozen retirement-age staff members who had week. Physicists and engineers will have to accelerator. Only the damaged magnet will
stayed in place to help out their departments. repair the damaged magnet and retrof it have to come out of the tunnel, he says.
Botany, for example, brought in new people others to correct the underlying design flaw, The faulty magnets are designed to
for the first time since the early 1990s. which could delay the start-up of the mam- focus the LHC’s beams of protons just
The natural history museum has received moth subterranean machine near Geneva, before they collide. The beams will run
some $70 million in outside funds in the past Switzerland, from November until the spring through three such quadrupoles on either
4 years, most for exhibits but some for of 2008. That would eliminate a 1-month side of each of f o u r c o l l i s i o n p o i n t s
research. There are now two endowed chairs, “engineering run” with which physicists had spaced around the 27-kilometer ring. The
one in ocean sciences and one in human ori- hoped to shake the bugs out of the machine LHC’s four massive particle detectors will
gins. Furthermore, “we’ve had a great infu- before shutting down for the winter, when sit at the collision points.
sion of attention to the mechanics of doing power becomes prohibitively expensive. Designed and built at Fermi National
good science here now,” says Fitzhugh. Laboratory officials aren’t giving up hope Accelerator Laboratory (Fermilab) in
Nonetheless, problems persist. “A lot of just yet, however. “We are pretty well along Batavia, Illinois, the magnet failed when
the scientists, like myself, think we have a on finding a fix that can be implemented in
▲
Continued on page 34
long way to go,” says Warren Wagner, a
botanist at the natural history museum. Small
did not push for a major research initiative
during his tenure; it’s been more than a decade
since the Smithsonian budget included one.
SAO, for example, is looking for $60 million
as its contribution to the Giant Magellan Tele-
scope but has yet to even get the request on the Quick fix. Researchers must
funding wish list the Smithsonian sends to the modify focusing magnets like
White House. The one science initiative in this one in place to keep the
many years to become part of the institution’s project on schedule.
budget proposal—for a global environmental
observatory focused on forests in 2008—was
nixed last year by the White House Office of
Management and Budget.
CREDIT: FERMILAB
SERC has made up for a decline in direct

support from the Smithsonian’s federal budget
over the past 10 years by seeking grants from
the National Oceanic and Atmospheric

Predicting the Future of
mRNA Capping
mScript ™
m
boost you RNA will
protein ex in vivo
r
pression
EPICENTRE’s mScript™ mRNA Production

mScript™ mRNA provides better in vivo translation
System combines the ScriptCap™ Capping
enzymes with our transcription and poly(A)
tailing technology to provide an optimized
solution for mRNA production.
• 100% capped mRNA transcripts—
versus 40% to 80% effective capping in co-
transcriptional cap analog systems.
• Cap 1 structure—
Cap 1 boosts in vivo translation by at least
20 to 50%.
• Saves time and money—

Superior mRNAs and higher yields all in less
time and at a lower cost.
P-702MS-A
mScript™ mRNA produces more Luciferase activity than co-transcriptionally capped

mRNA. Equal amounts of mScript, standard cap (Std CA), and ARCA capped and tailed
To save a fortune on your mRNA, mRNA were transfected into various mammalian cell lines. In vivo protein expression was
go to www.EpiBio.com measured by determining the RLU per μg of total protein 24 hours post-transfection.
and enter code: MSA14
SCIENCESCOPE
PHYSICS
Going Against the Flow
Attosecond Laser Pulses Illuminate Notwithstanding the laws of gravity, construc-
tion money this year at the National Science
Fleeting Dance of Electrons Foundation (NSF) is flowing from the bottom
of the ocean to the top of a 5-km mountain.
Like a prisoner trapped behind the wall of a wave then took them the rest of the way. NSF has shifted $15 million from the budgets
fortress, an electron faces a huge barrier in When the laser’s oscillating electric field of its fledgling oceans and ecological observa-
escaping the confines of an atom. Yet when reached its peak, it suppressed the atom’s tories networks to the Atacama Large Milli-
hit by a burst of intense light, it can set itself binding potential—in effect, thinning the meter Array in the Chilean Andes, in tune with
free in just a few hundred ALMA’s rising costs and the agency’s contin-
attoseconds (10–18 s), thanks ued tinkering with the two networks.
to a quantum-mechanical The changes have touched a nerve in
phenomenon known as tun- NSF’s oversight body, the National Science
neling. In essence, it seeps Board. Speaking up at last week’s board
through the bar rier—the meeting, several members said that the long
binding energy that normally time between approval and the start of a proj-
holds it in place. Now, for the ect has left them feeling out of the loop.
first time, scientists have seen “We’re just asking NSF to explain how things
this blindingly fast escape act have changed and whether the science still
happen in real time. justifies that level of support,” says Mark
This week in Nature, Ferenc Abbott of Oregon State University in Corvallis,
Krausz of the Max Planck noting that NSF now plans to spend $20 mil-
Institute of Quantum Optics lion less during the first 2 years of the ocean
in Garching, Germany, along observatories initiative than when the board
with researchers in Austria gave it the green light in 2002, for example,
and the Netherlands, reports whereas ALMA is costing $125 million more
watching electrons in neon than originally planned. The board has asked
atoms burrowing their way to Looking in. In the Garching experiments, atoms in the cylindrical NSF Director Arden Bement for more frequent
freedom. The team says the chamber were blasted with attosecond pulses and laser waves. updates on the $240-million-a-year account
findings—made possible by and better estimates of the lifetime costs of
the use of 250-attosecond pulses of ultra- wall holding the electron in. At precisely operating each facility. –JEFFREY MERVIS
violet (UV) radiation—confirm theoretical those points in the laser’s oscillation cycle,
predictions about the tunneling process. which lasted several hundred attoseconds, Pathology Institute Gets Lifeline
The researchers also report using tunnel- the researchers saw a marked increase in the Congressional supporters of the U.S. Armed
ing itself to image the acrobatics of electrons number of ionized atoms in the chamber as Forces Institute of Pathology (AFIP), which the
jumping from one orbital to another in neon the outer electrons tunneled their way Defense Department is planning to “de-
and xenon atoms that have been excited by through the lowered binding potential. establish,” are making a last-ditch attempt to
light. The work shows how “the powerful In other experiments, the researchers salvage its functions. Last week, the Senate
tools of attosecond science” can be used to used tunneling to probe the intra-atomic voted to delay the move until after the depart-
understand atomic-level phenomena, says dynamics of neon and xenon atoms. In the ment has responded to a pending report on the
Paul Corkum, a physicist at the Steacie Insti- xenon study, they blasted atoms with an impact of AFIP’s closing. Last month, the House
tute for Molecular Sciences in Ottawa, attosecond pulse powerful enough to knock voted to prevent the use of federal funds for
Canada, who did not take part in the work. an electron out of the element’s innermost the planned closing of Walter Reed Army Med-
To produce attosecond UV pulses, shell, causing electrons in the outer shells to ical Center, where AFIP resides, in its version of
researchers bombard a cloud of neon atoms rearrange themselves in an adjustment the bill, which funds military operations in Iraq
with a short burst of laser light that known as Auger decay. By targeting the and Afghanistan.
wrenches an electron out from deep inside atoms with the laser wave and noting how “This gives AFIP some breathing room,”
the atom and smashes it back toward the number of ions created by tunneling says a Senate staffer about legislation that
the atomic core. The most energetic pho- changed over time, the team was able to President George W. Bush has promised to veto
tons emitted in this process are f iltered trace the details of the Auger decay. because of the inclusion of nonmilitary items.
out t o yield a UV burst lasting a few Researchers say the ability to control Pathology groups oppose the dispersal of
hundred attoseconds. atomic-scale motion of electrons would have AFIP’s functions, particularly the possible
In their experiment, Krausz and his col- numerous applications. “Even simple-seeming mothballing of its renowned tissue repository
SOURCE: WWW.ATTOWORLD.DE
leagues trained an attosecond pulse as well processes such as laser surgery have atto- (Science, 20 May 2005, p. 1101), and Senator
as the laser wave used to generate it toward second phenomena at their core that have Edward Kennedy (D–MA) is also hoping to slow
a second chamber of neon atoms. First, the never been resolved,” says Corkum. In the the current outflow of talent. Advocates want to
attosecond pulse yanked electrons out longer term, Krausz says, such work could move the repository to the Uniformed Services
from the atoms’ inner shells to their outer lead to better compact x-ray light sources for University of the Health Sciences in nearby
edges, preparing the atoms for ionization biological imaging and radiation therapies. Bethesda, Maryland. –CONSTANCE HOLDEN
and the electrons for escape. The laser –YUDHIJIT BHATTACHARJEE

NEWS OF THE WEEK
Continued from page 31 Overlooked. Faulty sup- nets and will meet at
researchers tried to pressurize its cylindrical port was not designed CERN at the end of
casing to 25 times atmospheric pressure. the month to f inal-
to resist a force pushing
The test was supposed to simulate the the magnet’s innards ize the plan and start
buildup of helium gas during a “quench,” through its casing. the fix.
an event in which the superconducting The schedule for
wire in the magnet temporarily loses its starting the LHC in November was already
superconducting properties and starts act- extremely tight. Workers have lowered all
ing like a giant heating coil, boiling the liquid but a handful of the LHC’s 1624 main mag-
helium coolant that fills the volume between nets into the tunnel and are busy connecting
magnet and casing. The pressure pushed the the equipment. Even so, they are currently
innards of the magnet through the cylindrical about 5 weeks behind schedule and pushing
container like a piston as a key support from now when the LHC has its f irst to catch up, Evans says. “If it goes into
broke. The support was not designed to take quench,” Holmes says. But, he adds, “we 2008, then there is no question of having an
a lengthwise push, says Stephen Holmes, an should have caught this before we got this engineering run and 3-month shutdown,”
accelerator physicist at Fermilab. far.” Researchers at Fermilab and CERN Evans says. “We’ll have to do without it.”
“It’s better to catch it now than a year already have ideas for modifying the mag- –ADRIAN CHO
PALEOANTHROPOLOGY
Hobbit’s Status as a New Species Gets a Hand Up

PHILADELPHIA, PENNSYLVANIA—The diminu- some critics still think the bones could be Tocheri got permission to study high-
tive human who lived on the Indonesian those of a diseased H. sapiens, others who quality casts of the Flores bones, which
island of Flores 18,000 years ago has been heard Tocheri’s report were persuaded. “It’s were made for Stony Brook University
called many things: a pygmy, a diseased the most convincing evidence so far that it biological anthropologist William Jungers.
Homo sapiens, a hobbit. Now, in a report really is something different,” says paleo- What Tocheri saw confirmed his impression
that was the talk of the Paleoanthropology anthropologist Carol Ward of the University that three bones in the wrist closely resem-
Society’s annual meeting here last week, a of Missouri, Columbia. bled those of an ancient hominid, not
postdoctoral researcher claimed that the The roughly 1-meter-tall skeleton has modern humans.
shapes of the fossil’s wrist bones are so sparked heated debate. Its discoverers Tocheri ruled out that the primitive hand
primitive that it cannot be H. sapiens. “It is claim it as a new species of human called bones were altered by disease because their
definitely not a modern human. It’s not even H. floresiensis, whereas critics argue that distinctive shape develops in the f irst
close,” paleoanthropologist Matthew the tiny skull belonged to a modern human trimester, long before deformation from
Tocheri of the Smithsonian Institution in suffering from a disease such as micro- most diseases begins later in pregnancy or
Washington, D.C., said in his talk. Although encephaly, which leads to a small head. after birth. He also says known diseases do
When Tocheri first saw casts of the hand not reproduce the primitive bone shapes.
CREDITS (TOP TO BOTTOM): FERMILAB; REUTERS/HO/PETER SCHOUTEN/NATIONAL GEOGRAPHIC SOCIETY

bones at a lecture last fall, he was struck “This is not pathological,” Tocheri said.
Get a grip.
immediately by their primitive shape. In his That fits with emerging evidence from the
A study of
hand bones Ph.D. dissertation from Arizona State Univer- long limb bones, which show no pathology
suggests that sity in Tempe—which he is defending this either, says Jungers (Science, 19 May 2006,
the hobbit was week—he used three-dimensional imaging to p. 983). “The sick-hobbit scenario is
a primitive analyze an innovation in the modern human wrong,” he says.
species. hand. Living people and our most recent But until the hobbit bones can be com-
ancestors possess a complex of five bones pared with a wrist of a microencephalic
that mesh together to ease stress on the wrist human, some remain unconvinced. “The
when the hand is used forcefully, for example wrist bones don’t look like those of a normal
in pounding large tools or in precision work. modern human, but how can we rule out that
Neandertals had this derived shock-absorber it’s a pathological modern human until we
complex, too; it is first seen in the hand of get comparative evidence?” asks paleo-
an 800,000-year-old human ancestor, anthropologist Robert Martin of the Field
H. antecessor, from Atapuerca, Spain. Museum in Chicago, Illinois.
But the bony complex is not found in Although much work has focused on
apes or earlier human ancestors, including the fossil’s chimp-sized skull (Science,
H. habilis, which lived 1.75 million years 2 February, p. 583), the new skeletal data
ago in Africa. That species did use tools, are proving convincing to many. Says
but the shape of its hand bones does not dis- lower-limb expert Henry McHenry of
tribute force away from the base of the the University of California, Davis: “It
thumb and across the wrist as efficiently as clinches it for me that [the Flores fossil]
in modern humans. was not modern.” –ANN GIBBONS

SCIENCESCOPE
U.S. COMPETITIVENESS
Russian Academy Fights Plan
Chemistry Reports Warn of Eroding MOSCOW—The Russian government appears
to be backing away from its bid to strip the
American Research Lead autonomy of the Russian Academy of Sciences
(RAS) after members of the centuries-old
CHICAGO, ILLINOIS— The outlook for In education, the clouds appear even institution rejected plans last week to change
United States scientif ic leadership darker. According to panel member Sylvia how its money is spent and properties are
remains cloudy with a chance of showers. Ceyer, a chemist at the Massachusetts Insti- used. The Ministry of Education and Science
That’s the gist of two new reports * the tute of Technology in Cambridge, the num- suggested last month that RAS alter its charter
National Research Council released last ber of chemistry Ph.D.s awarded to native- to create a supervisory council that would
week on the future of U.S. research in born students has sunk roughly 25% since have the final word in managing the acad-
chemistry and chemical engineering, both 1970. Universities have made up the differ- emy’s 450 research institutes. Composed of
of which were detailed here for the first ence with foreign students, who now earn three academicians, three government offi-
time at the semiannual meeting of the nearly half of all chemistry Ph.D.s awarded cials, two legislators, and a Kremlin represen-
American Chemical Society. by U.S. universities. But Ceyer warned that tative, such a council would relegate scientists
The first report warned that American as industry jobs continue to move overseas largely to research.
preeminence in chemistry research is slip- and visas remain tight in the wake of the But in a rare act of defiance, RAS’s gen-
ping away as the country grapples with 11 September 2001 attacks, the percentage eral assembly voted almost unanimously to
declining numbers of homegrown doctoral of foreign-born Ph.D. chemists who have reject the council proposal. The plan “goes
degrees in chemistry and the rise against the spirit of science and the traditions
of competition from Western of science,” says Yuri Osipov, academy presi-
Europe and Asia. The second dent. The ministry appears willing to drop the
predicted sunnier skies for idea if further negotiations yield a version of
U.S. leadership in broad areas of the charter acceptable to the cabinet, which
chemical engineering research, must approve the document. “I do not
although it warned that the believe that we must strongly insist upon [the
heavy emphasis on biology, council],” said Andrei Fursenko, education
nanotechnology, and other hot and science minister.
f ields in research spending –BRYON MACWILLIAMS
threatens to undermine less-
sexy areas of the discipline. India Court Halts Quota Rise
The new reports are the latest NEW DELHI—The Indian Supreme Court has
in a series of disturbing fore- blocked a plan to more than double the num-
casts for U.S. scientific leader- ber of university slots reserved for disadvan-
ship. The strongest warning Brain drain. Falling numbers of homegrown Ph.D.s are one of sev- taged students, saying that the government is
came from a 2005 report from eral signs that U.S. preeminence in chemistry research is threatened. relying on outdated statistics. The plan,
the National Academies that adopted by Parliament, triggered protests last
claimed the United States faced a “gather- chosen to stay in the United States has year by students from privileged castes, who
ing storm” of dwindling educational per- declined slowly but steadily over the past worried that it would hinder their entry into
formance and lackluster federal commit- 5 years. The upshot: “The U.S. will remain some of the country’s most elite institutions.
ment to basic research, particularly in the a leader in chemistry for the next 5 years,” To overcome the evils of the caste system,
physical sciences (Science, 21 October Ceyer says. “But the U.S. lead will continue which relegates several groups to menial jobs
2005, p. 423). to shrink as the chemistry world becomes and subjects them to overt discrimination,
Although the United States remains the flatter and more competitive.” India has long had an affirmative action plan
single strongest country in a variety of Mark Wrighton, chancellor of Wash- that guarantees those groups 22.5% of public-
measures of chemistry research, the trends ington University in St. Louis, Missouri, sector jobs and slots at many universities. The
are largely pointing in the wrong direction, says that although such trends are cause new law would boost that figure to 49.5%.
says Charles Casey, a chemist at the Uni- for concern, the situation isn’t yet dire. “I But last week, the court said the government
SOURCE: ADAPTED FROM A REPORT BY S. T. CEYER/MIT
versity of Wisconsin, Madison, who think it would be a mistake to read too was basing its argument on data collected as
chaired the chemistry report. Today, for much into these trends,” Wrighton says. far back as 1931 and demanded fresher facts.
example, U.S. researchers publish only “We need to keep in mind that the United Pavagada Venkata Indiresan, former direc-
18% of the papers in the field, down from States is still the world leader.” Although tor of the Indian Institute of Technology in
23% a decade ago. Over that same period, the new reports didn’t offer solutions, Chennai, called the ruling “a defeat for cyni-
the output from Asian countries other than Casey says the Bush Administration’s cal politicians who tried to replace an essen-
Japan tripled and is now on par with the competitiveness initiative, which aims to tial service by unwarranted draconian regula-
U.S. output. double U.S. physical sciences research tion.” The government is weighing its options
over 10 years, is a step in the right direc- before a final verdict is issued in August.
* Benchmarking the Research Competitiveness of the
tion. “From the chemistry perspective, it –PALLAVA BAGLA
United States in Chemistry; Benchmarking the
Research Competitiveness of the United States in will help tremendously,” Casey says.
Chemical Engineering. –ROBERT F. SERVICE

eppendorf® is a registered trademark.
‡ Maximum protection
against aerosols
2
‡ Binds biomolecules
1
‡ ep Dualfilter T.I.P.S.
available in ten sizes
NEW!
ep Dualfilter
T.I.P.S.®
Stop aerosols!
Unique two-phase filter protection with ep Dualfilter T.I.P.S.®
The new Eppendorf ep Dualfilter T.I.P.S., with their Features of the ep Dualfilter T.I.P.S.
unique two-phase filter, provide the perfect shield Double protection provided by the two phase filter
against contamination. ‡ Provides maximum protection for both pipette
and sample
The filter consists of two visible phases, each with a ‡ Ultimate absorption of aerosols and biomolecules
different pore size. This two-phase filter protection ensures ‡ Free from PCR inhibitor additives
ultimate absorption of aerosols  and biomolecules , ‡ Patent pending two phase filter technology
outmatching all conventional filters. Rely on it. ‡ Supplied sterile, Eppendorf PCR clean and pyrogen-free
‡ IvD conformity
For more information go to ‡ Batch-related certificates available
www.eppendorf.com/dualfilter
Your local distributor: www.eppendorf.com/worldwide · Application Support: +49 180-3 66 67 89

Eppendorf AG · Germany · +49 40 538 01-0 • Eppendorf North America, Inc. 800-645-3050
NEWS OF THE WEEK
AVIAN INFLUENZA
Indonesia to Share Flu Samples Under New Terms

Indonesia has agreed to resume Indonesia’s authorization before
sharing samples of the H5N1 Shots for tots. Indonesia’s Health sharing any samples beyond
avian influenza virus with the Minister Siti Fadilah Supari (left) i t s labs, according to David
wants vaccines to protect citizens
World Health Organization in Heymann, head of WHO’s pan-
against bird flu in return for
return for a promised rewrite of sharing H5N1 samples. demic influenza efforts. Tashiro,
WHO’s rules governing the use a participant in the Jakarta meet-
of donated viral samples. The ing, says a similar provision may
new “Terms of Reference” for be written into the new terms of
handling viral samples, which reference for all countries that
will be hammered out over provide virus to WHO.
the next several months, may Heymann says WHO already
include a clause giving coun- has an initiative that would ensure
tries that provide flu samples developing countries a share of a
more control over how and pandemic vaccine when it is pro-
whether WHO can pass the duced, by providing them either
virus on to third parties, such as stockpiles of vaccines or an
companies making vaccines. Indonesia reference labs watch for mutations that advance purchase agreement. The initiative,
h a d h a l t e d s h a r i n g i t s s a m p l e s ove r might suggest the virus is mutating into a he hopes, will “provide the reassurance
concerns that it would not have access to form more easily transmitted among developing countries need to continue
any H5N1 vaccine ultimately produced humans. In return, WHO will request sharing viruses.” –DENNIS NORMILE
(Science, 23 February, p. 1065).
Although health officials and scientists ENDANGERED SPECIES ACT
are happy that Indonesia will once again
provide flu samples, some worry about a
possible new precedent. “My concern is that
Appointee ‘Reshaped’ Science, Says Report
if this rule [takes effect], some country may Environmental groups have long vilified status of tiger salamanders in California
in the future refuse to share the viruses or Julie MacDonald, the Bush Administration’s from endangered to threatened (Science,
vaccine seed virus strain outside” the WHO point person at the Interior Department on 10 September 2004, p. 1554)—a decision
network, jeopardizing vaccine production, endangered species. Last week, their com- that was later tossed out by the courts. It
says Masato Tashiro, director of the WHO plaints got some support from the agency’s also quotes the former director of the FWS
Collaborative Center for Influenza Surveil- in-house watchdog, who has concluded that Endangered Species Program saying that
lance and Research in Tokyo. the political appointee played fast and loose “MacDonald regularly bypassed managers
For more than 50 years, the WHO Global with research. to speak directly with field staff, often intim-
Influenza Surveillance Network has col- A report by the inspector general’s (IG’s) idating and bullying them into producing
lected seasonal flu viruses and provided office found that MacDonald not only has documents that had the desired effect.”
vaccine seed viruses to drug companies. been “heavily involved” in editing scien- Although the IG found nothing illegal
Virtually all the vaccines produced have tif ic documents by the U.S. Fish and in MacDonald’s actions, the report says she
been used in advanced countries in temper- Wildlife Service (FWS) but also leaked violated the federal code in two ways. She
ate zones to fight seasonal flu. some of that confidential material to indus- leaked internal agency documents to lobby-
WHO took the same approach in dealing try groups. Next month, Congress will hold ists for the California Farm Bureau Federa-
with H5N1, which so far has primarily hearings on her actions. tion and other groups. And she appeared to
affected developing countries in tropical MacDonald is a civil engineer who previ- give those lobbyists preferential treatment—
and subtropical Asia. Indonesia, which has ously worked on endangered species issues a charge that MacDonald denied to the
the highest number of human H5N1 fatali- for the California state government. Since investigators. (The Interior Department
ties—at least 63 so far—ceased sharing its 2004, she has been deputy assistant secretary declined to comment on the report, calling it
samples of the virus with WHO in January. for fish and wildlife and parks. The IG’s a personnel matter, and MacDonald has not
Indonesian officials said they feared the report was sent last week to Representative made any public statements.)
country would not be able to afford a vac- Nick Rahall (D–WV), now chair of the House Rahall says that next month’s hearing will
cine or get a share of limited supplies in the Natural Resources Committee, who had be “a sweeping review on whether politics is
event of a pandemic. At a meeting in Jakarta received an anonymous tip that MacDonald infiltrating decisions” about endangered
CREDIT: TARMIZY HARVA/REUTERS
organized by WHO to resolve the impasse had “bullied, insulted, and harassed” FWS species. The issue may also come up during
on 26 and 27 March, Siti Fadilah Supari, scientists to alter biological reports about the Senate confirmation hearing of Lyle
Indonesia’s minister of health, called the endangered species. The findings, which Laverty, director of Colorado’s Division of
current scheme “more dangerous than the haven’t been publicly released, were first Parks and Outdoor Recreation, whom the
threat of an H5N1 pandemic itself.” reported by the New York Times. White House on 23 March proposed to be
Under an interim agreement, Indonesia The report documents, for example, how MacDonald’s boss. The job has been vacant
will again provide samples, which WHO’s MacDonald told agency scientists to lower the since November 2005. –ERIK STOKSTAD

NEWSFOCUS
An Asian Tiger’s
Bold Experiment
As Singapore embarks on a billion-dollar second phase of its
makeover as a research hub, critics wonder whether the island
nation is really getting its money’s worth
SINGAPORE—From the crest of a low hill in a is heading in the right direction. Late last year,
southern corner of this island state, Philip Yeo in an opinion piece in the influential Straits
makes a sweeping gesture toward a scientific Times newspaper, Lee Wei Ling, head of Sin-
Emerald City: nine gleaming new research gapore’s National Neuroscience Institute,
buildings teeming with more than 1000 bio- wrote that “if the present approach is followed
medical scientists. “We’ve gone from nothing without modification, a coherent body of
to this in 5 years,” says Yeo, chair of Singa- research and success in a series of related
pore’s Agency for Science, Technology, and fields is unlikely to develop.” Among other
Research (A*STAR), a government agency things, Lee is skeptical of the reliance on
that runs Biopolis, as the campus is known. imported scientific talent and believes the
Thanks in no small measure to Yeo’s wiz- overall effort lacks a coherent focus. Her arti-
ardry at winning government support and cle triggered a rare spectacle in this prim city- Sustainable? Some Singaporeans are asking
wooing overseas talent, Biopolis has put this state: a public debate over research and devel- how a massive investment in biomedical talent
and facilities will play out.
tiny Southeast Asian nation on the biomedical opment (R&D) policy waged in dueling edi-
research map. As one indicator of success, the torials and opinion pieces.
number of papers produced at the flagship Yeo brushes off the criticism. “I’m not very Tapped to implement the strategy was
Institute of Molecular and Cell Biology good at listening,” he admits. “My forte is get- Yeo, an engineer with a Harvard University
(IMCB) zoomed from 82 in 2000 to 165 in ting things done.” But the debate has raised MBA who was named chair of the National
2006, according to Thomson Scientific. Cita- questions about when Singapore can expect to Science and Technology Board, which
tion rates rival those of institutions with receive an economic payoff from the 2 billion became A*STAR. A career civil servant,
longer histories. Other Biopolis centers are Singapore dollars ($1.3 billion) spent so far on Yeo is credited with having led Singapore’s
still coming up to speed. But in building up a building and staffing Yeo’s field of dreams. drive into semiconductors and specialty
research capacity from scratch, boasts Yeo, And A*STAR can expect closer scrutiny as chemicals while chair of the Economic
“no other country has it embarks on the Development Board. A colleague describes
ever moved so fast.” $1.3 billion second Yeo’s lifestyle as “ascetic” and giving new
That claim has a phase of its biomed- meaning to the word “workaholic.” He is
number of prominent ical initiative: another relentlessly cheerful, peppering facts and
backers. What’s hap- batch of institutes numbers with wisecracks.
pened in Singapore in with links to hospitals When the biomedical strategy was
just 5 or 6 years “is to extend the research launched, Singapore had a single life sciences
pretty darn remark- to patients. institute, IMCB, affiliated at the time with the
able,” says Edward National University of Singapore, plus a cen-
Holmes, formerly Whale hunting ter on pharmaceutical technologies under the
dean of the School of In June 2000, Sin- Economic Development Board. A*STAR
Medicine at the Uni- gapore unveiled a took charge of both and created three more
versity of California, National Biomedical institutes, building Biopolis to house them. To
San Diego (UCSD). Science Strategy to staff the labs, Yeo started luring scientific stars
By comparison, says make this research from abroad, in some cases spending years to
Holmes, deputy chair Getting things done. Biopolis visionary Philip Yeo area a central pillar of fill a strategic post.
of Singapore’s Bio- says he is too busy to listen to critics of Singapore’s a knowledge-driven A big catch early on was Liu, imported in
medical Research biomedical strategy. economy (Science, 2001 from the U.S. National Cancer Institute
Council, it took San 30 August 2002, in Bethesda, Maryland, to head Singapore’s
Diego 40 years to become a biomedical hub. p. 1470). The first phase called for creating newly minted Genome Institute. Researchers
The research enterprise has progressed a public research infrastructure that would there quickly made their mark, becoming the
“beyond my wildest expectations,” adds generate discoveries, train personnel for first in the world to sequence the SARS virus
CREDIT: A*STAR
molecular oncologist Edison Liu, director of big pharma R&D, spin off start-up firms, at the height of that crisis in 2003.
the Genome Institute of Singapore. and generally build up local expertise in Since then, Liu has been joined by an array
But some now question whether A*STAR biomedical sciences. of world-class scientists. For example, David

NEWSFOCUS
Lane, renowned for his work on the p53 tumor George Radda, former chief of the U.K.’s ocratic systems. “Look at how the guys in Cal-
suppressor gene, is on a sabbatical from the Medical Research Council. ifornia are fighting [over plans for] stem
University of Dundee, U.K., to head IMCB. In Yeo calls these senior figures “whales” cells,” Yeo says. “Nothing is moving!”
addition to an international standing, Lane who have schools of ambitious young A*STAR claims to be nearing its economic
brought to the job wide-ranging contacts and researchers—“guppies”—trailing in their goals of generating 25 billion Singapore dollars
industrial acumen—in 1996, he founded wakes. So far, roughly 75% of the 500 or so ($16.4 billion) in biomedical manufacturing
Cyclacel Pharmaceuticals, which is develop- Ph.D.-level Biopolis researchers are foreign- and 15,000 jobs in the sector by 2015. Last
ing novel cancer drugs. Lane says the contacts ers. Aiming for a 50–50 balance among year, manufacturing output hit S$23 billion,
are important for an institute so distant from A*STAR’s institutes, Singapore plans to having almost quadrupled in the past 6 years.
established research centers of the United send abroad and fund some 1000 students to Biomedical employment grew 3.9% to reach
States and Europe. And his Cyclacel experi- earn undergraduate 10,571. The agency
ence helps when exploring interactions with to Ph.D. degrees at “If the present approach is f igures that invest-
pharma executives. top foreign universi- followed, … a coherent body ment commitments
Yeo lured others to Singapore by dangling ties by 2015. The full in 2006 will add
irresistible research opportunities. Nancy ride costs more than of research and success in 1800 jobs when facil-
Jenkins and Neal Copeland, a wife-husband 900,000 Singapore a series of related fields is ities come online. And
team of mouse geneticists, say they opted for dollars ($590,000)— private spending on
Singapore to escape tightening budgets and his “million-dollar unlikely to develop.” biomedical R&D in
restrictions on consulting work at the U.S. kids,” Yeo says. The —Lee Wei Ling, Biopolis critic 2005 reached 35% of
National Institutes of Health. In the United presence of senior the nation’s total
States, says Copeland, “there wasn’t a lot of scientists in Singapore, Lane adds, ensures R&D spending, up from 28.5% in 2001.
new money to do new things.” At IMCB, he that scholarship students “will continue to
says, they are assured of generous funding for have outstanding mentoring when they come A voice in the wilderness
CREDIT: LUIS ENRIQUE ASCUI/REUTERS/LANDOV
their work developing mouse models for back here.” Not everyone buys that rosy picture. Lee’s
human cancers, and they’re encouraged to Building a research effort from scratch has broadside in The Straits Times last November
interact with companies. made it easy to create institutions with com- questioned the strategy of hiring “foreign
Yeo has also imported heavyweight plementary aims, says Lane. “In most coun- stars and then letting them decide for them-
administrators to run institutes and develop tries, the rivalries between institutions can selves what areas of research to engage.” She
policy. The roster includes the husband-wife hold them back from working together in a criticized the initiative as lacking coordina-
team of UCSD’s Holmes and Judith Swain, successful way,” he says. Another Singa- tion and called for a lead agency to take con-
who was the university’s dean of translational porean strength is a small, pragmatic govern- trol and identify niches in which Singapore
medicine; Philippe Kourilsky, former presi- ment to oversee the initiative, argues Yeo, who could excel. Examples she gave included
dent of the Pasteur Institute in Paris; and professes disdain for bigger and messier dem- hepatitis B, liver and stomach cancer,

INTERNATIONAL SCIENCE & ENGINEERING
VISUALIZATION CHALLENGE
COMPETITION DEADLINE
A P P ROAC H I N G
ENTRY DEADLINE: MAY 31, 2007
s ci en ce a n d en gi n eer in g’s m o st p owe r f u l stat e m e n ts
a re n ot ma d e f ro m wo rd s a lo n e
When the left brain The National Science Foundation (NSF) Award Cate g o r i e s
and the journal Science, published by the
collaborates with the right American Association for the Advancement • I l l u s t rat i o n
brain, science emerges with of Science, invite you to participate in the • I nformational G rap h i c s
fifth annual Science and Engineering
art to enhance communication Visualization Challenge. The competition • I nte rac t ive M e d i a
and understanding of research recognizes scientists, engineers, visualization • Non - Inte rac t ive M e d i a
results—illustrating concepts, specialists and artists for producing or
commissioning innovative work in
• Ph o to g rap h s
depicting phenomena and visual communication.
drawing conclusions. Winners in each category will be published
in the September 28, 2007 issue of Science
and Science Online, and will be displayed
on the NSF Web site.
Complete Entry Information:

www.nsf.gov/news/special_reports/scivis
NEWSFOCUS
autoimmune diseases, and head injury. initiative: Production at drug company plants Finance Minister Tharman Shanmugaratnam
“Smaller countries with limited resources reached S$6.4 billion in 2000. And most said, “It is too early to evaluate the results of our
have to be more focused on how those observers agree that pharma investment R&D initiatives. But from [the Ministry of
resources are used,” Lee wrote. would have continued to grow even in the Finance’s] perspective, I am satisfied that this is
The critique carried particular weight in absence of the biomedical strategy. A*STAR a good use of public funds.”
Singapore, given Lee’s membership in what officials counter that their bootstrapping That’s A*STAR’s reading as well. It’s forg-
one researcher refers to as Singapore’s “ruling efforts have boosted the value of the manufac- ing ahead full-speed with phase two. A pair of
family.” She’s the daughter of Lee Kuan Yew, turing, moving from simple molecules to bio- new centers, the Institute for Clinical Sciences
Singapore’s revered first prime minister. logics: drugs cultured from living cells. And and the Singapore Immunology Network, will
Lee’s piece “created a stir in the entire they maintain that the growing pool of trained link bench researchers and staff at local hospi-
A*STAR community,” says IMCB’s Copeland. researchers is attracting additional interest tals to pursue clinical studies. The Ministry of
But neither A*STAR nor Yeo made a formal from big pharma. Within the last few weeks, Health is developing programs to enable clini-
response. So a week before A*STAR held its GlaxoSmithKline opened a $13 million cians to devote part of their time to research.
annual press briefing on the biomedical initia- medicinal chemistry outfit at Biopolis that And it plans a new medical school in coopera-
tive on 6 February, Lee repeated her claims in will double the firm’s research corps in Singa- tion with Duke University.
an interview with Reuters. Not surprisingly, pore to 60; and Eli Lilly announced a 5-year, Swain, head of the Institute for Clinical
questions about Lee’s comments dominated $150 million plan to boost its drug-discovery Sciences, believes Singapore’s unique mix of
the briefing. At the time, Yeo said that he efforts in Singapore in part by tripling its Indians, Malays, and Chinese “could be a
intended to “just ignore” criticism from “one R&D staff to 150. competitive advantage” for studies of how
voice in the wilderness.” And he mocked Last November, the World Bank published different ethnicities respond to drugs. One
Lee’s recommendations. Childhood vacci- a report examining how six Asian cities— disadvantage, however, is a small population
nations have vanquished hep- size. Alan Colman, CEO of ES
atitis B among Singaporeans, Foreign, senior scientists Cell International, says his
Yeo says. And rather than f irm is likely to go to the
spend money on head-injury
ensure that Singaporeans United States or Europe with
research, he told Science, “it returning from training their cardio stem cell therapy
would be cheaper to give every abroad “will continue to when it is ready for trials.
child a crash helmet.” Whether Singapore can
Lee declined to comment have outstanding mentoring sustain its rapid development
further, writing in an e-mail to when they come back here.” in biomedical science is
Science, “The points have another open question. Much
been put across to the small —David Lane, Institute of may depend on the success of
number of individuals I was Molecular and Cell Biology Biopolis managers in keeping
targeting.” Her views have got- senior scientists rooted to the
ten oblique support from Ting Choon Meng, a Bangkok, Beijing, Seoul, Shanghai, Singa- island. Lane says he will move back to
physician and founder of medical device pore, and Tokyo—are seeking new strategies Dundee at the end of 2007, although he plans
maker HealthSTATS. In a January Straits for economic growth. World Bank economist to spend “considerable time” in Singapore for
Times article, Ting argued that Singapore’s Shahid Yusuf says that he and co-author research and to advise A*STAR.
researchers are “putting the cart before the Kaoru Nabeshima are impressed at how One looming uncertainty is whether
horse” by overlooking the practical payoffs of quickly Singapore has put together an infra- Biopolis can continue on its present trajectory
research. “As a nation and as individuals, we structure resembling that of San Diego and without the energy of Yeo, who stepped down
have begun to showcase our innovations. But other hot spots. But he notes that research as A*STAR’s chair on 1 April. Yeo is not going
we may still end up not fully reaping the budgets are rising across Asia, and other rivals far, however. He will chair an arm of the Min-
rewards of our IP ideas,” he wrote. have biotech strategies. “When all of them get istry of Trade and Industry that promotes small
Yeo may have little time for critics. But his into this business, how will that affect the oth- and medium-sized businesses. He will also
star scientists, perhaps more used to defend- ers’ prospects?” he asks. As for Singapore, serve as a policy adviser to the prime minister.
ing science policies, are keen to make the case which has invested more heavily than others in Striding across the hill near Biopolis, Yeo
that research in Singapore can be both glob- biotech, Yusuf says, the questions are: “How doesn’t sound as though his interest in bio-
ally significant and locally relevant. “Every- much longer do they need to wait, and will medicine is waning. He points to two just-
body agrees, it’s a small place and you need to [the returns] be large enough to provide a completed Biopolis buildings now being fit-
focus,” says Copeland. But “people are focus- major engine of growth for Singapore?” ted out for new labs. Nearby, several low-rise
ing,” he says. Cancer is one target, and a Yeo dismisses the report. “I don’t believe buildings will soon be demolished to make
majority of Yeo’s recruits work on themes World Bank people are competent to make way for a Biopolis daycare center. A bit far-
related to cancer. Swain adds that as transla- recommendations to Singapore,” he says. ther, cranes are topping out the two towers of
tional medicine extends to work with patients, Fusionopolis, a S$550 million Biopolis clone
CREDIT: D. NORMILE/SCIENCE
it is imperative to align with local needs. One Safe for now in which A*STAR is gathering six institutes
example is gastric cancer, which for genetic In the wake of the debate touched off by Lee’s that work on information and communica-
and dietary reasons is prevalent in Asia. article, Singapore’s leaders have signaled their tions technologies. Yeo can’t contain his
Whether the initiative is giving the econ- confidence in the National Biomedical Science enthusiasm. “Come back in another few years
omy the desired kick is trickier to assess. Sin- Strategy. Most recently, in a 14 February speech and see what’s here,” he says.
gapore had big pharma investment before the unveiling the fiscal 2007 budget, Second –DENNIS NORMILE

Hot money. Researchers are gathering currency
across Europe and testing its cocaine content.
Follow the money

Money has a peculiar life of its own. When
not folded into a wallet or crumpled in a
pocket, the typical €20 bill can pass between
hundreds of hands for about a year before
getting recycled at a bank. In this time, it
moves through every part of society, from the
wealthy to the unemployed. But where most
scientists see a symbolic unit driving social
phenomena, Sörgel sees a cotton-paper filter
ideal for sponging up chemicals. And
because of the way that electrons are strung
on cocaine’s carbon frame, he says, it “binds
perfectly to the fibers.”
One explanation for the widespread con-
tamination of paper currency is that cocaine
is often snorted up the nose through rolled-
PUBLIC HEALTH up bills, and that sorting machines in banks
cause cross-contamination. “We really don’t
Hard Data on Hard Drugs, know for sure yet,” says Sörgel, but the evi-
dence so far supports this story. In a typical
Grabbed From the Environment sample of bills from European banks these
days, he finds that the majority of euros carry
detectable amounts of cocaine. Among the
Fieldwork in new and fast-growing areas of epidemiology requires wads of cash and contaminated bills, about 1 in 20 is typically
a familiarity with sewer lines loaded with around 10 micrograms of
cocaine, while the rest usually have a hun-
BARCELONA— It’s almost midnight when German euros for Spanish ones. Back at the dredth of that. (These amounts are minuscule
Fritz Sörgel and Verena Jakob walk into a lab in Germany, they’ll extract the chemical compared with the typical 100-milligram
chic cocktail bar. Still on the early side, the residues that have adsorbed to each bill—a line that goes up a nose.)
place is barely beginning to fill with the typ- process that destroys the money, but more For 7 years, Sörgel has been playing the
ical clientele of young, hip Spaniards. on that later. Among the thousands of part of the annoying tourist, buying bottles of
Installing themselves on low couches, the compounds that can be detected, Sörgel is water with €100 bills in every European
pair scan the drinks menu. “What I really looking for one: methylbenzoylecgonine, country, building a continent-wide map of
want is a piña colada,” says Sörgel with feel- better known as cocaine. cocaine use. There have been some close
ing. Returning from the bar, he looks It’s been known since the mid-1980s that shaves on this trip, such as when Jakob was
defeated. “Only daiquiris.” cocaine residue contaminates paper curren- suspected of shoplifting because of a suspi-
You probably wouldn’t guess that Sörgel cies, but Sörgel and others are taking advan- cious lump under her shirt—which was the
and Jakob, environmental chemists who tage of a natural experiment that began in money. (Sörgel managed to talk his way out
have been working since dawn, are still on 2000 with the simultaneous introduction of of that one.)
the job. Indeed, despite the tragic absence of the euro currency across Europe. Each coun- Banks have at times been suspicious when
piña coladas, Sörgel gets what he’s really try’s circulating stock of bills is becoming Sörgel asks to exchange wads of bills for his
after: Spanish bills in exchange for a crisp contaminated with cocaine at a different rate. “study of cocaine,” but they also have been
German €100 note. Jakob carefully squir- Measuring cocaine on the money is part of extremely helpful. The entire project would
rels away the change in a plastic tube. With a new effort to study the phenomenon of illicit have been a nonstarter if a German bank had
the f inal sampling of the day done, they drug use by turning to the environment. not agreed to redeem the entire €30,000 after
breathe a sigh of relief. Epidemiologists have struggled to get a quan- laboratory testing. The cocaine is detected
“It’s so stressful always having to worry titative view of drug use for decades. But the using a device called a mass spectrometer, but
CREDIT: BRETT PAULL/DUBLIN CITY UNIVERSITY
about the money,” says Sörgel, director of the traditional data—tons of drugs seized, the the first step is a methanol bath to extract the
Institute for Biomedical and Pharmaceutical number of people seeking treatment for addic- chemical residues. That makes the bills look
Research in Nuremberg, Germany. He’s tion, drug-related mortality, and responses to crisp and clean at the end, but it also loosens
referring to the brick of new German bills drug-use questionnaires—are biased and the metallic foil used to check against coun-
worth €30,000 ($40,000) that Jakob, his patchy, says Roberto Fanelli, a toxicologist at terfeit money. Sörgel exchanges the bills for
Ph.D. student, has been carrying in a secret the Mario Negri Institute for Pharmacological crisp new money, and the bank recycles the
pocket under her shirt since they arrived in Research in Milan, Italy. By interrogating the treated bills.
Spain a few days ago. (If it goes missing, the environment rather than the people, he says, Although Sörgel’s study of money is the
institute is out of luck, says Sörgel.) In a few “you can obtain data in real time” that are not biggest and longest-running, it is not the only
days, they will have exchanged all of the only objective but also “rather affordable.” one. Parallel projects are under way elsewhere

NEWSFOCUS
in Europe, and the collective data are adding through the city center, and there meets the European researchers say they are putting
up to a worrying picture. In Ireland, for two municipal wastewater treatment plants. the technique on firm experimental ground.
example, “people have been in denial that For their final samples, Sörgel dipped right Sörgel notes that about a ton of cocaine is
there’s a cocaine problem,” says Jonathan into the output of one of these plants, a trickle seized annually in Germany, a country
Bones, an environmental chemist at Dublin in a scummy gulley. thought to have a “moderate” drug problem
City University (DCU). But he and fellow Sörgel aims to administer a drug test to compared to others in Europe. Based on his
DCU chemist Brett Paull have been finding the entire city. The metabolic byproduct of sampling from rivers and wastewater at
some of the highest levels of cocaine con- cocaine, benzoylecgonine, is chemically 29 locations across Germany, he estimates
tamination on euros from Dublin’s banks. In unique in the environment and breaks down that Germans now consume on the order of
one case, 100% of a sample of 45 bills was slowly. Using the mountain stream water as 20 tons of cocaine per year. Sörgel’s data
coated in cocaine. They have recently ana- his baseline, he can estimate the amount of suggest an upward trend, and indeed, the
lyzed a sample of 75 bills and again found cocaine that passes through the entire popu- country’s traditional indicators of drug abuse
them all to be contaminated. lation. Repeating the procedure at intervals have all increased in recent years. “The
The main advantage of using money is should reveal drug consumption in a fixed methods are working,” he says.
that it’s quick and dirty: Instead of Fanelli has now hunted for cocaine
running around an entire country to get Check the source. Researchers in residues in the wastewaters of London
data, “the money does it for us,” says Spain aim to drug-test an entire city. and of Lugano, Switzerland, a popular
Sörgel. Paull is conf ident that his party destination for Italian tourists.
data are at least a “warning light” that He estimates that London’s daily
Ireland has a serious drug problem, but cocaine consumption is on the order
he says that many unknowns make it of 1 kilogram for every 1 million peo-
difficult to translate the data into quan- ple. He says this “reasonably trans-
titative statements about drug use. He lates” to cocaine use among 4% of
and Bones are trying to nail some of Londoners 15 to 30 years old. Official
them down. For example, to put a rate estimates put that figure at 2%. “So
on the natural degradation of cocaine we know we’re close to the real fig-
on money, Paull and Bones are spiking ure,” he says. Fanelli’s team found
euro bills with varying amounts of pure similar per capita cocaine loads in
cocaine and incubating them under Lugano’s wastewater, but there they
controlled conditions. also extended the sampling over sev-
One encouraging fact is that the eral months, revealing the variation
rank of average amounts of cocaine by day of the week. Monday was
found on euros from different countries consistently the low point of cocaine
roughly matches the ranking of national consumption, says Fanelli, whereas
drug problems by the E.U.’s traditional weekends were typically 30% to
survey-based statistics. Spain is in the 40% higher than the weekday aver-
lead, followed closely by Italy, with age, and sometimes double that.
Ireland now catching up. U.S.-based researchers are hot on
But tracking contaminated money the trail as well, but some are running
is only one part of the epidemiology into barriers. Jörg Rieckermann, an
story. After cocaine enters the nostril of environmental chemist at San Diego
a drug user and messes with the brain’s State University in California, has won
chemistry for about an hour, it is modi- a research grant from the Swiss
fied by enzymes in the liver and washed out geographic area in full detail, from seasonal National Science Foundation to survey
of the blood by the kidneys. You can guess dips to weekend spikes. cocaine contamination in wastewater. He
where it ends up next. Fanelli pioneered this technique in a 2005 selected San Diego for his analysis, but the
study of water from the Po River near Milan. city has forbidden him from taking samples.
The sewers don’t lie His group was studying the persistence of Controversy has been brewing since
One morning last month, Sörgel and Jakob legal pharmaceuticals in the aquatic environ- September 2006, when city politicians
went high up on a narrow, winding road in ment, he says, “but then we realized that we learned that a representative of the White
the Sierra Nevada mountains, dodging vil- could detect other drugs as well.” It is “com- House’s Office of National Drug Control
lagers and wood-hauling donkeys to reach pletely proven” that cocaine can be detected Policy (ONDCP) wanted samples of San
the pristine, presumably cocaine-free in the environment, he says, and now the Diego’s wastewater. ONDCP press secretary
snowmelt streams that feed the Spanish city more difficult task is “how to use these data Jennifer de Vallance said that the study started
CREDIT: J. BOHANNON/SCIENCE
Granada to the south. At a small bridge over for drug epidemiology.” Translating a minute about a year ago and is costing the office
a glassy brook, they dangled a plastic-lined and fluctuating signal in the environment to about $20,000. Samples have been collected
net into the water, bringing up two samples its ultimate source requires many assump- from about 100 participating wastewater
that Jakob sealed in bottles and labeled. tions, he says, “such as the percentage of the facilities across the United States, she says,
From there they sampled their way back cocaine that is metabolized in the body and generating about 500 samples, which are
down to Granada, following the Genil River the amount that is degraded before it reaches being analyzed at the Office of the Armed
as it wends through suburban sprawl, arcs the sampling site.” Forces Medical Examiner in Rockville,

NEWSFOCUS
Maryland. Others have heard about ONDCP’s Beyond the lab, sewer epidemiologists the right places,” says Fanelli.
project. “People from the White House con- will need the help of social scientists to draw “I have no doubt that these data are mean-
tacted me soon after my 2005 study of the Po meaningful conclusions from their data. ingful,” says Norbert Frost, chief drug epi-
River,” says Fanelli. “They plan to sample Computer models already track shifts in demiologist at the European Monitoring Cen-
wastewater from 100 sites and publish a report.” crime patterns, income, and pollution in tre for Drugs and Drug Addiction in Lisbon,
If public concerns can be overcome and large urban centers—as well as the daily flow Portugal, “but we must bring this to the next
these methods can be scaled up to monitor of water through pipes and sewers. Plugging level, where the techniques are standardized
“several thousand” wastewater treatment in environmental drug data could allow and producing peer-reviewed reports.”
plants across a country, says Fanelli, “sewer researchers to “score” communities in terms of The first formal opportunity to compare
epidemiology” will become a field in its own “drug-abuse levels,” says Barbara Tempalski, a notes will come later this month. Frost is gath-
right. But several technical hurdles must first social geographer at the Center for Drug Use ering a small group of international drug-abuse
be cleared. For one, researchers use slightly and HIV Research in New York City. And researchers from various fields in Lisbon on
different methods. Whereas Sörgel uses hunting for correlations between drug load 16 April to discuss environmental drug moni-
upstream river water for control samples, and other social, public health, and economic toring, the first meeting of its kind. It will be
Fanelli uses sterile, deionized water. “Those factors may reveal useful risk predictors that “an open discussion,” says Frost, covering
differences can have significant effects on so far have been obscured by the noise in the everything from analytical techniques to inte-
the results,” says Sörgel, so “standardizing available data. “Finding the hot spots of drug gration with the social sciences.
the methods is critical.” consumption can let us focus resources in –JOHN BOHANNON
ANIMAL BEHAVIOR The meeting, held from 23 to 25 March,

covered a broad range of topics from coop-
The World Through a Chimp’s Eyes eration and communication to tool use and
culture, experimental design, and conserva-
tion of this endangered species. “It’s a whole
A novel meeting assembled the world’s leading thinkers about chimp culture, tools, different quality of science from the exciting
cooperation, reasoning, and other heady topics cowboy era of maybe 2 decades ago,” said
Richard Wrangham, an anthropologist at
CHICAGO, ILLINOIS—It’s not every day that a tory. And surprisingly, it was the first one to Harvard University who for 20 years has
scientific meeting opens with a roomful of focus solely on the cognitive abilities of our studied wild chimps in Uganda’s Kibale
eminent researchers pant-hooting like nearest animal relatives. “It’s amazing,” Forest. “It’s a sign of a maturing field. You
chimpanzees, but then “The Mind of the said pioneering f ield researcher Jane have technical brilliance and tremendous
Chimpanzee” conference held here at the Goodall, one of approximately 300 partici- innovation in a wide range of areas.”
Lincoln Park Zoo last week marked a rare pants at the meeting. “We’re talking about The cumulative effect of the talks—many
occasion in itself. For only the third time in things now that I couldn’t talk about in the of which included videos that few people had
20 years, the zoo hosted a meeting that ‘60s. We couldn’t even talk about the seen—powerfully demonstrated that new
brought together researchers who study chimpanzee mind because chimpanzees insights are continuing to redraw the dividing
chimpanzees in the wild and in the labora- didn’t have one.” line between “us” and “them.” And one clear
theme emerged from the blending of labora-
tory and field studies: More effort than ever is
being made to perceive the world the way that
chimpanzees do, as opposed to simply asking
how closely their behavior mirrors our own.
Beyond compare
After the zoo’s Elizabeth Lonsdorf, a confer-
ence co-organizer, kicked off the meeting by
having the participants give each other a
“proper chimp greeting,” she introduced Kyoto
University’s Tetsuro Matsuzawa, one of the few
CREDIT: TETSURO MATSUZAWA/KYOTO UNIVERSITY
researchers who studies both wild and captive

chimpanzees. Matsuzawa’s talk kept the audi-
ence participation level high, eliciting loud
“oohs,” “ahhs,” and guffaws. Matsuzawa
described the numerical skills of a chimpanzee
named Ai and her son Ayumu, who live at the
university’s Primate Research Institute in
Testing, 1, 2, 3. Tetsuro Matsuzawa Kyoto. Building on work he first reported in
taught the chimp Ayumu to touch Nature 7 years ago, he showed videos of
numbers in sequence. Ayumu using a touch-screen monitor to select
the randomly displayed numbers 0 through 9,

NEWSFOCUS
in ascending order. He then repeatedly per- senters at the meeting offered intriguing developed field sites that are sure to trigger yet
formed a more difficult variation on this task, in clues to some of these riddles—and urged more investigations into cultural versus eco-
which the numbers were masked with white their colleagues to keep a chimp-centric view logically determined behaviors.
blocks shortly after they were flashed on the when designing experiments. Crickette Sanz of the Max Planck Institute
screen. “No one can do this,” he said, proving Kyoto University’s Satoshi Hirata showed for Evolutionary Anthropology in Leipzig,
the point with a hilarious clip of his graduate videos of a cooperation test he designed with Germany, described three different large com-
students failing the exercise with only four captive chimps. He placed fruit in a hole in the munities of chimpanzees she has extensively
masked numbers. “Our common ancestors ground, and then covered it with large stones. studied with her husband, David Morgan, in the
might have had immediate memory, but in the
course of evolution, they lost this and acquired
languagelike skills,” posited Matsuzawa.
As difficult as it is to assess a chimpanzee’s
memory, researchers similarly have a shaky
handle on how they communicate with each
other. “There could be a whole ’nother level of
chimp communication that we don’t have the
capability of understanding,” said psycholo-
gist Lisa Parr, who studies chimpanzee facial
expressions at Yerkes National Primate
Research Center at Emory University in
Atlanta, Georgia.
Parr described an objective metric she has
helped develop called the Chimp Facial
Action Coding System to understand better
what they are saying to each other with their Do as I do. Two chimps in Bossou, Guinea, watch an elder crack nuts, a skill that appears to pass from
expressions. “People have only looked at generation to generation by observation rather than teaching—and is considered part of their “culture.”
peak-intensity expressions,” says Parr, such as
the bared teeth that have been compared to the Chimps failed to work together to move the Goualougo Triangle in the Republic of Congo.
human smile. “Expressions are graded in stones, but when he placed himself in the Aided by 18 remote video cameras, Sanz and
intensity and force. No one has looked at experiment, a chimp solicited his help—possi- Morgan have documented 22 different tool
whether those have meaning.” bly because it knew he would not compete for uses since 1999, including various types of
In a similar vein, Katie Slocombe of the the food. In a different test that required two honey gathering, termite fishing, and leaf-
University of St. Andrews in Fife, U.K., has chimps to pull ropes cooperatively and move a sponging for water. “Crickette has done a mar-
begun parsing chimpanzee vocalizations to plank holding food close enough for them to velous job of looking at tool use in a systematic
see whether they have meanings that we have reach, they would cooperate but never solicit way,” said Jill Pruetz, an anthropologist at the
yet to recognize. “It’s a very neglected area of help. When he stood in the room, one of the University of Iowa, Ames, who recently
chimpanzee cognition,” said Slocombe. “Up chimps came and took his hand, again solicit- received much attention for describing chim-
until now, everyone’s been so dismissive. They ing his help. “Experimental arrangements panzees’ use of spears to trap bush babies at a
say, ‘It’s stimulus-response; it’s hardwired; it’s should be considered very carefully,” he said. site she has developed in Fongoli, Senegal
boring.’ I don’t think that’s the case.” Many researchers have long assumed that (Science, 23 February, p. 1063).
As Slocombe and Klaus Zuberbühler chimpanzees in the wild cooperate when they Goualougo and Fongoli are two recently
reported in the February 2005 Journal of hunt for red colobus monkeys, one of their developed field sites that Andrew Whiten, an
Comparative Psychology, they analyzed favorite meats. Harvard University’s Ian Gilby evolutionary psychologist at the University of
vocalizations she recorded during aggressive said think again—and see it through chimp St. Andrews, included in an update of what’s
interactions between 14 chimpanzees at the eyes. In a study he conducted at Kibale Forest, known as the Collaborative Chimpanzee
Budongo Forest Reserve in Uganda. They he found that “impact” males that were good Cultures Project. In 1999, Whiten, Goodall,
found that aggressors and victims gave dis- hunters attracted other males. “Is it collabora- Wrangham, and colleagues published a
tinct screams. Slocombe is now planning to do tion or byproduct mutualism taking advantage landmark paper in Nature, “Culture in
what she said will be the first ever “playback” of key hunters?” asked Gilby. “Impact males Chimpanzees,” that focused on six field sites,
experiments in the wild of recorded screams. may act as a catalyst for hunting.” documenting 39 different behaviors (most of
Similar studies in monkeys have revealed that Researchers face equally vexing conun- which were tool use) not due to ecological
they use calls to identify specific predators. drums when they try to tease out cultural (that’s forces. Since then, said Whiten, the number of
“Vocalizations can tell us a lot more than we what others in the community do) versus eco- sites has doubled, and researchers have docu-
currently think,” said Slocombe. logical (that’s what the environment dictates) mented 571 potentially unique behaviors.
determinants of tool use. Matsuzawa and “Fifty years ago, we knew nothing about wild
CREDIT: ETSUKO NOGAMI
Cultural sensitivities Tatanya Humle famously reported in 2002 that chimpanzees,” said Whiten, praising the
In chimpanzee research circles, incendiary chimps in Bossou, Guinea, used sticks of dif- “richness and complexity” of the data at the
debates revolve around the degree to which ferent lengths to dip for ants based on the risk meeting. “Look at us now.” No one vocalized
the animals cooperate, reason, teach, imitate, of being bitten—suggesting ecological rather in response, but the human facial actions—
and have culture. The debates burn on than cultural roots. Now there’s a deluge of smiles and nods—spoke volumes.
because there are no firm answers, but pre- new observations of unique tool use at recently –JON COHEN

MEETINGBRIEFS>>
AMERICAN PHYSICAL SOCIETY | 5–9 MARCH | DENVER, COLORADO
for the streaming of 20% of the atoms.

Experimenters Agree: You Can The bigger question may be, will
researchers continue to pursue the phenome-
non? “Clearly, this is a real effect,” says
Cross Off Flowing Crystals Sébastien Balibar, an experimenter at the
École Normale Supérieure in Paris. “This is
It was exciting while it lasted, but the claim ing suddenly increased, indicating that some really very new physics, even if it isn’t the
that crystalline solid helium can flow like the helium atoms had let go of their brethren and spectacular idea originally proposed.” But
thinnest liquid has seeped away. Such bizarre were standing stock-still. That could happen the messy details of defects may not gener-
“supersolidity” would have been perhaps the only if they slipped through the crystal with- ate as much excitement as the prospect of an
strangest manifestation of quantum mechan- out any resistance. oh-so-cool flowing crystal.
ics among things bigger than atoms and mol- Some theorists quickly argued that such
ecules. Alas, experimenters now agree that flow was simply impossible in an orderly
crystalline helium itself does not budge.
Instead, the resistance-free “superfluid” flow
crystal. Instead, they said, it likely arose from
more-conventional superfluid liquid helium
Ultrashort Laser Pulses
emerges as helium atoms wend along faults
and defects in the crystal.
percolating through defects in the crystal.
That interpretation was bolstered last year
See Inside the Body
when Reppy and Cornell’s Ann Energetic x-rays zip through flesh and set the
Sophie Rittner reproduced the standard for quickly seeing inside the body,
effect but found that it went away but flashes of gentler visible and near-
if they gently heated the crystal to infrared light can also reveal what lies under
eliminate defects, a process called the skin. Using laser pulses a few millionths
annealing (Science, 24 March of a nanosecond long, Warren Warren, a
2006, p. 1693). However, Chan chemist at Duke University in Durham,
and colleagues countered that North Carolina, and colleagues can trace
they saw no evidence that anneal- biomolecules such as melanin within tissue
ing stanched the flow. to make a three-dimensional image of their
Now, Chan and colleague microscopic distribution. The new technique
Anthony Clark, Reppy and Rittner, might be used to detect without biopsy a
and Keiya Shirahama and col- type of skin cancer called melanoma.
leagues at Keio University in “This is absolutely first-rate work,” says
Yokohama, Japan, all have man- David Jonas, a chemist at the University of
aged to shrink the apparent flow Colorado, Boulder. “The very small level of
by annealing their crystals. What’s signal they can detect is very impressive,
more, all reported that they can and that seems to be the key to making this
increase the signal by freezing useful for medical applications.”
helium quickly to make defect- Red and near-infrared light can pass
Frozen. Eliminate defects such as the grain boundaries visible in laden crystals consisting of many through flesh, which is why your fingers
this photo, and solid helium won’t flow. tiny grains. In fact, Reppy and glow luridly when you hold them over a
Rittner found that up to 20% of the flashlight. You cannot make out the insides
“The hypothesis that fits all the experi- atoms can flow in such a helium snowball. of your fingers, however, because the light
ments is that the crystal does not support The concordance of results shows that scatters off inhomogeneities in flesh,
superfluidity and that any experiment that defects are the key ingredient. “It will pro- obscuring the details. To get around that
shows flow can be explained by remnant dis- bably change the direction of research,” Chan problem, Warren and his team used light
order” in the crystal, says John Reppy, a says. Norbert Mulders of the University of pulses a few femtoseconds long and
physicist at Cornell University. Delaware in Newark says experimenters can exploited subtle interactions between the
Flowing helium crystals have been on expect a little gloating from their theorist light and material that vary in a nonlinear
CREDIT: S. SASAKI ET AL./SCIENCE 313, 1908 (2006)
shaky ground for a while. The first signs of colleagues. “They will absolutely love it,” way with the intensity of the light.
the elem e n t ’s s u p e r s o l i d ity emerged in Mulders says. “They can run around for a Researchers can trace a fluorescent sub-
2004 when Moses Chan of Pennsylvania State couple of years saying ‘We told you so!’ ” stance by scanning a sample with tightly
University in State College and Eunseong Precisely how the disordered solid flows focused femtosecond pulses whose wave-
Kim, now at the Korea Advanced Institute of remains unclear. Theorists Lode Pollet and lengths are two times too long to trigger the
Science and Technology in Daejeon, set a Matthias Troyer of the Swiss Federal Institute fluorescence. Because of the mismatch, a tar-
small can of solid helium twisting back of Technology in Zurich presented simula- get molecule fluoresces only when it is tickled
and forth on the end of a thin shaft (Science, tions confirming that atoms can glide along simultaneously by two photons, which will
1 July 2005, p. 38). Below a certain tempera- the boundaries between grains without resist- happen only where the light is most intense.
ture—a few ten-thousandths of a degree ance. But some experimenters argue that By monitoring the fluorescence while mov-
above absolute zero—the frequency of twist- flow along grain boundaries cannot account ing the laser’s focal spot through the sample,

NEWSFOCUS
Pulling Strings to Untangle Catastrophe stretch of string infinitely weak. Discounting

the improbable accumulations, however,
About the simplest experiment imaginable That’s because the average is inflated by shows that a finite string has a strength that
may yield insights into calamitous events such extremely rare events. For example, although decreases in a particular way as its length
as the sudden failure of cables on a suspension the chances of flipping 25 tails in a row are increases—namely, with the logarithm of
bridge. Taking a break from work on liquid just 1 in 33,554,432, the payout for such a run the length.
crystals, physicist Peter Palffy-Muhoray and is also a whopping $33,554,432, which still To test this model, the researchers pulled
graduate student Jake Fontana of Kent State drives the average winnings up. The paradox on strands of thread and fishing line ranging
University in Ohio spent a few months tugging can be avoided by recognizing that, in any from a millimeter to a kilometer long—
on string to see how the force required to finite number of tosses, such runs are so stretching the longer lengths along a bike
break it varied with its length. Their prelimi- improbable they should be ignored. If the path. Their logarithmic prediction fit the data
nary results fit a model based on a gambling game is repeated until the coin has been better than two other well-studied models: one
puzzle that stumped Daniel Bernoulli that treats the string as a chain and
and other mathematicians in the early assumes an exponentially decreasing
18th century. distribution for the weakness of the
“Rare events are by their very nature links, and another that focuses on
hard to find but also extremely impor- how stress is shared by fibers in a
tant because they can lead to catastro- multifiber line.
phe,” says Mark Warner, a theorist at the Others have probed how strength
University of Cambridge in the U.K. varies with length for the short fibers
“You have to find a system in which you used in composite material, but few
can explore a large range of [length] have examined extremely long ones,
scales, and a long string seems to be just says William Curtin, who studies theo-
the thing.” Size plays a key role in fail- retical mechanics at Brown University.
ure: A big rock is typically weaker than a “It’s a nice data set,” he says. Curtin
small one because it has longer cracks. cautions that the relation should
The string experiment embodies the Snap judgment. Jake Fontana took his string-pulling rig outdoors depend on the specific microscopic
tricky mathematics of the Petersburg to gain insight into catastrophic failure. structure of the line. “There’s not a
paradox, a game in which a gambler’s universal form” for the length-
winnings inevitably fall far short of his reason- tossed 100 times, there probably won’t be a strength relation, he says.
able expectations. After paying an entry fee, run of more than six tails, and even that would Fontana says he enjoyed the chance to
the player flips a coin until it comes up heads. pay out only $64. work outdoors, but the experiment still had its
If he tosses tails once before that happens, he To apply this scheme of ignoring exceed- trials. “The most stressful part was trying to
gets $2. If he tosses tails twice, he gets $4. With ingly improbable events to string, Palffy- keep people on the path from hitting the
every additional tails, the payout doubles. In Muhoray and Fontana assumed that the string string, trying to be as polite as possible,” he
principle the average winnings are infinite, so would break wherever unspecified defects says. The researchers hope to pull even longer
CREDITS (TOP TO BOTTOM): THOMAS PALERMO; TONG YE/DUKE UNIVERSITY; DAVID ELDER/WISTAR INSTITUTE
a gambler ought to pay any amount to play. In accumulated. These accumulations corre- strings, perhaps using pulleys to wrap them
reality, however, the game never pays out more sponded to runs of tails in the coin tossing, back on themselves.
than a few dollars. and on average, they should make any long –A.C.
researchers can map the target substance. A The amount of absorption is still tiny, how- The technique lets researchers detect
commercial system already images skin using ever, and to see it, the researchers employ absorption of as little as one 10-millionth
such two-photo fluorescence. another trick. They make the intensity of the of the original pulses. “We’re trying to
But the fluorescence technique has draw- first color’s pulses oscillate up and down and l o o k a t p r o c e s s e s wh e r e t h e r e ’s j u s t
backs, Warren says. Many biomolecules, such check whether this causes the absorption barely enough signal so that you can
as melanin, fluoresce only weakly, and light from the second beam to vary in a similar way. access them using less average power
from the fluorescence itself scatters within the That technique enables them to convert a tiny than a laser pointer,” Warren says. Jonas
flesh. So Warren and his team instead measure intensity variation into a much clearer fre- says that “by being able to detect such
the amount of light absorbed by specific sub- quency signal. To make the conversion, they small effects, you’re able to get the power
stances. For example, track the pulses of the down enough that you’d feel safe having
they apply pulses of second color as they this done to you.”
two different colors are reflected from the Warren presented an image of a melanoma
chosen so that when a sample. They break removed from human skin that showed the
melanin molecule the entire train of telltale streaks and clumps of accumulating
absorbs a photon of pulses into its com- melanin. As a step toward clinical applica-
the first color, it will ponent frequencies. tions, he and his colleagues have proposed a
more readily absorb a The tiny oscillation trial in which they will analyze irregular moles
photon of the second Flashy. A new laser technique (left) reveals details of then shows up as an in patients that doctors intend to remove
color as well. melanoma ordinarily seen with biopsy. additional frequency. shortly anyway. –ADRIAN CHO

COMMENTARY
Thinking about Molecular motoring Understanding bonding
medicine
55 58 61
LETTERS I BOOKS I POLICY FORUM I EDUCATION FORUM I PERSPECTIVES
LETTERS
edited by Etta Kavanagh
Making Articles Available afford to subscribe to certain scien-

tific journals and databases.
for Flu Planning The more we can do to reduce arti-
ficial and unnecessary barriers to info-
rmation sharing and collaboration—
RECENT DISCUSSION ABOUT THE SHARING OF H5N1 SAMPLES BY THE at every level—the better off all of us will be in the long term as we
Indonesian government with the World Health Organization highlights collectively prepare for a worldwide flu epidemic, and other potential
the importance of intellectual property issues in vaccine development public health emergencies. Enhanced access to scientific journals will
[“Indonesia, WHO patch up,” M. Enserink, ScienceScope, 23 Feb., help better ensure that community planning and preparedness efforts
p. 1065; (1)]. It is also worth noting the barriers to collaboration and reflect the latest and most authoritative and substantive scientific
information sharing represented by another form of intellectual prop- knowledge and insights. It also will help broaden involvement in dis-
erty: copyrighted articles about avian flu and pandemic flu prepared- cussions about critical and often unresolved scientific, medical, regu-
ness contained within medical and scientific publications. latory, and ethical questions.
It is disappointing that, at a time when many political and govern- Researchers and publishers should do their part to help make sure
mental officials are emphasizing the importance of collaboration, that important information about this public health threat reaches pro-
information sharing, and community involvement in pandemic flu fessionals and members of the public in a timely manner (6). Allowing
planning and preparedness, interested professionals and members of unrestricted access to relevant articles, letters, and correspondence
the public are compelled to pay high fees or wait for weeks for an inter- about avian influenza and pandemic flu preparedness would be an
library loan request to be filled when articles from medical and scien- excellent start.
tific journals are readily available online and could be easily shared MITCHELL BERGER*
with a broad audience. Public Health Planner, DiPiero Center, Camden County Department of Health and Human
Articles from major scientific publications are available, but in Services, 512 Lakeland Road, 2nd floor, Blackwood, NJ 08012, USA.
many cases, there is a cost. For instance, to read an interesting article *The opinions expressed in this article are solely those of the author and should not be
about the utility of blood transfusions from the Journal of the American imputed to any public or private entity.
Medical Association costs $15 (2). It costs $10 to access relevant arti-
References and Notes
cles in Science online (3), while Nature charges $30 (4). A recent dis- 1. D. G. McNeil Jr., “Indonesia may sell, not give, bird flu virus to scientists,” N.Y. Times, 7
cussion of the 1918 influenza pandemic co-authored by a high-ranking Feb. 2007, p. A6.
U.S. government official costs $15 for 24-hour access (5). To their 2. T. Hampton, JAMA 296, 1827 (2006).
3. For instance, M. Enserink, Science 313, 1555 (2006).
credit, some journals do occasionally make relevant articles available 4. D. Butler, J. Ruttimann, Nature 441, 137 (2006).
for free upon initial publication or at other times. Others can be found 5. D. Morens, A. Fauci, J. Infect. Dis. 195, 1018 (2007).
on various blogs and Web sites (e.g., Flu Wiki). However, this informa- 6. One free service, funded through unrestricted grants by several pharmaceutical compa-
nies, already collects and distributes to subscribers on a weekly basis journal abstracts
tion is often difficult to access for those not affiliated with the large about influenza and other topics. Perhaps this or a similar site also could provide relevant
academic institutions, corporations, or governmental entities that can articles about pandemic influenza (see http://www.amedeo.com/).
Pneumococcal Vaccines tion played a major role in the 1918 in-

fluenza pandemic is substantial, but seems
toms (8). Blood culture and time of death
both suggest a role for the pneumococcus in
and Flu Preparedness to have been forgotten (3). In two studies, a substantial fraction of the deaths of these
culturable pneumococci could be found in young soldiers.
INFLUENZA-ASSOCIATED MORTALITY IN THE the peripheral blood of 50 of 105 living sol- The role of conjugate pneumococcal vac-
next five decades is likely to exceed that of diers with influenza, during the pandemic in cine in reducing influenza-associated mor-
any other global catastrophe (1, 2). The role the United States and the UK (4, 5), and bidity has recently been demonstrated (9).
of secondary bacterial infections and the from 55 of 89 heart blood cultures taken Children who received the vaccine and then
CREDIT: PETER HOEY
need for bacterial vaccines are not men- from U.S. soldier influenza victims immedi- developed laboratory-confirmed influenza
tioned in the U.S. Department of Health and ately after death (6, 7). Roughly 1/3 of were at 45% less risk of hospitalization due
Human Services Pandemic Influenza Plan deaths during the 1918 pandemic occurred to the influenza-associated pneumonia than
(2). The evidence that pneumococcal infec- more than 2 weeks after the onset of symp- were children who had not received the

LETTERS
www.eppendorf.com/advantage
Get Eppendorf quality today!
vaccine in a prospective double-blind ran-
domized trial (9). These data suggest that
conjugate vaccine given to infants may
greatly reduce influenza-associated morbid-
ity, but also, coupled with data on herd im-
munity of conjugate pneumococcal vaccine
in the United States (10), suggest that im-
munization of children may protect adults
as well.
The reasons for increased pneumococcal
morbidity and mortality after influenza have
been reviewed recently (11). Practical meas-
ures to be taken to prevent and treat second-
ary bacterial infections include antibiotic
stockpiling; the enhancement of conjugate
pneumococcal vaccine coverage of children
to ensure optimal protection for them and
high levels of herd immunity among adults;
and provision to adults of 23 valent pneu-
mococcal vaccine to prevent pneumococcal
bacteremias. Research studies evaluating
the impact of conjugate pneumococcal vac-
cine introduction on influenza-associated
eppendorf ® is a registered trademark.
morbidity in adults and children in the

United States and other countries contem-
plating the introduction of these vaccines
are needed as well.
KEITH P. KLUGMAN1 AND SHABIR A. MADHI2
1William H. Foege Professor of Global Health, Rollins School
of Public Health and Professor of Infectious Diseases,

School of Medicine, Emory University, Atlanta, GA 30322,
USA. E-mail: [email protected]. 2Associate Pro-
fessor, Respiratory and Meningeal Pathogens Research
Unit, Medical Research Council and University of the
Promotion Witwatersrand, Johannesburg 2000, South Africa.
April 1– References
June 30,
1. V. Smil, Pop. Dev. Rev. 31, 201 (2005).
2. HHS Pandemic Influenza Plan (U.S. Department of
2007
Health and Human Sciences, Washington, DC, 2005).
3. J. F. Brundage, Lancet Infect. Dis. 6, 303 (2006).
4. R. Muir, G. H. Wilson, Br. Med. J. 1, 3 (1919).
5. E. F. Hirsch, M. McKinney, JAMA 71, 1735 (1918).
6. L. H. Spooner, L. H. Scott, E. H. Heath, JAMA 72, 155
(1919).
7. J. N. Hall, M. C. Stone, J. C. Simpson, JAMA 71, 1986
Gentle & Easy!

(1918).
8. C. E. Mills, J. M. Robbins, M. Lipsitch, Nature 432, 904
(2004), supplementary fig 2.
9. S. A. Madhi, K. P. Klugman, Vaccine Trialist Group, Nat.
Med. 10, 811 (2004).
10. CDC, Morb. Mortal. Wkly. Rep. 54, 893 (2005).
With Eppendorf Advantage offers it is now easier than ever to add 11. J. A. McCullers, Clin. Microbiol. Rev. 19, 571 (2006).
Eppendorf premium quality centrifuges to your lab.
Gentle & Easy: Centrifuge 5702/5702 R Bonus Packs Timing of a Back-

The Eppendorf 5702 family of centrifuges offers an outstanding combination
of gentle treatment of sensitive clinical samples and ease-of-use.
Migration into Africa
INDIGENOUS NORTH AFRICANS ARE GENETI-
Take advantage now! cally quite distinct from sub-Saharan Afri-
From April 1–June 30, 2007, Centrifuge 5702 and Centrifuge 5702 R are available cans (1), and this difference is reflected in
as attractive Bonus Packs at special prices that are too good to miss*. their lighter skin and European/Middle
Eastern physical features. We have previ-
Our exciting offers are just a few clicks away! ously suggested, on the basis of the distri-
For more details visit www.eppendorf.com/advantage bution of mtDNA type M1, that North
Africans are largely descended from a back-
*Offers may vary by country.
LETTERS
migration into Africa within the last 2000 to colleagues would conveniently fit with the
15,000 years, resettling the temporarily lush arrival of farming in the European Med-
Sahara and spreading the Afro-Asiatic iterranean.
language family (2). In their Report “The In conclusion, we suggest that more
* Offers may vary by country. ep-points are not available in all countries. For details see www.ep-points.com
mtDNA legacy of the Levantine early Upper recent influx from Asia, possibly since the
Palaeolithic in Africa” (15 Dec. 2006, Last Glacial Maximum 20,000 years
p. 1767), A. Olivieri and colleagues used ago, may better explain some of the major
high-resolution mtDNA data to propose that genetic and linguistic patterns in North
the migration from Asia back to North Africa Africa and adjacent areas [cf. (4, 5)]. We
happened much earlier, and they link the set- nevertheless believe that future archaeo-
tlement of North Africa with the settlement genetic research on Ice Age Africa and sub-
of Europe 40,000 to 45,000 years ago. sequent periods will benefit greatly from the
Three points lead us to believe that our complete mtDNA sequencing approach
younger chronology for the back-migration taken by Olivieri and colleagues.
5702/5702 R Bonus Packs
into northern Africa still merits consideration. PETER FORSTER1* AND VALENTINO ROMANO2
First, the mtDNA trees reconstructed by 1Department of Forensic Science and Chemistry, Anglia Centrifuge
Olivieri and colleagues are less than conclu- Ruskin University, Cambridge CB1 1PT, UK, and New Hall,
University of Cambridge, Cambridge CB3 0DF, UK. + swing-bucket rotor
sive because they consist of phylogeographi- 2Dipartimento di Oncologia Sperimentale e Applicazioni
+ 4 round buckets
cally mixed branches, which cause uncer- Cliniche Università di Palermo, Palermo, Italy, and Assoc- in one package!
tainty in identifying the relevant founder iazione OASI Maria SS. (I.R.C.C.S.), Troina, Italy.
*To whom correspondence should be addressed. E-mail:

[email protected]
References
1. L.-L. Cavalli-Sforza, P. Menozzi, A. Piazza, The History and
Geography of Human Genes (Princeton Univ. Press,
Princeton, NJ, 1994).
2. P. Forster, Philos. Trans. R. Soc. London Ser. B 39, 255
(2004).
3. A. Muzzolini, L’Art Rupestre Prehistorique des Massifs
Centraux Sahariens (BAR, Oxford, 1986).
4. C. Renfrew, Cambr. Archaeol. J. 1, 3 (1991).
5. I. Diakonoff, J. Semit. Stud. 43, 209 (1998).
Response
THE PRINCIPAL PROBLEM WITH GREAT SYN- Promotion
theses of languages, genes, and figurines
(or pots) is that they lump together different
April 1–
migrational and cultural processes and June 30,
Neolithic cave art from the central Sahara Desert.
This example is from the Sefar site in Algeria.
especially overstretch recent events of the
Holocene, thereby downplaying or swamp- 2007
[Image after (3)] ing the genetic signals that point to much
earlier events of the Pleistocene (1, 2).
nodes for genetic dating. Second, in our view Forster and Romano propose a recent
the fact that the North African mtDNA marker arrival—within the last 2000 to 15,000
types still correspond so closely with the Afro- years—of haplogroup M1 in North Africa Your additional bonus
Asiatic language zone argues against the exis- from western Asia, linked to the spread of Each Centrifuge 5702/5702 R
tence of that correlation for tens of thousands Afro-Asiatic languages. This would entail a Bonus Pack comes standard with:
of years. Third, cave art in the Sahara shows Near Eastern origin of the Afro-Asiatic lan- l an extra 100 ep-points*
that in Neolithic times (around 5000 B.C.), guage family and thus would be in agree- registration bonus
the population of the Sahara was still of ment with Bellwood (3), provided that one l the chance to win one of 50 gift
sub-Saharan African ancestry (see figure), subscribes to such a tight link between genes certificates (e. g., Amazon, American
whereas “Europoid” figures documenting the and languages. Afro-Asiatic scholarship (4), Express, or equivalent) to the amount
arrival of west Eurasians appear later in the as well as the coalescence times of both M1a of approx. 50 Euro each
cave art record (3). and M1b and the diverse basal distribution l one unique Eppendorf pipette pen
Within the framework of our younger of M1a lineages especially in East Africa,
chronology, the occurrence of low fre- however, militate against this interpretation. More product details and contact
quencies of M1 types in the European As we proposed in our Report, the arrival of information at
Mediterranean can be explained by diffu- M1 in Africa is most likely contemporary www.eppendorf.com/advantage
sion from the Middle East and North Africa with that of U6, but if one alternatively
during and since the Neolithic. The Sar- hypothesized that only M1a originally went
dinian M1 mtDNA founder date of 7700 ± into the Northeast African Mediterranean
3100 years years calculated by Olivieri and coast, then 25,000 to 30,000 years ago
www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007

LETTERS
would be the realistic time frame. and Romano has no impact on the issue of DORON M. BEHAR,4
The latter hypothesis is valid when one the late Near Eastern influx because hap- JEAN-MICHEL DUGOUJON,5
CLOTILDE COUDRAY,5
assumes the less parsimonious scenario that logroup U6 very clearly testifies to an early A. SILVANA SANTACHIARA-BENERECETTI,1
only haplogroup U6 was involved 40,000 presence in North Africa of Near Eastern lin- ORNELLA SEMINO,1 HANS-JÜRGEN BANDELT,6
to 45,000 years ago in the early Upper eages, which must have proceeded to as far ANTONIO TORRONI1*
Palaeolithic diffusion of Levantine popula- as Northwest Africa with the ancestors of 1Dipartimento di Genetica e Microbiologia, Università di
tions into North Africa and that a diffusion the Iberomaurusians before the Late Glacial Pavia, Via Ferrata 1, 27100 Pavia, Italy. 2Department of
Biotechnology, College of Science, University of Baghdad,
of M1a lineages marked a new phase in Maximum (8). The anthropological evidence Iraq. 3Dipartimento di Genetica e Biologia Molecolare,
the Nile Valley Complex, 25,000 to 30,000 from North Africa, pointing to the auto- Università “La Sapienza,” Piazzale Aldo Moro 5, 00185
years ago (5). It is then also more plausible chthonous Mechta-Afalou physical type, Rome, Italy. 4Molecular Medicine Laboratory, Rambam
to see the development and emergence of with continuity well into the Capsian of the Health Care Campus, Efron 9 street, Bat Galim, 31096
Haifa, Israel. 5Centre d’Anthropologie, FRE 2960 CNRS,
proto–Afro-Asiatic languages there, in the mid-Holocene, gives clear support to the Université Paul Sabatier, Toulouse III, 37, allées Jules
Nile Valley (6, 7). Later migrations and gene ancient presence of Upper Palaeolithic peo- Guesde, 31073 Toulouse Cedex, France. 6Department of
flow, which undoubtedly took place, have ple in North Africa (5). Moreover, the pres- Mathematics, University of Hamburg, Bundesstrasse 55,
20146 Hamburg, Germany.
certainly complicated phylogeographic ence of figurines of sub-Saharan type in the
patterns. For instance, one may also envi- cave art of the Sahara may simply be indica- *To whom correspondence should be addressed. E-mail:
sion some mutual contacts between the tive of resettlement of the region by groups [email protected]
Levantine Natufian culture and contem- from the south, already adapted to savannah
References
porary autochthonous cultures of the Lower ecology, after the early Holocene arrival of 1. H.-J. Bandelt, V. Macaulay, M. Richards, in Examining the
Nile Valley (~15,000 years ago). Later monsoon rains changed the Sahara into a Farming/Language Dispersal Hypothesis, P. Bellwood,
Neolithic influence then brought a whole habitable region (10). Thus, the argument is C. Renfrew, Eds. (McDonald Institute Monographs,
McDonald Institute for Archaeological Research,
package of Near Eastern mtDNA lineages not informative on the antiquity of a “Euro- Cambridge, 2003), pp. 99–111.
into all of North Africa, as attested, for poid” settlement in North Africa. 2. M. Richards, Annu. Rev. Anthropol. 32, 135 (2003).
instance, by the relatively high frequency of ANNA OLIVIERI,1 ALESSANDRO ACHILLI,1 3. P. Bellwood, Science 306, 1681 (2004).
4. C. Ehret, S. O. Y. Keita, P. Newman, Science 306, 1680
mtDNA haplogroups H, J, and T in modern MARIA PALA,1 VINCENZA BATTAGLIA,1
(2004).
North African populations (8, 9). SIMONA FORNARINO,1 NADIA AL-ZAHERY,1,2 5. D. W. Phillipson, African Archaeology (Cambridge Univ.
The cave art argument adduced by Forster ROSARIA SCOZZARI,3 FULVIO CRUCIANI,3 Press, Cambridge, ed. 3, 2005).
FOCUS ON CAREERS
Careers in Cancer Research
IN THIS ISSUE:
M
any different roads can lead to a career in
cancer research. Which path will you take? Find
some direction in the latest Careers in Cancer
Research feature in this issue, page 131.
UPCOMING FEATURES:
April 20— Postdoctoral Careers: Transferable Skills
April 27 —Biotech and Pharma
May 4 — Interdisciplinary Research
Also available online at www.sciencecareers.org/businessfeatures

LETTERS
6. C. Ehret, A Reconstruction of Proto-Afroasiatic (Proto- ments will apply immediately to the review RESPONSE TO COMMENT ON
Afrasian): Vowels, Tone, Consonants and Vocabulary
of the ozone standard. This is not the case. “Divergent Induced Responses to
(Univ. of California Press, Berkeley/Los Angeles/London,
1995). The ozone review is pretty far down the an Invasive Predator in Marine
7. M. Brett, E. Fentress, The Berbers: the Peoples of Africa pipeline, and the EPA concluded some time Mussel Populations”
(Blackwell, Oxford, 1996). ago that any significant changes to this
8. J. C. Rando et al., Ann. Hum. Genet. 62, 531 (1998).
review would likely delay getting this stan- Aaren S. Freeman and James E. Byers
9. V. Macaulay et al., Am. J. Hum. Genet. 64, 232 (1999).
10. R. Kuper, S. Kröpelin, Science 313, 803 (2006). dard completed. Preliminary DNA analysis indicates that only a few Mytilus
MARCUS PEACOCK trossulus mussels were present in our study of M. edulis.
Excluding these M. trossulus did not influence the
Deputy Administrator, U.S. Environmental Protection Agency,
Speeding Up the EPA Washington, DC 20460, USA. outcome of our analyses. Our study provides essential
evidence that populations of M. edulis respond differently
Review Process to the two crab predators, and the adaptive significance of
shell thickening in mollusks is well established.
TECHNICAL COMMENT ABSTRACTS
AS ERIK STOKSTAD’S ARTICLE “EPA DRAWS Full text at www.sciencemag.org/cgi/content/full/316/
fire over air-review revisions” (News of COMMENT ON “Divergent Induced 5821/53c
the Week, 15 Dec. 2006, p. 1672) notes, Responses to an Invasive Predator
everyone agrees that the U.S. Environmental in Marine Mussel Populations”
Protection Agency’s (EPA) process for deve- Paul D. Rawson, Phillip O. Yund,
loping ambient air quality standards is slow Sara M. Lindsay
Letters to the Editor
Letters (~300 words) discuss material published
and cumbersome. Steve Johnson, the first Freeman and Byers (Reports, 11 August 2006, p. 831) in Science in the previous 3 months or issues of
career scientist ever to head the EPA, has presented evidence for the rapid evolution of antipredator general interest. They can be submitted through
also found that this process doesn’t deliver defenses in the mussel Mytilus edulis. However, their the Web (www.submit2science.org) or by regular
up-to-date science and could be more trans- analysis is confounded by three issues. Samples from mail (1200 New York Ave., NW, Washington, DC
parent to the public. The revisions to this some sites are likely to have included a second species, 20005, USA). Letters are not acknowledged upon
M. trossulus; their manipulation of chemical cues does not receipt, nor are authors generally consulted before
process will result in more timely and trans- preclude other interpretations; and they failed to establish
parent reviews that do deliver up-to- publication. Whether published in full or in part,
an adaptive significance to shell thickening.
letters are subject to editing for clarity and space.
date research. Full text at www.sciencemag.org/cgi/content/full/316/
The article states that these improve- 5821/53b
Off-The-Shelf Peptide Microarrays Customized Peptide Arrays Applications

Kinase Peptide Microarrays PepSpot™ Systematic study of protein
> 5 000 annotated kinase substrate Peptide scans or collections of peptides protein interactions
peptides on glass slides on cellulose membranes Antibody epitope mapping,
Fast (≤ 2 weeks turnaround) mapping of immunodominant
PhosphoSite Detector
Affordable (< $ 5/peptide) regions
Peptide scans through > 70 kinase
Flexible (≤ 5 000 PepSpots/membrane)
substrate proteins on glass slides Identification of substrates
Protease Peptide Microarrays PepStar™ for kinases, proteases, and
Economical access to identical peptide phosphatases
> 2 000 fluorescent peptides from
microarrays on glass slides Profiling of substrate
annotated protease cleavage sites
Fast (≤ 3 weeks turnaround) specificities
Phosphatase Peptide Microarrays Affordable (from < $ 200/chip)
> 2 000 phosphopeptides on glass slides Flexible (≤ 5 000 peptides/slide) Testing of inhibitors
derived from annotated phosphosites and many more
JPT Peptide Technologies | [email protected] | www.jpt.com

T +49-30-6392-7878 | F +49-30-6392-7888 | USA/Canada T 1-888-578-2660 | F 1-888-578-2666
BOOKS ET AL.
HISTORY OF SCIENCE monograph to other contemporary scientific
work on the Cirripedia and concludes by
Seeing Is Believing? comparing them to the illustrations in books
that inspired and sustained the mid-Victorian
Harriet Ritvo craze for aquaria and tide pool naturalizing.
Perhaps the most interesting chapter is the
A
s Jonathan Smith notes at the begin- sexual selection. Otherwise, Smith focuses on final one, which analyzes the images in The
ning of Charles Darwin and Victor- Darwin’s early monograph on barnacles, his Formation of Vegetable Mould Through the
ian Visual Culture, Darwin’s most late experimental works on plants, and his Action of Worms, published the year before
famous book is almost completely devoid of final volume on worms. Darwin’s death. In it, Darwin returned to his
illustration. There is only one illustration in On The chapter titles (all of the same form: early fascination with geology, combining
the Origin of Species, a powerfully abstract “Darwin’s Barnacles,” “Darwin’s Birds,” the epochal and the domestic scales as
diagram of branching evolutionary descent. In etc.) belie the complexity and he described the role of earth-
this, as in much else, the Origin was unusual. richness of Smith’s interpre- worms in producing soil
Illustration was a highly valued feature of tation. Smith anchors each Charles Darwin and and burying the evidences of
19th-century work in zoology, botany, and chapter in the explication of Victorian Visual Culture human civilization. The book
geology, whether it was intended for special- specific images. He does a by Jonathan Smith contained no earthworm por-
ists or for a broader public. Most naturalists very good job of explaining Cambridge University
trait and only one diagram of
(although not Darwin) were adept draftsmen, their significance, strongly Press, Cambridge, 2006. the annelid digestive system.
and there was a ready market even for expen- supported by the generous 374 pp. $100, £60. According to Smith, this omis-
sive volumes with lavishly produced plates. design of the book: not only ISBN 9780521856904. sion highlights the functional
Like most of his contemporaries, Darwin are there plenty of illustra- Cambridge Studies in or utilitarian emphasis of
understood that images could function as argu- tions, but most of them are Nineteenth-Century Darwin’s argument: what is
ment as well as description. It is well known large enough for their details Literature and Culture. interesting about the worms is
that he considered the Origin to be a mere to be discernable. Smith’s that by passing earth through
abstract of the much longer and more fully explanations require him to their digestive system they
substantiated treatise that he would have writ- integrate many separate threads of scholar- transform the landscape—both by turning it
ten if he had not feared being scooped by ship, including the history of the book, art over and by enriching it with their castings
Alfred Russel Wallace. In his more leisurely history, and the history of science, as well as (excrement). Several of the illustrations are
and typical works, Darwin made frequent and Victorian literary and cultural studies. For rather monumental representations of worm
subtle use of visual images. example, the chapter on barnacles begins by castings, which itself gave offense to those
Smith (a professor of English at the comparing the illustrations in Darwin’s committed to a more rarefied ideal of the art
University of Michigan, Dearborn) examines of natural history. The rest are of objects—
many of these illustrations in detail, discussing whether monuments like Stonehenge or more
them both in relation to the written texts they modest efforts like Roman villas or random
accompanied and in relation to the larger sci- stones in cultivated fields—gradually being
entific and popular visual cultures from which submerged under the slowly accumulated by-
CREDIT: FIG. 3 FROM C. DARWIN, THE FORMATION OF VEGETABLE MOULD THROUGH THE ACTION OF WORMS
they were drawn. He argues persuasively that products of earthworm metabolism.
Darwin’s images should not be considered Carefully considered, none of these images
merely as adornment, or even as illustration in would have given much comfort to the cultural
the rhetorical sense, but as a thoroughly inte- critics (of whom John Ruskin was the most
grated component of the work in which they prominent) who had long complained about
appear. He thus refers to Darwin’s works as what they considered the degrading influence
“imagetexts” (he did not coin this rather pon- of the materialist aesthetic implicit in Darwin’s
derous neologism). work. In particular, his arguments about sexual
Perhaps as a way of emphasizing the per- selection suggested that the human sense of
sistence of Darwin’s concern with the visual beauty was shared by animals (or at least that it
dimension of his books, Smith devotes most of was very similar to a faculty that animals
his attention to the beginning and the end of also possessed) and that it had developed in
Darwin’s career. Of the series of works in response to the exigencies of reproductive
which Darwin elaborated the schematic argu- competition rather than as a reflection of some
ments of the Origin, only two receive much higher mode of perception. Perhaps still more
attention: The Expression of the Emotions in disturbing to Victorian convention, Darwin
Man and Animals, which is extensively dis- suggested that in many cases the definitive
cussed in the two chapters on faces, and The aesthetic judgment of strutting males was exer-
Descent of Man, which figures in recurrent cised by females (although he endorsed other
considerations of the aesthetic implications of conventional ideas about human gender).
One of the many strengths of the book is
The reviewer is at the Department of History, Massachusetts “Grotesque realism.” Darwin used three engrav- that, in addition to placing Darwin’s use of
Institute of Technology, Cambridge, MA 02139, USA. ings from photographs to support his argument that imagery in several contemporary contexts,
E-mail: [email protected] worms pass large quantities of castings. Smith traces Darwin’s impact on the popular

BOOKS ETAL.
visual culture of his time. Of course this medical establishment such as The claim that medicine is golem-like
impact increased dramatically after 1859. Its uncertainties over the diagnosis is far from novel, and as the authors
most conspicuous products were not the of tonsillitis. It is likely that put it: “The really hard question that
heartfelt laments of the artistic elite, but sev- our perceptions of medicine, remains is, ‘Knowing medicine
eral still-familiar stock caricatures or car- unlike most other branches is fallible, what should we do?’”
toons such as the ape or monkey with a of science and technology, Although Dr. Golem indeed
human face (in the early days the face often already include some knowl- may lead readers to reflect on
bore Darwin’s own easily recognizable fea- edge of its successes and their experiences with medicine
tures) and the staggered evolutionary trans- failures and of the gaps and its limitations (particularly
formation from a monkey (or, sometimes, in its existing theories. None- with regard to prognosis and
from a fish or lizard) to an ape to a caveman theless, documenting ex- therapy), it unfortunately fails
to a modern human. The fact that these amples of these failures and to provide much guidance about
images continue to provoke a range of reac- gaps is a useful exercise, what in fact patients should do
tions very similar to those they raised a cen- in part as an antidote to in light of this knowledge. For
tury and a half ago illustrates the importance increasing public confi- example—perhaps because the
of studies like Smith’s, which can help us to dence that cures and an- book arose implicitly in parallel
understand our own time as well as Darwin’s. swers are just around the to considerations of science
10.1126/science.1141442 corner. This has been evi- and technology—there is little
dent in much of the hype engagement with issues of per-
MEDICINE surrounding genetic, stem sonal values and ethics in rela-
cell, and cancer research. tion to medical decision-making
Placebo or Protector? The book explores eight topics,
beginning with the placebo effect. Collins and
or with the political, social, and eco-
nomic contexts within which medicine (par-
Rachel A. Ankeny Pinch describe it as “the hole in the heart of ticularly in underdeveloped countries) is prac-
medicine” as it encapsulates the problematic ticed. Nor do the authors examine the art or
T
he traditional Jewish myth of the golem nature of nonscientific aspects of medicine craft of medicine in any detail. Hence they
centers on a human-like creature cre- and their impact on any attempts to scientize ignore long-standing debates in the philoso-
ated by humans out of clay and water. It medicine. A provocative chapter covers the phy of medicine over the other skills (such as
will follow orders and is very powerful yet dubious acceptance of cardiopulmonary resus- communication and diagnostic talent) that are
also is a bit stupid and clumsy and hence dan- citation (CPR). They note that critics have clearly central to good medical practice. In
gerous. The golem has become a familiar argued that CPR at best is nothing more than a fact, they set up an odd dichotomy between
trope in popular culture, making appearances “passing ritual” (whose primary use is to allow medicine as science and medicine as “succor,”
in literary novels such as Umberto Eco’s families more time with a dying loved one) and which implicitly denies that nonscientific
Foucault’s Pendulum (1) and even in the at worst is dangerous and a substantial waste of forms of knowledge might play important
Pokémon games. It serves as an especially resources in terms of training and equipment. roles beyond providing comfort when science
appropriate framing metaphor A less successful chapter on cannot cure—a striking omission for scholars
for the series of books focused Dr. Golem “alternative medicine” focuses quick to recognize alternative forms of expert-
on science and technology by How to Think About on the early-1970s controversy ise in other settings. They leave underexplored
the sociologists and science Medicine that surrounded Linus Paul- the hybrid nature of medicine as a pragmatic,
studies scholars Harry Collins ing’s championing of large scientific, artistic, and social undertaking.
(Cardiff University) and Trevor by Harry Collins and doses of vitamin C as a cure for In a sense, Collins and Pinch appear to have
Trevor Pinch
Pinch (Cornell University) cancer. This case study fails to used Dr. Golem to confront their own personal
(2, 3). In this latest installment, University of Chicago do justice to the original schol- demons: faced with the need to make personal,
Dr. Golem, we are taken on a Press, Chicago, 2005. arly research on which it is life-and-death decisions, they seem to want to
tour of the “messiness” that 258 pp. $25, £17.50. based. Nor does it provide an hold medicine to higher standards than we
ISBN 9780226113661.
characterizes medicine in order adequate examination of the might hold other forms of science. The prob-
to reflect on the complexities complex issues associated with lem is that when viewed in this limited and ide-
inherent in medical science and practice and today’s complementary and alternative med- alized way, medicine falls so far short that it is
on how we should make medical choices ical theories and practices (which are only unclear how we should understand the impli-
given these uncertainties. briefly touched within the placebo chapter). cations of these case studies for any medical
CREDIT: COURTESY UNIVERSITY OF CHICAGO PRESS
To illustrate medicine’s recognized fallibil- Collins and Pinch also integrate some of choices with which we might be faced.
ity, the authors present a series of case studies their own experiences with medicine into their
that popularize existing scholarly accounts. narrative. Although these personal threads References
These range from familiar debates over the may sometimes annoy a more scholarly- 1. U. Eco, Foucault’s Pendulum (Secker and Warburg,
London, 1989).
definition of death and contested disease cate- minded reader, they do help illustrate dis- 2. H. Collins, T. Pinch, The Golem: What Everyone Should Know
gories such as chronic fatigue and fibromyal- agreements between the authors (for instance, About Science (Cambridge Univ. Press, Cambridge, 1993);
gia to more mundane interactions with the about measles, mumps, and rubella vaccina- reviewed by U. Segerstråle, Science 263, 837 (1994).
3. H. Collins, T. Pinch, The Golem at Large: What You Should
tion). The authors emphasize that this book Know About Technology (Cambridge Univ. Press,
The reviewer is at the School of History and Politics,
forced them to consider not only what to think Cambridge, 1998).
University of Adelaide, Adelaide, SA 5005, Australia. but also what to do—what choices to make
E-mail: [email protected] when faced with difficult medical decisions. 10.1126/science.1124931

POLICYFORUM
SCIENCE AND SOCIETY
Framing Science To engage diverse publics, scientists must

focus on ways to make complex topics
personally relevant.
Matthew C. Nisbet1 * and Chris Mooney2
I
ssues at the intersection of science and pol- is attributable to human activity, compared demonstrates, many scientists not only fail to
itics, such as climate change, evolution, and with 75% of Democrats (6). Regardless think strategically about how to communicate
embryonic stem cell research, receive con- of party affiliation, most Americans rank on evolution, but belittle and insult others’
siderable public attention, which is likely to global warming as less important than over religious beliefs (8).
grow, especially in the United States as the a dozen other issues (6). Much of this On the embryonic stem cell issue, by com-
2008 presidential election heats up. Without reflects the efforts of political operatives parison, patient advocates have delivered a
misrepresenting scientific information on and some Republican focused message to
highly contested issues, scientists must learn to leaders who have em- the public, using “soc-
actively “frame” information to make it rele- phasized the frames of ial progress” and “eco-
vant to different audiences. Some in the scien- either “scientific un- nomic competitive-
tific community have been receptive to this certainty” or “unfair “… citizens do ness” frames to argue
message (1). However, many scientists retain economic burden” (7). not use the that the research offers
the well-intentioned belief that, if laypeople In a counter-strategy, news media as hope for millions of
better understood technical complexities from environmentalists Americans. These mes-
scientists
news coverage, their viewpoints would be more and some Democratic sages have helped to
like scientists’, and controversy would subside. leaders have framed assume.” drive up public support
In reality, citizens do not use the news global warming as for funding between
media as scientists assume. Research shows a “Pandora’s box” of 2001 and 2005 (9, 10).
that people are rarely well enough informed or catastrophe; this and However, opponents of
motivated to weigh competing ideas and argu- news images of polar increased government
ments. Faced with a daily torrent of news, cit- bears on shrinking ice floes and hurricane funding continue to frame the debate around the
izens use their value predispositions (such as devastation have evoked charges of “alarm- moral implications of research, arguing that
political or religious beliefs) as perceptual ism” and further battles. scientists are “playing God” and destroying
screens, selecting news outlets and Web sites Recently, a coalition of Evangelical lead- human life. Ideology and religion can screen
whose outlooks match their own (2). Such ers have adopted a different strategy, framing out even dominant positive narratives about
screening reduces the choices of what to pay the problem of climate change as a matter of science, and reaching some segments of the
attention to and accept as valid (3). religious morality. The business pages tout public will remain a challenge (11).
Frames organize central ideas, defining the economic opportunities from developing Some readers may consider our proposals
a controversy to resonate with core values and innovative technologies for climate change. too Orwellian, preferring to safely stick to the
assumptions. Frames pare down complex Complaints about the Bush Administration’s facts. Yet scientists must realize that facts will be
issues by giving some aspects greater empha- interference with communication of climate repeatedly misapplied and twisted in direct pro-
sis. They allow citizens to rapidly identify why science have led to a “public accountability” portion to their relevance to the political debate
an issue matters, who might be responsible, frame that has helped move the issue away and decision-making. In short, as unnatural as it
and what should be done (4, 5). from uncertainty to political wrongdoing. might feel, in many cases, scientists should
Consider global climate change. With its As another example, the scientific theory strategically avoid emphasizing the technical
successive assessment reports summarizing of evolution has been accepted within the details of science when trying to defend it.
the scientific literature, the United Nations’ research community for decades. Yet as a
Intergovernmental Panel on Climate Change debate over “intelligent design” was launched, References
1. T. M. Beardsley, Bioscience 56, 7 (2006). www.aibs.org/
has steadily increased its confidence that antievolutionists promoted “scientific uncer- bioscience-editorials/editorial_2006_ 07.html.
human-induced greenhouse gas emissions are tainty” and “teach-the-controversy” frames, 2. S. L. Popkin, The Reasoning Voter (Univ. of Chicago Press,
causing global warming. So if science alone which scientists countered with science-inten- Chicago, IL, 1991).
3. J. Zaller, Nature and Origins of Mass Opinion (Cambridge
drove public responses, we would expect in- sive responses. However, much of the public Univ. Press, New York, 1992).
creasing public confidence in the validity of likely tunes out these technical messages. 4. W. A. Gamson, A. Modigliani, Am. J. Sociol. 95, 1 (1989).
the science, and decreasing political gridlock. Instead, frames of “public accountability” that 5. V. Price, et al., Public Opin. Q. 69, 179 (2005).
Despite recent media attention, however, focus on the misuse of tax dollars, “economic 6. Pew Center for the People and the Press (2007); http://
pewresearch.org/pubs/282/global-warming-a-divide-on-
many surveys show major partisan differ- development” that highlight the negative causes-and-solutions.
ences on the issue. A Pew survey conducted repercussions for communities embroiled in 7. A. M. McCright, R. E. Dunlap, Soc. Probl. 50, 3 (2003).
in January found that 23% of college- evolution battles, and “social progress” 8. Film promotion, www.flockofdodos.com/
9. Virginia Commonwealth University Life Sciences Survey
educated Republicans think global warming that define evolution as a building block for (2006); www.vcu.edu/lifesci/images2/ls_survey_2006_
CREDIT: PHOTOS.COM
medical advances, are likely to engage report.pdf

1School of Communication, American University, Wash- broader support. 10. Pew Center for the People and the Press (2006); http://
ington, DC 20016, USA. 2Washington correspondent, Seed peoplepress.org/reports/display.php3?ReportID=283.
The evolution issue also highlights another
magazine (seedmagazine.com). 11. M. C. Nisbet, Int. J. Public Opin. Res. 17 (1), 90 (2005).
point: Messages must be positive and respect
*Author for correspondence. E-mail: [email protected] diversity. As the film Flock of Dodos painfully 10.1126/science.1142030

PERSPECTIVES
NEUROSCIENCE
We may be able to learn new things very
quickly, and incorporate them into memories,
Rapid Consolidation if representations of related information have
already been stabilized in the brain.
Larry R. Squire
Neocortex
W
e learn and remember better when
new material can be related to what
we already know. Professional ath-
letes can remember details of particular plays
that occurred in a long match. Experienced RAT BRAIN
poker players can reconstruct the card distri- Hippocampus
bution and betting sequence that occurred in
previous hands. This is possible because these
individuals have a rich background of relevant
experience and therefore can organize new
material into meaningful and orderly patterns.
On page 76 of this issue, Tse et al. (1) make
use of these ideas to explain their new find-
ings in rats.
In his classic 1932 monograph on remem-
bering (2), British psychologist Frederic Bartlett
developed the concept of “schemas” to refer to
preexisting knowledge structures into which
newly acquired information can be incorpo-
rated. Although the schema concept is funda-
mental to the psychological science of human
memory, it has been difficult to bring the con- Banana flavor Pear flavor Melon flavor New flavors
cept to biology and especially to studies with
experimental animals. Tse et al. show in rats
how the schema concept is relevant to the phe- GRADUAL LEARNING ONE-TRIAL LEARNING
nomenon of memory consolidation. Memory Good schemas wanted. When a rat learns associations between flavors and spatial locations, as studied by
consolidation refers to the gradual process Tse et al. (1), the associations are initially learned as individual facts (left). With extended training, the
of reorganization by which new memories animal develops an organized structure or schema for flavors and places (middle). This organized knowledge
become remote memories (3, 4). Initially, the structure (bold lines) can then support rapid learning of new associations in a single trial and the rapid
learning of facts and events (declarative mem- consolidation of information into the neocortex (right).
ory) depends on the hippocampus, a structure
deep in the temporal lobe of the mammalian correct location in the arena. Animals learned vor-place combinations rearranged every two
brain. As time passes after learning, the impor- the six flavor-place associations gradually sessions (the inconsistent arena). In the con-
tance of the hippocampus gradually diminishes across 6 weeks of training; each flavor-place sistent arena, animals could then learn new
and a more permanent memory is established pair was presented once per session for flavor-place associations in a single trial, as
in distributed regions of the neocortex. This training and three sessions were scheduled before. By contrast, in the inconsistent arena,
process typically takes a few years in humans each week. Not surprisingly, animals with animals failed at one-trial learning, presum-
and at least a month in rodents. According to hippocampal lesions failed to learn the ably because they had not established a stable
one influential model (5), the process is slow associations. schema that could guide rapid learning.
because if changes were made rapidly, they Evidence that flavor-place training af- The most surprising finding by Tse et al.,
would interfere with the preexisting framework forded the development of a schema came and what connected the schema concept to
of structured knowledge that has been built up from finding that animals were subsequently memory consolidation, was that removal of
from other experiences. able to learn new flavor-place associations in a the entire hippocampus as early as 48 hours
In the Tse et al. study, rats learned to asso- single trial and could remember the new asso- after the rapid learning of two new flavor-
ciate six flavors with six places in a familiar ciations for at least 2 weeks (see the figure). place associations fully spared memory of the
testing arena. A rat was first cued with a par- The extended training had helped because in a associations (these animals had first been
ticular flavor (a 0.5-g morsel of flavored food) similar task in which rats were trained on a given extensive training on six other flavor-
in one of four start boxes and then could new flavor-place association each day (6), place associations, thus establishing a schema).
receive more of the same food by going to the memory was only weakly established and per- It was not the case that memory of the new
CREDIT: P. HUEY/SCIENCE
sisted for less than a day. Tse et al. also tested associations was never dependent on the hip-
other rats that learned six flavor-place associ- pocampus, nor that memory was somehow
The author is at the VA Medical Center, San Diego, CA ations in one arena (the consistent arena) and formed directly in the neocortex, because hip-
92161, USA, and the Departments of Psychiatry, Neuro-
sciences, and Psychology, University of California, San concurrently learned six different flavor-place pocampal lesions made 3 hours after learning
Diego, CA 92093, USA. E-mail: [email protected] associations in another arena but with the fla- abolished memory of the new associations. In

PERSPECTIVES
short, the neocortex was able to incorporate and rapid consolidation of new associations? not that memory is literally transferred from
new information rapidly. This is unexpectedly Answers to these questions would help hippocampus to neocortex, but rather that the
rapid for a process that, on the basis of as sharpen the notion of schema and clarify the hippocampus guides gradual changes in the
many as 20 studies in experimental animals, conditions under which rapid memory consol- neocortex that increase the complexity, distri-
ordinarily takes at least a month (7). idation occurs. bution, and interconnectivity of memory stor-
It is tempting to suppose that memory con- The study also casts fresh light on an issue age sites. This new study is the first to show
solidation proceeded rapidly because new of long-standing interest. As the authors point that this process can occur rapidly.
information was fully compatible with what out, the fact that storage and recall of spatial
had already been learned—in other words, a memory can occur independently of the hip- References
1. D. Tse et al., Science 316, 76 (2007).
good schema was available. If so, questions pocampus runs counter to the proposal that the 2. F. C. Bartlett, Remembering (Cambridge Univ. Press,
naturally arise about the minimum require- hippocampus forms and stores cognitive maps Cambridge, 1932).
ments for an effective schema. Perhaps con- (8). Thus, the new findings, as well as other 3. L. R. Squire, P. Alvarez, Curr. Opin. Neurobiol. 5, 169
(1995).
tinued training with two new flavor-place work (9), give no special emphasis to spatial
4. P. W. Frankland, B. Bontempi, Nat. Rev. Neurosci. 6, 119
pairs every day would be sufficient (not just cognition and suggest instead that the hip- (2005).
extended training with the same set of pairs, as pocampus is a general-purpose learner of new 5. J. L. McClelland, B. L. McNaughton, R. C. O’Reilly,
was done by Tse et al.). Although new associ- facts and events, both spatial and nonspatial. Psychol. Rev. 102, 419 (1995).
6. M. Day, R. Langston, R. G. M. Morris, Nature 424, 205
ations trained in this way ordinarily persist for The larger question concerns the nature of (2003).
less than a day (6), with extended experience memory consolidation itself. Recent studies of 7. L. R. Squire, R. E. Clark, P. J. Bayley, in The Cognitive
they might persist much longer and also brain metabolism and activity-related genes in Neurosciences III, M. Gazzaniga, Ed. (MIT Press,
Cambridge, MA, 2004).
become rapidly independent of the hippocam- mice describe the decreasing importance of 8. J. O’Keefe, L. Nadel, The Hippocampus as a Cognitive
pus. And what might happen if animals simply the hippocampus as time passes after learning Map (Oxford Univ. Press, Oxford, 1978).
explored the same arena day after day? Would and the increasing importance of several corti- 9. L. R. Squire, P. J. Bayley, Curr. Opin. Neurobiol., in press.
merely having strong familiarity with a con- cal regions, including the prefrontal, temporal,
sistent environment support the rapid learning and anterior cingulate cortex (4). The idea is 10.1126/science.1141812
BIOCHEMISTRY
A mechanism that controls the stepwise
movement of a molecular motor along a
Processive Motor Movement filamentous microtubule track in the cell does
not seem to require the track itself.
David D. Hackney
P
ermeating throughout a eukaryotic cell motility occurs. On page 120 of this issue, duce the nonequivalence of the heads that is
is a lattice of filamentous tracks called Alonso et al. (2) show that a microtubule needed for processive movement.
microtubules, upon which molecular track is not necessary for such gating to The first indication of a gate with either
motors travel, moving cargo about. In occur. This throws a surprising wrench into a monomer or dimer of kinesin (one or two
this transport system, the molecular existing models of kinesin movement and heads, respectively) was that steady-state ATP
motor kinesin-1 carries relatively large loads suggests that the track is not required to pro- hydrolysis rates are low unless microtubules
(molecular complexes, membranous vesicles,
and organelles), its motion powered by the
SOLUBLE TUBULIN GATES
energy liberated from hydrolyzing adenosine KINESIN DIMER
triphosphate (ATP) (1). In fact, kinesin-1 can ADP
ADP
move processively for long distances (over
a micrometer) along a microtubule without
falling off. This feat requires coordinating the This gate is closed and
two motor domains (“heads”) of kinesin so TUBULIN ADP cannot be released. ATP H2O
that one head is always attached to the micro- TETHERED
tubule while the other is detached and moving INTERMEDIATE
further along the track, in the direction of ADP ATP binding to the ADP Pi
movement. The proposed molecular mecha- nucleotide-free
head opens the ADP
nism underlying this processive motion has ADP
gate and allows
been highly debated. One controversial point ADP release from
the other head.
has been how an operation called “gating” (–) (+)
proceeds, that is, how the ATPase cycle of a MICROTUBULE
kinesin head is stalled until a specific bind- MICROTUBULE GATES
ing or conformational change needed for Kinesin’s gates. Kinesin has two motor domains (heads; red and yellow) joined by a coiled coil. A tethered inter-
mediate of kinesin (shown in one possible conformation) is gated if release of ADP from one head is blocked. ATP
The author is in the Department of Biological Science, can bind and trigger interchange of heads that bind to a microtubule track. This is coupled to release of ADP,
Carnegie Mellon University, Pittsburgh, PA 15213, USA. hydrolysis of ATP, and release of phosphate (Pi). Alonso et al. find that soluble tubulin also has a gate that blocks
E-mail: [email protected] binding of another tubulin molecule and release of the second ADP. This gate can also be opened by ATP.

PERSPECTIVES
are present (3). This gate, much like a check- albeit weakly and reversibly, permitting rapid Additionally, gate-limiting ADP release
point, occurs because adenosine diphosphate dissociation if not tethered to the microtubule does not apply to ATP synthesis. The tethered
(ADP) release from kinesin is very slow (gate by the strongly attached head. Rice et al. (6) intermediate synthesizes ATP from ADP and
closed) until binding of a microtubule to the reported that the position of the neck linker inorganic phosphate at a rate that is 20 times
kinesin-ADP complex opens the gate (4). region that joins the head to the coiled coil as fast as that of a monomer (12). ATP syn-
Alonso et al. show that this gate can also be dimerization region of kinesin is coupled to thesis requires microtubule contact, and
opened by soluble tubulin heterodimers, the nucleotide binding. In that analysis, the lead the more rapid synthesis by dimers argues
protein constituent of microtubules. head (closest to the plus end of the micro- against an intermediate state in which a teth-
Another gate coordinates the actions of tubule) can release ADP because its neck ered head with ADP is not in contact with a
both heads. When kinesin, with ADP bound to linker is directed backward. The trailing head microtubule for at least part of the time in the
each head, associates with a microtubule, only cannot release ADP because its neck linker presence of phosphate. This favors state I,
half the ADP is released (5) and a “tethered is directed forward (state I). A less likely con- because the Rice et al. (6) model is consistent
intermediate” forms, with one head attached formation (state IV) orients the neck linkers with more facile ATP synthesis when the
tightly to the microtubule in the nucleotide- in opposite direction. Gating occurs in this neck linker is directed forward.
free form and the other head retaining model because the head in the trailing position The experiments underlying these con-
its bound ADP. This gate prevents further is forced to have its neck linker directed flicting conclusions regarding the confor-
progress until ATP binds to the nucleotide- toward the plus end of the track, and this ori- mation of the tethered dimer each have their
free head and allows ADP release limitations. Fluorescence polariza-
from the tethered head to occur—a so- I II III IV tion experiments (13) favor state I,
called “ATP waiting state” (see the ADP ADP or IV during processive movement
first figure). ATP hydrolysis returns KINESIN DIMER at saturating ATP concentrations,
ADP
the dimer to the same ATP waiting ADP but the conformation of the
state, but with the roles of the heads ATP waiting state may be different.
(–) (+)
reversed and with net movement of MICROTUBULE Measurements by fluorescence
one binding site in the direction of resonance energy transfer are con-
POSSIBLE CONFORMATIONS OF TETHERED INTERMEDIATE
movement (toward the plus end of the sistent with state I (14), but do not
polarized microtubule track). In waiting. Shown are four possible conformations for the tethered inter- exclude states II or III. Structural
A reasonable explanation for the mediate of kinesin. Each state awaits binding to ATP. studies by electron microscopy
failure of the tethered intermediate to require that the microtubule lattice
release the second ADP is that constraints of entation is unfavorable for ADP release. be fully saturated with kinesin heads.
kinesin’s dimer interface prevent the tethered Alonso et al. suggest this cannot be the mech- However, heads often bind cooperatively (9,
head from also interacting productively with anism because the gate is still present with sol- 15). Thus, the conformation of an isolated
the microtubule to induce rapid ADP release. uble tubulin, which could bind to both heads dimer on a bare tubulin lattice may be differ-
But Alonso et al. show that even soluble tubu- without forcing the neck linker of either one ent from that in a localized region of inter-
lin heterodimers that are freely available for into a conformation that is unfavorable for acting dimers packed together on the lattice.
binding (not restricted within a track struc- ADP release. Rather, the authors favor the Clearly, kinesin has more stories to tell, and
ture) cannot open this gate. However, the gate conformation in which the tethered head, further work will be required to fully distin-
can still be opened by the addition of ATP or a bound to ADP, does not contact the micro- guish between the different possible inter-
nonhydrolyzable ATP analog. tubule but is docked against the other head mediary states.
These new results bear on unresolved (state III). In this conformation, they ascribe
issues about the conformation of the tethered the failure of kinesin to bind two soluble tubu- References
1. C. J. Lawrence et al., J. Cell Biol. 167, 19 (2004).
intermediate and the mechanism that controls lin molecules to either a conformational 2. M. C. Alonso et al., Science 316, 120 (2007).
the gate. Four possible conformations for the change in the tethered head or to a steric con- 3. S. A. Kuznetsov, V. I. Gelfand, Proc. Natl. Acad. Sci. U.S.A.
tethered intermediate can be considered (see flict that prevents binding two tubulins in the 83, 8530 (1986).
the second figure). All have one nucleotide- absence of nucleotide on both heads. 4. D. D. Hackney, Proc. Natl. Acad. Sci. U.S.A. 85, 6314
(1988).
free head tightly associated with the micro- The analysis of Alonso et al. is heavily 5. D. D. Hackney, Proc. Natl. Acad. Sci. U.S.A. 91, 6865
tubule but differ in the position of the tethered influenced by electron microscopy of (1994).
head, which contains ADP and is free kinesin bound to microtubules in which 6. S. Rice et al., Nature 402, 778 (1999).
or weakly bound to the microtubule. In two density observed off the microtubule sur- 7. K. Hirose, J. Lowe, M. Alonso, R. A. Cross, L. A. Amos, Mol.
Biol. Cell 10, 2063 (1999).
cases, the tethered head with ADP is held off face is attributed to the tethered head (state 8. I. Arnal, R. H. Wade, Structure 6, 33 (1998).
the surface of the microtubule, with either III) (7, 8). Others, however, report that both 9. A. Hoenger et al., J. Mol. Biol. 297, 1087 (2000).
considerable diffusional freedom (state II) or heads bind to a microtubule (states I or IV) 10. A. Hoenger et al., Biol. Chem. 381, 1001 (2000).
docked against the other head that is attached and that any extra density off the micro- 11. A. Yildiz, M. Tomishige, R. D. Vale, P. R. Selvin, Science
303, 676 (2004).
to the track (state III). In both cases, the microtubule surface is due to binding of a second 12. D. D. Hackney, Proc. Natl. Acad. Sci. U.S.A 102, 18338
tubule should not stimulate release of ADP layer of kinesin (9, 10). Evidence specifi- (2005).
from the tethered head because there is no cally for state I or IV also comes from 13. A. B. Asenjo, N. Krohn, H. Sosa, Nat. Struct. Mol. Biol.
10, 836 (2003).

contact between them. the observed alternation of 16- and 0-nm
14. M. Tomishige, N. Stuurman, R. D. Vale, Nat. Struct. Mol.
Reasons for proposing why the two other “steps” that a head makes along a micro- Biol. 13, 887 (2006).
possible conformations cannot release ADP tubule (11). This is consistent with state I or 15. A. Vilfan et al., J. Mol. Biol. 312, 1011 (2001).
must be more subtle because the tethered head IV but not with state III, which would have
is actually in contact with the microtubule, more steps of similar length. 10.1126/science.1141549

PERSPECTIVES
CHEMISTRY
Terpenoid synthases generate an extraordinarily
diverse set of natural products. Evolution of these
Roots of Biosynthetic Diversity enzymes from a common α-helical ancestor has
resulted in profound changes in activity.
David W. Christianson
W
ith more than 50,000 compounds 75% sequence identity to that of FPPase from the generation of 15-carbon to 10-carbon prod-
serving myriad functions in all forms the same species, yet catalyzes a different ucts, and chimeras that are predominantly
of life, the terpenoids or isoprenoids chemical reaction. CPPase-like generate 10-carbon cyclopropana-
are the most structurally and stereochemically The CPPase-FPPase chimeras exhibit re- tion and branching products. When incubated
diverse family of natural products found on markable trends in biosynthetic versatility. For solely with DMAPP, cyclobutanation products
Earth (1). Despite the complexity and magni- example, as the chimeras become increasingly are generated by chimeras that are predomi-
tude of this chemical library—referred to as the CPPase-like and are incubated with IPP and nantly FPPase-like, a cyclopropanation prod-
terpenome—all terpenoid compounds ulti- DMAPP, chain-elongation activity shifts from uct is generated by chimeras that are predomi-
mately derive from one or both of two simple
precursors: isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP) (see the
first figure) (2). These molecules can be linked
together in head-to-tail fashion by enzymes that
catalyze chain-elongation reactions, or in a MULTISTEP CYCLIZATION CASCADES
more irregular fashion by enzymes that catalyze
cyclopropanation, branching, or cyclobuta-
nation reactions. On page 73 of this issue,
Thulasiram and co-workers provide compelling
evidence that the metal-dependent synthases H H
that catalyze these fundamental coupling reac- Taxadiene
tions diverged from a common ancestor early in
the evolution of terpenoid biosynthesis (3). PPO
The authors created a series of “chimeric” Geranylgeranyl diphosphate
proteins—artificial enzymes constructed by CH3
replacing segments of a synthase involved in SQSase + FPP TSase
PPO
+IPP
chain elongation with corresponding seg- CH3
ments from another synthase involved in Presqualene diphosphate Trichodiene
cyclopropanation. The synthases in question PPO
are farnesyl diphosphate synthase (FPPase), Farnesyl diphosphate
which catalyzes the chain-elongation reaction
with IPP and DMAPP, and chrysanthemyl +IPP BPPase
OPP
diphosphate synthase (CPPase), which cat- Limonene Bornyl diphosphate
alyzes the cyclopropanation reaction between PPO
two DMAPP molecules (see the first figure). Geranyl diphosphate
The structures of FPPases shed light on
how terpenoid synthases are able to generate FPPase Chain elongation
and manipulate highly reactive carbocation
intermediates in catalysis. The first structure, PPO
of avian FPPase, showed that this distinctive IPP
+
chemistry is facilitated in a hydrophobic
active-site cleft nested within an α-helical fold PPO
DMAPP
(4). Further insight into the mechanism of CPPase
chain elongation has been gained from the Cyclobutanation Branching Cyclopropanation
+ DMAPP + DMAPP + DMAPP
structure of Escherichia coli FPPase com- H
plexed with IPP and an unreactive DMAPP H
PPO PPO
PPO
analog, showing that the active-site contour is
a template that binds the flexible isoprenoid
reactants with the proper orientation and con- Maconellyl diphosphate Lavandulyl diphosphate Chrysanthemyl diphosphate
formation for catalysis (5). The α-helical fold
Complexity from simple roots. Family tree of terpenomic diversity (2), showing examples of 10-, 15-, 20-,
of CPPase from snowfield sagebrush shows and 30-carbon natural products generated by terpenoid synthases that share the FPPase fold. Isoprene groups
are colored to trace their biosynthetic fates; newly formed carbon-carbon bonds are in green. Thulasiram and
The author is in the Roy and Diana Vagelos Laboratories, co-workers show that the α-helical FPPase fold can be mutated in CPPase-FPPase chimeras to catalyze all four
Department of Chemistry, University of Pennsylvania, coupling reactions at the base of the tree. BPPase, bornyl diphosphate synthase; PPO, diphosphate; SQSase,
Philadelphia, PA 19104, USA. E-mail: [email protected] squalene synthase; and TSase, trichodiene synthase.

PERSPECTIVES
reshape the active-site template and change

the resulting reaction product or products.
Moreover, the promiscuous activities of ter-
penoid synthases introduced by nature (10) or
by design (3, 11, 12) should enable further
evolution—both in the test tube and in
nature—of an ever-expanding array of natural
FPPase BPPase TSase SQSase products, with virtually limitless potential in
A shared fold. FPPase from E. coli (5) is colored violet-red, representing the successively larger domains of
chemistry, biology, and medicine.
CPPase substituted for FPPase in the sagebrush CPPase-FPPase chimeras reported by Thulasiram et al. For References and Notes
example, the c69f chimera contains the violet domain of CPPase and the blue-red domains of FPPase, and so 1. D. E. Cane, Ed., Isoprenoids, Including Carotenoids and
on. The α-helical fold of FPPase is also shared by terpenoid synthases that generate larger and more complex Steroids, vol. 2, Comprehensive Natural Products
products (6–9). The chemistry (3) and structural biology (6–9) of these synthases suggest divergence from a Chemistry, D. H. R. Barton, K. Nakanishi, O. Meth-Cohn,
common α-helical ancestor in the evolution of terpenoid biosynthesis. Eds. (Elsevier, Oxford, 1999).
2. J. C. Sacchettini, C. D. Poulter, Science 277, 1788 (1997).
3. H. V. Thulasiram, H. K. Erickson, C. D. Poulter, Science
nantly CPPase-like, and a branching product is tion cascade that typically proceeds through 316, 73 (2007).
generated by all CPPase-FPPase chimeras. multiple carbocation intermediates. Some ter- 4. L. C. Tarshis, M. Yan, C. D. Poulter, J. C. Sacchettini,
Biochemistry 33, 10871 (1994).
The CPPase-FPPase chimeras are biosyn- penoid cyclases are high-fidelity templates 5. D. J. Hosfield et al., J. Biol. Chem. 279, 8526 (2004).
thetically more promiscuous than either native that chaperone reactive substrate and interme- 6. J. Pandit et al., J. Biol. Chem. 275, 30610 (2000).
CPPase or FPPase as a result of a reshaped tem- diate conformations to generate a single prod- 7. D. W. Christianson, Chem. Rev. 106, 3412 (2006).
plate for substrate binding, which permits alter- uct, but others tolerate a surprising degree of 8. D. A. Whittington et al., Proc. Natl. Acad. Sci. U.S.A. 99,
15375 (2002).
native trajectories for intermolecular carbon- biosynthetic promiscuity and thereby reflect 9. M. J. Rynkiewicz, D. E. Cane, D. W. Christianson, Proc.
carbon bond formation. Such promiscuity por- significant evolutionary potential. For exam- Natl. Acad. Sci. U.S.A. 99, 13543 (2001).
tends evolution: Given that the FPPase fold is ple, γ-humulene synthase generates 52 differ- 10. C. L. Steele, J. Crock, J. Bohlmann, R. Croteau, J. Biol.
Chem. 273, 2078 (1998).
readily evolvable to catalyze all four of the fun- ent products from substrate farnesyl diphos- 11. Y. Yoshikuni, T. E. Ferrin, J. D. Keasling, Nature 440,
damental isoprenoid coupling reactions, native phate (10). However, mutation of different 1078 (2006).
synthases catalyzing these reactions such as groups of three to five active-site residues 12. B. T. Greenhagen, P. E. O’Maille, J. P. Noel, J. Chappell,
squalene synthase (SQSase, see the figures) (6) reshapes the template, thereby decreasing the 13. I thank the NIH (grant GM56838) and the Biotechnology
likely diverged from an FPPase-like ancestor. overall number of products and altering the and Biological Sciences Research Council (UK) for the
A family of terpenoid cyclases that gener- main product formed (11). The products of Underwood Fellowship supporting my study at the
ate cyclic and polycyclic compounds also other sesquiterpene cyclases can also be University of Cambridge. I am grateful to D. E. Cane and
T. Blundell for stimulating scientific discussions and to L.
shares the FPPase fold (see the second figure) changed through active-site mutations (12). Di Costanzo, H. A. Gennadios, E. Y. Shishova, L. S. Vedula,
(7–9). These enzymes bind a flexible iso- Thus, a recurring theme among the greater and T. Zakharian for assistance with the figures.
prenoid diphosphate substrate with the proper family of terpenoid synthases is that only a
conformation for an intramolecular cycliza- few amino acid substitutions are sufficient to 10.1126/science.1141630
CHEMISTRY
High Bond Orders in Metal-Metal The recent discovery of high-order bonds

between metal atoms raises fundamental
Bonding questions about the nature of chemical bonding.
Frank Weinhold and Clark R. Landis
W
hat is a chemical bond, and how principles articulated by G. N. Lewis more At a superficial level, bonding might
many bonds can be made between than 90 years ago. describe anything that holds atoms together.
any two atoms? Each generation of The advances in question are the quintuple However, chemists generally reject this term
chemists readdresses and refines such ques- bond of a recently synthesized dichromium for weak or nonspecific atom-atom attractions
tions in the framework of its best current species (1) and the hextuple bond proposed for (such as those resulting from gravitational or
experimental and theoretical methods. Recent W2 (2). As was the case following Cotton’s dispersion forces) that cannot withstand the
synthetic and computational advances in report in 1973 of the first metal-metal quadru- jostlings of ambient thermal collisions or that
metal-metal bonding seem to extend the con- ple bond and West’s synthesis in 1981 of the do not exhibit the regularity of atomic valency
cept of bond order to surprising new levels. first Si-Si double bond, these species challenge patterns of the periodic table. “Putting grams
Such expansion of chemical valency is fueling anew our understanding of the meaning and in a bottle” is no longer a must for a chemi-
a reconsideration of fundamental bonding limits of chemical valency and bond order. To cally bonded species, but chemists generally
chemists this is no minor matter. In Shaik’s demand sufficient robustness to permit char-
The authors are in the Department of Chemistry, University
words, the chemical bond is a fundamental ter- acterization of its chemical and electronic
of Wisconsin, Madison, WI 53706, USA. E-mail: weinhold@ ritory of chemistry, “the element from which an structure. In Cotton’s words, bond order is
chem.wisc.edu, [email protected] entire chemical universe is constructed” (3). “how many electron pairs . . . play a signifi-

PERSPECTIVES
cant role in holding the atoms together” (4). but they were often plagued with nonorthogo- suggests a linear geometry, unusually strong
The notions of chemical valency and struc- nality artifacts and unphysical limiting behav- XH bonds, and high reactivity of the “unsatu-
tural bonding were first developed by Kekulé ior. In some cases, bond order was simply a rated” triple bond. No wonder the consterna-
and others in the 19th century. In 1916, these surrogate for bond length—an assumption tion when the first stable Si≡Si species was
developments culminated in Lewis’s formula- based on a presumed mathematical relation reported to adopt a planar, but nonlinear,
tion of his remarkable “shared electron pair” between bond order and bond length derived trans-bent geometry (13)! Is this a true triple
concept (5). Empirical data at the time indi- from a few molecules (such as ethane, ethene, bond? Theoretical studies (14) support the
cated that two atoms may share up to six or ethyne). In this extreme, the bond-order notion that three pairs of electrons contribute
electrons to form single, double, or triple concept ceases to have independent meaning, significantly to holding the atoms together.
bonds. This concept was further enriched by limits, or intellectual content. Rather than form one σ bond and two equiva-
Robinson, Ingold, and others with the intro- More general and powerful tools of analy- lent π bonds as in carbon-carbon triple bonds,
duction of two or more Lewis structures sis were required to successfully recover the high preference for p-atomic orbital
that “resonate” to describe a single molecular chemical bonding and bond-order concepts character in the Si-Si σ bond leads to one
structure. A famous example is from modern delocalized ab normal π bond, one “slipped” π bond, and a
the dual-Kekulé representation initio wave functions. The trans-bent geometry (see the figure, top panel).
of benzene as two resonating density matrix techniques Transition metals, which have six valence
“cyclohexatriene” bond patterns pioneered by Löwdin (9) atomic orbitals (one s and five d), offer greater
with net 1.5 carbon-carbon provided a rigorous basis opportunities for electron pair sharing.
bond order. The HMMH bimetallic
In the 1920s, Schrödinger’s hydrides (where M = Cr,
discovery of the wave equation High-order bonding. Bonding orbital surfaces for the Mo, or W) were theoreti-
unusual “slipped” π bond of trans-bent HSi⬅SiH (top)
and its application by Heitler cally predicted (15) to
and the side-on δ bond of trans-bent, quintuply
and London to the H2 molecule bonded HWWH (bottom). These bonds exemplify exhibit quintuple bond-
laid the basis for a rational the- unconventional orbital shapes that result from compe- ing and strong trans-bent
ory of chemical bonding and tition between forming the strongest σ bonds and geometry, as was later
associated phenomena. Yet de- retaining optimal orbital directionality for the higher- found in the synthesized
spite these theoretical triumphs, order bond components. chromium derivative (1).
the connection between quan- From the 14 total valence
tum wave functions and the “pair- for expressing any wave electrons of HMMH, five shared electron pairs
ing” and “sharing” of electrons function (including, in hold the two metals together and the remaining
that underlie Lewis-like chemi- principle, exact solutions two pairs make the M-H bond. The quintuple
cal bonding were not immed- of Schrödinger’s equa- bond comprises familiar σ, 2π, and δ bond
iately resolved. tion) in terms of intrinsic orbitals. The final bond is constructed from a
Pauling initially combined the “natural” orbitals. (Natural side-on bonded pair of sd-hybridized orbitals
rather inaccurate Heitler-London orbitals are optimized combi- (see the figure, bottom panel). Again, the σ-
ansatz with Lewis structure diagrams to create nations of basis set functions that provide skeletal requirements enforce the trans-bent
the valence bond (VB) method. In VB theory, maximum parsimony and minimal depend- geometry and significant barriers to rigid rota-
two atoms share electron pairs in localized ence on the details of numerical basis set tion about the M-M bond.
wave functions that are constructed from selection in constructing molecular wave Is there a limit to how many electron
atomic valence orbitals, or combinations (“hy- functions.) In addition, Pople and Lennard- pairs may hold two atoms together? For the
brids”) of atomic orbitals. Thus, a connection Jones (10) demonstrated how one could rigor- transition metals, the limit seems to be six as
between the Lewis and quantum viewpoints ously transform the confusing delocalized a result of the dominance of the s and d
was forged. The directional features of molecular orbitals into equivalent localized atomic orbitals in bonding interactions. In
orbitals limit the maximum number of molecular orbitals that recover the expected principle, actinide elements might use a
bonds; although the carbon atom has four Lewis bond pattern. larger set of s, d, and f valence atomic
valence electrons and four valence orbitals, In the 1980s, the more general natural orbitals, suggesting as many as 13 possible
the directional qualities of the orbitals bond orbital (NBO) (11) and natural reso- bonds. However, Roos et al. (2) find that
that lead to the strongest electron pair nance theory (NRT) (12) methods were devel- weak bonding interactions and promotion
bond prevent a quadruple bond in dicarbon. oped for extracting the optimal Lewis struc- effects limit the effective bond orders
As numerical accuracy improved in the ture(s) and quantifying the density error in this (which maximize at diatomic Pa2) to five or
1970s, the VB wave functions were found (6) (or any chosen alternative) bonding pattern. less. Poor matches in the radial extents of the
to be inferior (except for H2) to delocalized For a wide range of organic and inorganic s, d, and f orbitals make use of all actinide
molecular orbital wave functions that appear species, NRT bond orders are found to be valence orbitals in the formation of bonds
to abandon the localized chemical bonding in excellent agreement with expected empiri- unfavorable. It seems safe to say, at least
concept. As two leading practitioners ex- cal values, including the near-integer total until new experiments or improved calcula-
pressed it, “The more accurate the calcula- valency values associated with periodic table tions prove otherwise, that six is the maxi-
tions become, the more the concepts tend to assignments. mum number of electron pairs that hold two
vanish into thin air” (7), and “the supercom- Assigned bond orders conjure up powerful atoms together.
puter has dissolved the chemical bond” (8). imagery in the minds of chemists. For exam- The discovery of new high-order metal-
Early bond-order metrics were devised to ple, the formula HX≡XH (where X is a group metal bond motifs signals a landmark in
extract chemical meaning from computations, 14 element such as carbon or silicon) strongly synthetic methodology and a renaissance of

PERSPECTIVES
Lewis-like structural concepts in inorganic 3. S. Shaik, J. Comput. Chem. 28, 51 (2007). (1988).
4. F. A. Cotton, in Multiple Bonds Between Metal Atoms, F. A. 12. E. D. Glendening, J. K. Badenhoop, F. Weinhold, J.
chemistry. Future synthetic and computa- Cotton, C. A. Muttillo, R. A. Walton, Eds. (Springer, New Comput. Chem. 19, 628 (1998).
tional explorations should be guided by closer York, 2005), pp. 707–795. 13. A. Sekiguchi, R. Kinjo, M. Ichinohe, Science 305, 1755
attention to the maximally matched donor- 5. G. N. Lewis, J. Am. Chem. Soc. 38, 762 (1916). (2004).
6. J. M. Norbeck, G. A. Gallup, J. Am. Chem. Soc. 96, 3386 14. C. R. Landis, F. Weinhold, J. Am. Chem. Soc. 128, 7335
acceptor interactions that lead to favorable (1974). (2006).
Lewis-like bonding patterns. 7. R. S. Mulliken, J. Chem. Phys. 43, S2 (1965). 15. F. Weinhold, C. R. Landis, Valency and Bonding
8. B. Sutcliffe, Int. J. Quantum Chem. 58, 645 (1998). (Cambridge Univ. Press, Cambridge, 2005), pp.
References 9. P.-O. Löwdin, Phys. Rev. 97, 1474 (1955). 555–559.
1. T. Nguyen et al., Science 310, 844 (2005). 10. J. A. Pople, J. Lennard-Jones, Proc. R. Soc. London Ser. A
2. B. O. Roos, A. C. Borin, L. Gagliardi, Angew. Chem. Int. 202, 166 (1950).
Ed. 46, 1469 (2007). 11. A. E. Reed, L. A. Curtiss, F. Weinhold, Chem. Rev. 88, 899 10.1126/science.1140756
PHYSICS
An analysis of currents confined to layers in
So Small Yet Still Giant a semiconductor structure reveals information

about electron-electron interactions.
Igor V. Lerner
A
lthough electronic devices keep shrink- fined as the scale over which the phase coher- in the active layer scatter from electrons in the
ing toward the nanometer scale of ence of electron quantum waves is main- passive layer, transferring momentum to them
atoms, physicists still deal with tained. This scale increases as the temperature and thus dragging them in the same direction
many-particle systems in which tracing the T decreases, reaching hundreds of nanometers until the resulting intralayer electrostatic force
paths of individual particles is beyond the at T ~ 1 K. For these temperatures, the wave equals the dragging force. Much experimental
reach of theory and experiment. Because of nature of electrons reveals itself in the inter- effort has been spent to study it under different
this, we have to rely on a statistical approach. ference between waves going along different conditions, although up to now these studies
Conventional wisdom, inherited from 19th- paths as a result of scatter-
century statistical physics, says that physical ing from impurities. This
measurements on a given sample are well leads to an irregularly oscil- B
described by averaging over an ensemble of lating but reproducible de-
identical samples. pendence of the sample
This notion became obsolete more than conductance on magnetic
two decades ago, however, with the prediction field or electron concen-
of reproducible conductance fluctuations (i.e., tration.
variations from sample to sample) in “meso- In metallic materials,
scopic” structures with dimensions intermedi- these conductance fluctua-
ate between atoms and bulk matter (1, 2). tions are always very small.
These fluctuations do not decrease with sam- Price et al. have made
ple size (as they should in classical physics) an experimental break- + –
– V +
but still remain much smaller than the average through by measuring the
conductance. On page 99 of this issue, Price et Coulomb drag at a temper- Coulomb drag and its fluctuations. An electric current in the upper layer
al. (3) report the observation of the Coulomb ature so low that this effect drags electrons and holes in the bottom layer, resulting in the electron and
hole currents in opposite directions. The net flow, which is due to the elec-
drag (4) in a bilayer system at very low tem- is suppressed on average
tron-hole asymmetry, is detected by a voltmeter V. At low temperatures, the
peratures where the reproducible fluctuations and is governed by the main reason for the asymmetry is the wave nature of electrons revealed in
of the drag turn out to be much larger than its fluctuations. The dominant random interference patterns in the local densities of states due to scatter-
average value. Thus, the authors have discov- role of the fluctuations in ing in both layers. This makes the direction of the drag force unpredictable,
ered mesoscopic fluctuations that, in contrast the Coulomb drag at very leading to its random but reproducible fluctuations in an external magnetic
to the conductance fluctuations, fully govern low temperatures was re- field B that changes the electron interference pattern in both layers.
the effect rather than give corrections to it—a cently predicted theoreti-
very unusual situation in statistical physics. In cally (5); however, the observed effect turns were largely limited to the drag effect at rela-
carrying out this work, they have developed a out to be four orders of magnitude higher than tively high T where its fluctuations were
new tool for studying the wave-like behavior the prediction. Thus, these fluctuations can unobservable.
of electrons in solids. truly be called giant, although they are still an The first experimental observations of
Mesoscopic fluctuations exist because order of magnitude smaller than the intralayer Coulomb drag (6–8) took place more than 10
quantum mechanics reigns not only at micro- conductance fluctuations. years after it had been theoretically predicted
scopic scales, as had always been expected, The Coulomb drag effect studied by Price three decades ago (9). One of the reasons for
but at the much larger mesoscopic scale, de- et al. occurs between two close but spatially such a long delay is that the drag effect is
separated layers of electrons, when an electri- very small. Partly, this is due to a very
cal current flowing through the “active” layer weak Coulomb coupling between the layers:
The author is at the School of Physics and Astronomy,
University of Birmingham, Birmingham B15 2TT, UK. induces a voltage in the second “passive” Momentum transfer between the layers is very
E-mail: [email protected] layer. It works via Coulomb friction: Electrons inefficient. But quantum mechanics is the

PERSPECTIVES
main culprit and takes the blame for the sup- being in the same direction as the current in aged over the entire sample. The drag temper-
pression of the effect. the active layer. ature dependence is very specific for such a
The reason is that electrons are fermions, However, the density of states (the num- mechanism, and the authors show that it is in a
and the Pauli exclusion principle at the heart ber of energy levels per unit of energy) also good agreement with the measurements.
of quantum mechanics tells us that two identi- fluctuates in the mesoscopic regime (10). The fluctuational Coulomb drag effect
cal fermions cannot coexist. At zero tempera- This led to the suggestion (5) that at low results from the interplay of the interlayer
ture, therefore, each state can either hold one enough temperatures the Coulomb drag electron-electron interactions and the inter-
electron or remain empty; because lower- force could become random, governed by the layer quantum coherence effects. This phe-
energy states are filled up first, all the states electron-hole asymmetry due to the fluctua- nomenon is a sensitive tool to help us learn
are occupied up to a certain level, the Fermi tions. The net sign of the effect then becomes more about the electron-electron interactions
energy εF, while the states above this level random (see the figure). This was expected in different materials and structures. Without
remain empty. Thus, the drag effect is possible to be observable only for quite small samples doubt, the first observation of this effect by
only at a finite temperature T, when in both with considerable disorder, where the magni- Price et al. opens a new direction in studying
layers the states around εF become only par- tude of the fluctuations in conductance and the fundamental properties of electrons in
tially occupied (that is, electrons are kicked density of states within one layer approaches solids at very low temperatures.
out by thermal energy and can be scattered the average.
among the states). Furthermore, charge carri- Price et al. courageously ventured to mea- References and Notes
1. B. L. Altshuler, JETP Lett. 41, 648 (1985).
ers in the active layer from both positively sure the effect in a relatively large and relatively 2. P. A. Lee, A. D. Stone, Phys. Rev. Lett. 55, 1622 (1985).
charged holes below εF and negatively charged clean sample where the intralayer fluctuations 3. A. S. Price et al., Science 316, 99 (2007).
electrons above εF in the passive layer, are tiny. The random drag resistance measured 4. This effect is so-named because it refers to a current of
dragging both in the same direction. Had the by Price et al. is small, but it is still four orders electrons in one layer “dragging” a current in the other
layer through electrical (Coulomb) forces.
electron and hole states been totally sym- of magnitude higher than predicted. The 5. B. N. Narozhny, I. L. Aleiner, Phys. Rev. Lett. 84, 5383
metric, positive and negative flows would authors have proposed a plausible qualitative (2000).
exactly cancel each other, resulting in no drag explanation for such a dramatic enhancement. 6. P. M. Solomon, P. J. Price, D. J. Frank, D. C. La Tulipe,
effect whatsoever. Phys. Rev. Lett. 63, 2508 (1989).
In their samples, the electron mean free path
7. T. J. Gramila, J. P. Eisenstein, A. H. MacDonald, L. N.
Thus, the drag effect exists only as a result for impurity scattering in each layer is much Pfeiffer, K. W. West, Phys. Rev. Lett. 66, 1216 (1991).
of the electron-hole asymmetry. On average, larger than the separation between the layers, 8. U. Sivan, P. M. Solomon, H. Shtrikman, Phys. Rev. Lett.
the asymmetry is due to a slightly different so that only large momentum transfer from the 68, 1196 (1992).
9. M. B. Pogrebinskii, Sov. Phys. Semicond. 11, 372 (1977).
energy distance of the electron and hole states active to the passive layer is effective for the 10. B. L. Altshuler, B. I. Shklovskii, Sov. Phys. JETP 64, 127
from the bottom of the Fermi sea. Thus, the drag. As a result, the electron-hole asymmetry (1986).
asymmetry is small and so is the Coulomb is contributed only by fluctuations in the local 11. I. V. Lerner, Phys. Lett. A 133, 253 (1988).
drag. The sign of the effect is positive with the density of states known to be much bigger
net flow of charge carriers in the passive layer (11) than those in the density of states of aver- 10.1126/science.1141972
APPLIED PHYSICS
Searching for a Solid-State Advances in device fabrication are facilitating

production and detection of electromagnetic
Terahertz Technology radiation at terahertz frequencies.
Mark Lee and Michael C. Wanke
T
he range of frequencies around 1 tera- sors for fast, high-specificity chemical de- ble THz technology infrastructure has
hertz (THz = 1012 cycles per second) tection and new modes of biological and arguably never been stronger than it is now.
is like the neglected middle child medical imaging. As reviewed by Borak (1), the characteristic
in the electromagnetic spectrum. Both Microwave electronics ultimately fail at interactions of THz radiation with various
microwaves (<0.1 THz) and infrared radia- higher frequencies because of fundamental forms of matter can lead to new applications.
tion (>20 THz) are used widely, thanks to the electron velocity limits, causing transistor Laboratories worldwide have carried out
combination of high technical performance performance to degrade rapidly above proof-of-principle demonstrations to show
and mass-produced solid-state microelec- ~0.1 THz. At the other end of the spectrum, how THz can be used in rapid-but-precise
tronics. Caught in between, the THz spec- infrared photonics cannot be extended down hazardous chemical sensing, concealed
trum has yet to be used in a mature solid-state to frequencies less than about 20 THz. weapon detection, noninvasive medical
device. The pace of recent advances gives Perversely, atmospheric attenuation of THz and biological diagnostics, and high-speed
hope, however, for a viable THz technology radiation is also much stronger than for telecommunications. To get such THz appli-
that would permit such applications as sen- microwave or infrared, leading to far more cations out of the laboratory and into com-
stringent requirements on signal-to-noise mon use will require elevating the THz
performance in this THz technology gap. microelectronic technology base to be on
The authors are at Sandia National Laboratories,
Albuquerque, NM 87185–1082. E-mail: mcwanke@ Nevertheless, the impetus to develop a a par with microwave electronics and in-
sandia.gov technically practical and economically feasi- frared photonics.

PERSPECTIVES
A generic THz application requires two require 5 to 10 mW from a THz source to Competing all-solid-state THz sources
main components: a coherent THz source function optimally. cannot currently meet the several milliwatt
and a THz detector. This THz source/detector This milliwatt power requirement lies at the average power threshold but may exhibit
combination must provide sufficient signal- heart of the THz solid-state technology prob- other useful features. Frequency multipliers
to-noise and speed performance to detect, lem. Realistic estimates of the THz source for use with high-power microwave sources
usually at “real-time” rates (100 Hz to 1 power needed to deliver acceptable signal-to- are a mature and compact technology that
kHz), a signal that has propagated through noise ratios outside a controlled research envi- operates at room temperature and can be eas-
the atmosphere. As a result, the primary ronment usually lead to the conclusion that tens ily tuned over wide frequency ranges from
goals of solid-state THz source and detector of milliwatts are desired. Until 2002, the only roughly 0.1 to 1 THz. However, intrinsic con-
device research are increasing average coherent THz oscillators capable of average version losses for large frequency multipli-
source power and decreasing detector noise. power much greater than 1 mW were vacuum cation factors and difficulties in handling
Solid-state THz detector devices have tube–based or accelerator sources, such as large input powers cause a multiplier’s power
proven to be quite capable in many regards. If molecular gas lasers and backward-wave oscil- output to drop rapidly with increasing output
one is willing to operate at temperatures near lators that are large, finicky, and expensive. frequencies, so that only about 10 μW
liquid helium (~4 K), are available near 1 THz (8).
several types of super- Similarly, obtaining lower fre-
conductor and semicon- quencies by mixing two solid-
ductor detectors have state near-infrared lasers on a
been developed for re- photoconductive semiconductor
search situations. How- switch at room temperature pro-
ever, the need for such duces very broadly tunable power
cooling is generally seen up to a few THz but currently
as an insurmountable ob- 10 mm exhibits only microwatts or less
stacle to broad accept- of average power above 1 THz
ance outside the research (9). Recently, a record peak pulse
lab. For this reason, re- power of 100 W was demon-
searchers are keen to strated in a p-doped germanium
develop THz detectors (p-Ge) laser near 2.7 THz. Un-
that operate at or closer to fortunately, current p-Ge lasers
room temperature. Front of thick packing “See-through” image as The real image in the back typically require large magnetic
For example, current foam seen using a 2.9-THz QCL of the packing foam fields, temperatures below 15 K,
work in high electron Cutting-edge imaging. Terahertz imaging could be used for detecting concealed and a very low duty cycle to
mobility transistors has weapons, among other applications. (Left) Front of packing foam. (Middle) Image of operate (10).
sought to circumvent elec- hidden razor blade taken with 2.7-THz quantum cascade laser operating at a few milli- Current research in solid-state
tron speed limits by using watts. (Right) Back of foam with concealed object. THz technology emphasizes indi-
collective charge-density vidual component development.
oscillations, known as plasmons, instead of A revolutionary advance in THz solid- The present focus is on improving detector
individual electrons as the charge carriers. state source technology came in 2002 when sensitivity, source power, and operating tem-
Plasmon wave velocities in a semiconductor Kohler et al. reported successful operation of perature with microelectronic materials and
channel can be 10 times as high as electron a quantum cascade laser (QCL) at THz fre- methods that are ultimately amenable to large-
velocities, similar to how water waves travel quencies (5). The QCL evades semiconduc- scale production. Should this work progress
much faster than the water molecules in the tor band-gap limitations on photonic devices at its current pace, this part of the spectrum
wave. THz response has recently been by using sophisticated semiconductor het- will become as useful as the microwave and
observed in various forms of plasmonic tran- erostructure engineering and fabrication infrared frequency bands are today.
sistors up to room temperature in submi- methods to create synthetic electron energy
crometer channel length devices and near gaps at frequencies much smaller than those 1. A. Borak, Science 308, 638 (2005).
liquid nitrogen temperature (77 K) in mil- that nature provides. Since 2002, THz QCLs 2. E. A. Shaner et al., IEEE Photonics Tech. Lett. 18, 1925
limeter-length devices (2, 3). have progressed rapidly in frequency cover- (2006).
In another example, NASA’s Jet Pro- age, increased power output, and increased 3. R. Tauk et al., Appl. Phys. Lett. 89, 253511 (2006).
4. P. H. Siegel et al., IEEE Trans. Microwave Theory Tech. 47,
pulsion Laboratory applied modern nano- operating temperature. Currently, they are
CREDIT: I. BRENER/SANDIA NATIONAL LABORATORY
596 (1999).
lithography methods to a classic microwave the only solid-state source capable of gener- 5. R. Köhler et al., Nature 417, 156 (2002).
device, the metal-semiconductor Schottky ating >10 mW of coherent average power 6. B. S. Williams et al., Elec. Lett. 42, 89 (2006).
diode, to fabricate mixers (which can be above 1 THz, with record continuous wave 7. B. S. Williams et al., Optics Express, 13, 3111 (2005).
8. See, for example, www.virginiadiodes.com/multipliers.
used to measure power spectra around a ref- power of 138 mW near 4.4 THz and an oper- htm.
erence frequency) that operate to at least ation temperature of 10 K (6, 7). The output 9. J. E. Bjarnason et al., Appl. Phys. Lett. 85, 3983 (2004).
3 THz (4). These receivers operate at ambi- power of QCLs drops as the temperature 10. R. E. Peale et al., J. Nanoelectron. Optoelectron. 2, 1
ent temperature, have low enough noise increases, but milliwatts can still be obtained 11. (2007). Sandia is operated by Sandia Corporation, a Lockheed
for most conceivable applications, and at liquid nitrogen temperature, and submilli- Martin Company, for the U.S. Department of Energy’s
have proven robust enough to travel onboard watt laser operation has been achieved up to National Nuclear Security Administration under contract
NASA’s Earth-observing Aura satellite. 164 K. To date, THz QCLs have spanned the DE-AC04-94AL85000.
The chief drawback is that these devices frequency range between 1.5 and 4.5 THz. 10.1126/science.1141012

REVIEW
future changes in the Atlantic MOC resulting
from increasing levels of carbon dioxide in the
atmosphere. Here we review the currently avail-
Atlantic Meridional Overturning able paleoceanographic data for the LGM and
find support for an active circulation in the deep
Circulation During the Last Atlantic, but not for the strong, deep MOC in-
volving enhanced production of NADW in the
Glacial Maximum northern North Atlantic.
Density Gradients in the Main

Jean Lynch-Stieglitz,1* Jess F. Adkins,2 William B. Curry,3 Trond Dokken,4 Ian R. Hall,5 Thermocline and Below
Juan Carlos Herguera,6 Joël J.-M. Hirschi,7 Elena V. Ivanova,8 Catherine Kissel,9 The tilt of the surfaces of equal seawater density
Olivier Marchal,3 Thomas M. Marchitto,10 I. Nicholas McCave,11 Jerry F. McManus,3 between the western and eastern sides of the
Stefan Mulitza,12 Ulysses Ninnemann,13 Frank Peeters,14 Ein-Fen Yu,15 Rainer Zahn16 basin provides tangible evidence for a meridional
overturning in the Atlantic Ocean today [e.g.,
The circulation of the deep Atlantic Ocean during the height of the last ice age appears to have (14)] (Fig. 2A). The very fact that these surfaces
been quite different from today. We review observations implying that Atlantic meridional are not strictly horizontal suggests the existence
overturning circulation during the Last Glacial Maximum was neither extremely sluggish nor an of a vertical shear in the average meridional flow
enhanced version of present-day circulation. The distribution of the decay products of uranium in in the basin: For a fluid on a rotating planet, a
sediments is consistent with a residence time for deep waters in the Atlantic only slightly greater zonal density contrast must be balanced by a
than today. However, evidence from multiple water-mass tracers supports a different distribution vertical shear in the meridional motion to lowest
of deep-water properties, including density, which is dynamically linked to circulation. order (the “thermal wind relationship”). The
shear reflects a northward flow near the surface
n today’s Atlantic Ocean, water more shallow period during which the factors controlling the and southward flow below (i.e., a MOC) (15).
I than about 1 km flows northward while

deeper water flows south, forming what is
called a meridional overturning circulation
structure of the Atlantic MOC (e.g., freshwater
budgets and atmospheric circulation in the
northern and southern high latitudes) appear to
Seawater density depends on temperature,
salinity, and pressure. Density at a given depth on
either side of the Atlantic Basin in the past can
(MOC) (Fig. 1A). The northward-flowing sur- have differed from today. be estimated from the ratio of oxygen isotopes,
18 16
face and intermediate waters lose buoyancy in In the 1980s, a prominent community effort O/ O, in the fossil carbonate tests of benthic
the North Atlantic, where they are transformed in the emerging field of paleoceanography foraminifera, bottom-dwelling protists (16). This
into the southward-flowing North Atlantic Deep provided evidence suggesting that this Atlantic ratio reflects both the temperature and the 18O/16O
Water (NADW). This meridional circulation is overturning circulation may not have existed in ratio (which generally covaries with salinity in the
considered an important element of the climate its current form during the LGM. The nutrient- upper ocean) of the seawater in which the
because of its attendant meridional heat flux; the poor NADW seemed to have been replaced by foraminifera calcify (16, 17).
northward-flowing surface waters are warm, higher-nutrient waters like those formed in the The density contrast across the width of the
whereas the southward-flowing deep waters are Southern Ocean today (2–5). Both theoretical entire ocean basin reflects the vertical shear in
cold, so the MOC is accompanied by a net studies (6) and ocean general circulation model the integrated meridional flow across the basin
northward transport of heat in the Atlantic. The experiments (7) suggested that the MOC may (18–20). In the South Atlantic at 30°S, density is
possibility that this overturning circulation could have multiple equilibria and that transitions be- higher along the eastern margin in the upper
change in the future motivates us to understand tween the equilibria may be triggered by anom- 2 km, reflecting the vertical shear in mass trans-
how it may have differed in the past. The Last alies in the freshwater fluxes at the sea surface. port associated with the MOC (Fig. 2A). The
Glacial Maximum (LGM), a time interval A new generation of coupled ocean- benthic foraminifera from surface sediments re-
centered at about 21,000 years ago and with a atmosphere general circulation models has since flect this contrast, with higher 18O/16O on the
duration of a few millennia [e.g., (1)], was a been developed. When forced with estimated higher-density eastern margin (Fig. 2B). During
LGM atmospheric CO2 concentration and con- the LGM, however, the cross-basin gradient in the
1
Georgia Institute of Technology, Atlanta, GA 30332, USA. tinental ice sheets, these models produce widely oxygen isotopes of the foraminifera was reduced
2
California Institute of Technology, Pasadena, CA 91125, different Atlantic circulation scenarios. Some and perhaps even reversed (21) (Fig. 2C). The
USA. 3Woods Hole Oceanographic Institution, Woods Hole,
MA 02543, USA. 4Bjerknes Centre for Climate Research, show a stronger overturning cell associated with circulation that we see today (flow to the north
5007 Bergen, Norway. 5Cardiff University, Cardiff CF10 3YE, NADW formation either extending slightly above 1 km and to the south below this depth), or
UK. 6CICESE, Oceanologia, 22860 Ensenada, Baja Califor- deeper (8) or unchanged in vertical extent (9); a similar overturning circulation with a shallower
nia, Mexico. 7National Oceanography Centre, University of others show a weaker and shallower overturning southward flow of NADW as in some of the
Southampton, Southampton SO14 3ZH, UK. 8Shirshov
Institute of Oceanology, Russian Academy of Sciences,
cell associated with NADW production (10, 11). model simulations for the LGM (10, 11), would
Moscow 117997, Russia. 9Laboratoire des Sciences du Only when perturbed with enhanced freshwater require higher densities along the eastern margin
Climat et de l’Environnement, CEA/CNRS/UVSQ, 91198 Gif- fluxes to the North Atlantic, as presumably oc- than along the western margin in the upper 2 km
sur-Yvette Cedex, France. 10University of Colorado, Boulder, curred in the past during brief periods of ac- of the South Atlantic. If the LGM relationship
CO 80309, USA. 11University of Cambridge, Cambridge
celerated decay of the continental ice sheets, do between the oxygen isotope ratio in the forami-
CB2 3EQ, UK. 12Forschungszentrum Ozeanränder/MARUM,
Universität Bremen, 28334 Bremen, Germany. 13Bjerknes the coupled models produce a shutdown of nifera and density was similar on both sides of
Centre for Climate Research, University of Bergen, 5007 NADW (12, 13). The large freshwater fluxes the South Atlantic Basin (as it is today), these
Bergen, Norway. 14Vrije Universiteit, 1081HV Amsterdam, capable of shutting down the Atlantic MOC in scenarios would be incompatible with the oxygen
Netherlands. 15National Taiwan Normal University, Taipei these models would not have been sustainable for isotope data. Water masses with very different
116, Taiwan, Republic of China. 16Institució Catalana de
Recerca i Estudis Avançats, Universitat Autónoma de the entire LGM. It is of more than academic temperatures, salinities, and oxygen isotope
Barcelona, E-08193 Bellaterra (Cerdanyola), Spain. interest to determine which models (if any) show ratios occupying the same density horizon on
*To whom correspondence should be addressed. E-mail: the correct response to ice age conditions, as either side of the ocean basin could potentially
[email protected] these are the same models that are used to predict cause a change in the oxygen isotope ratios

REVIEW
across the South Atlantic without a change in the open ocean (28). Nonetheless, the agreement be rich in zinc, which supports the idea that
density contrast. These properties need to be between the reconstructions based on these two some of this water mass comes from the South-
better constrained before any circulation scenar- water-mass tracers provides increased confidence ern Ocean (29). However, the deep (>2 km)
ios can be ruled out on the basis of the oxygen in the overall picture. water mass does show higher 13C/12C and lower
isotope data alone (21, 22). Cd in the deep North Atlantic
than in the deep South Atlantic;
Deep-Water Nutrient Properties this finding suggests that waters
Surface waters sinking in the originating in the North Atlan-
Greenland, Iceland, Norwegian, tic also contributed to the deep
and Labrador seas collectively (>2 km) water mass in the LGM
form the NADW, which is readily Atlantic. This contribution may
identifiable as a tongue of nutrient- be simply the result of mixing
poor water extending to great between GNAIW—which may
depths in the Atlantic and even- form as far south as the Labrador
tually reaching the Southern Sea or the subpolar open North
Ocean (Fig. 1A). Some high- Atlantic—and the deeper water
nutrient water from the Southern mass originating in the south, or
Ocean (Antarctic Bottom Water, it may consist of the addition of
AABW) can be seen penetrating small amounts of a distinct,
northward beneath the NADW. denser water mass forming in the
The nutrient distributions for far North Atlantic (30).
times in the past are reconstructed Although the nutrient tracers
by measuring the carbon isotopic provide a coherent picture of the
composition (13C/12C) of fossil distribution of water masses, they
shells of benthic foraminifera provide little information about
buried in the sediments. Primary the absolute rates of flow in the
producers in the surface ocean deep ocean. A fundamental rea-
take up both nutrients and carbon, son is that the remineralization of
discriminating against the heavy organic matter has a relatively
isotope of carbon as they do so, small effect on the nutrient con-
leading to high 13C/12C ratios in centration of deep waters in the
surface waters and low-nutrient Atlantic (at least today) and is
water masses (e.g., NADW). relatively poorly understood.
Higher nutrient concentrations
and lower 13C/12C in AABW Deep-Water Radiocarbon Activities
reflect the longer time these The rate of radioactive decay of
14
waters have spent away from the C is well understood and, in
surface, collecting nutrients and principle, could provide a mea-
13
C-poor carbon from the decay sure of the rate of deep-water
of organic matter transported to renewal or ventilation. Two ap-
depth in particulate and dissolved proaches have been developed to
forms. Past nutrient distributions correct past 14C ages from car-
have also been reconstructed from bonate that grew in the deep
the ratio of cadmium to calcium ocean for the time since their
in tests of benthic foraminifera. deposition. Radiocarbon mea-
Like the major nutrients, Cd is surements on benthic forami-
taken up by organisms at the sea nifera can be corrected using ages
surface and released at depth as of contemporaneous planktonic
the organic material is decom- –1
(surface-dwelling) foraminifera
posed. Both of these nutrient Fig. 1. (A) The modern distribution of dissolved phosphate (mmol liter )—a (31–33), and radiocarbon mea-
proxies (13C/12C and Cd/Ca) biological nutrient—in the western Atlantic (61). Also indicated is the southward surements on deep-sea corals can
show that during the LGM there flow of North Atlantic Deep Water (NADW), which is compensated by the be adjusted using independent
was a low-nutrient water mass northward flow of warmer waters above 1 km, and the Antarctic Bottom13Water ages derived from uranium and
(AABW) below. (B) The distribution of the carbon isotopic composition ( C/12C,
above a depth of about 2 km 13 thorium isotopes (34–36). Today,
expressed as d C, Vienna Pee Dee belemnite standard) of the shells of benthic
(often referred to as Glacial North the radiocarbon distribution in the
foraminifera in the western and central Atlantic during the LGM (23, 24). Data
Atlantic Intermediate Water, from different longitudes are collapsed in the same meridional plane. (C) deep Atlantic mostly reflects the
GNAIW) and high-nutrient wa- Estimates of the Cd (nmol kg–1) concentration for LGM from the ratio of Cd/Ca in relative contributions of water
ters below 2 km (23–27) (Fig. 1, B the shells of benthic foraminifera from (25). Today, the isotopic composition of from the north with high 14C
and C). There are multiple factors dissolved inorganic carbon and the concentration of dissolved Cd in seawater activity and water from the south
(air-sea exchange of carbon, car- both show “nutrient”-type distributions similar to that of PO4. with low 14C activity, with only a
bonate saturation state, oxidation small decrease in 14C activity due
of organic matter in sediments) controlling the The LGM configuration is often interpreted to in situ decay within the deep North Atlantic.
isotopic and chemical compositions of the as a shoaling of NADW and a northward Similar to its use in the modern ocean, past 14C
foraminifera tests that can potentially decouple extension of AABW in the Atlantic Ocean. The could be combined with other water-mass tracers
the nutrient proxies from nutrient distributions in high-nutrient water below 2 km also appeared to to account for this overprint of water-mass

REVIEW
mixing and thereby estimate the contribution due centuries for 231Pa. Because the residence time nutrient-poor, radiocarbon-rich water mass
to radiocarbon decay within the Atlantic (37). To of deep waters in the Atlantic is also on the (GNAIW) dominant above 2 km and more
use radiocarbon to directly constrain the LGM order of centuries, most of the 230Th produced nutrient-rich, radiocarbon-poor water below
circulation in the Atlantic would require a large in the Atlantic is buried in Atlantic Ocean 2 km (perhaps a glacial analog of AABW). The
number of precise measurements (38, 39). sediments, whereas some of the 231Pa is ex- maintenance of these two distinct water masses in
However, the existing measurements of 14C ported to the Southern Ocean. This leaves the the face of mixing processes similar to those in
activity in benthic foraminifera suggest a water- modern 231Pa/230Th ratio in Atlantic sediments the modern ocean that would tend to erase these
mass structure in the LGM Atlantic that is below the ratio at which these isotopes are boundaries requires one or both water masses to
consistent with the nutrient proxies, with 14C- produced by the decay of U (42). A longer be renewed fairly rapidly (24). (ii) 231Pa/230Th
rich waters indicating recent exchange with the residence time of water in the Atlantic should ratios in deep-ocean sediments deposited during
atmosphere above 2 km depth and 14C-poor cause the 231Pa/230Th ratio in the underlying the LGM are comparable to those deposited
waters below this depth (40). The interpretation sediments to increase, but there was no signif- under modern conditions. This also argues
of the 14C is complicated by assumptions about icant difference in the mean 231Pa/230Th of against a much slower circulation for both deep-
the surface-ocean radiocarbon activity and the Atlantic sediments deposited during the LGM water masses in the Atlantic. (iii) The cross-basin
mixing of shells of different ages by organisms relative to those laid down more recently (42). contrast in oxygen isotopes in benthic foraminif-
living within the sediments, so the agreement be- These data could be consistent with a range of era in the South Atlantic collapsed during the
tween the reconstructions based on 14C and the past circulation states, from a slight increase in LGM. This would argue against a strong MOC
nutrient tracers gives us further confidence in the MOC to a decrease of up to 30% (43). More that imports surface and intermediate waters to
the overall picture of the deep-water masses in recently, with detailed downcore records, slight- the Atlantic compensated by the export of deeper
the LGM Atlantic. ly higher ratios were found for the LGM sed- waters. Hence, we have good evidence for a
iments relative to the Holocene at a water depth circulation of deep waters in the LGM Atlantic
Radioisotopes of Protactinium and Thorium of 4.5 km near Bermuda (44), consistent with an that was not completely sluggish. The data also
The contrasting chemical behavior of uranium overall small increase in the residence time of do not support a MOC with the same extent and
decay-series nuclides provides a means for overlying deep waters (GNAIW and AABW). structure as today. Sea surface temperature
assessing the rate of deep circulation. Uranium Lower ratios were found at 1.7 km depth in the reconstructions in the North Atlantic (49, 50)
is highly soluble in seawater and thus has a northern North Atlantic (45) and 3.4 km depth also appear inconsistent with a stronger version
residence time of several hundred thousand years on the Iberian margin (46), allowing for the of today’s overturning cell characterized by
in the ocean. Because this is far longer than the possibility of vigorous renewal of GNAIW. Be- surface water inflow compensating deep-water
overall oceanic mixing time (a millennium or cause variations in particle flux (47) and com- export from the polar seas of the North Atlantic.
two), uranium is approximately evenly distrib- position (48) can also influence sedimentary These observations seem to rule out at least some
231
uted throughout the ocean. This means that the Pa/230Th, a better understanding of these of the more extreme circulation states produced
radioactive decay of U isotopes, and hence the factors will strengthen our ability to quantify by climate models for the LGM.
production of its daughter isotopes, is spatially past ocean circulation with the use of these
uniform to a good approximation. By contrast, nuclides. Outlook
these radioactive-decay products, thorium and What would be required to provide a reliable
protactinium, are extremely particle-reactive (41) Glacial Circulation of the Atlantic observational estimate of the Atlantic MOC
and are rapidly removed from seawater by At this point, the most robust observations that during the LGM? Paleoceanographic methods
settling particles and subsequently buried on the can help to constrain scenarios of past variability inherently are associated with larger errors and
sea floor. The resulting oceanic residence times in the Atlantic MOC are as follows: (i) There was have a sparser geographic coverage than modern
are on the order of decades for 230Th and a water-mass divide in the LGM Atlantic, with a oceanographic measurements, and using this
type of data to produce a conclu-
sive quantitative reconstruction of
past ocean circulation is a chal-
lenge. It will almost certainly
require a combination of different
paleoceanographic proxies such
as those discussed here (38, 51).
Ocean margin density reconstruc-
tions and 231Pa/230Th measure-
ments may be particularly useful,
as they may integrate the effects
of circulation in the ocean interior
where paleoceanographic mea-
surements are not possible; mea-
surements of 14C may also be
useful as a direct measure of
elapsed time. But work remains
to be done in order to (i) better
Fig. 2. (A) Density (sq) of seawater across the South Atlantic at 30°S (61). The waters are denser on the eastern understand the processes by
side of the basin, reflecting the presence of the meridional overturning circulation. (B) Oxygen isotopic which Pa and Th are removed
composition of the shells of benthic foraminifera (d18O) in recent sediments on the eastern (red symbols) and from the water column; (ii) better
western (blue symbols) side of the basin, reflecting the density contrast across the basin today, with higher d18O assess past ocean temperatures
for the higher-density waters on the eastern side. The solid lines are the predicted d18O values based on modern and salinities for paleodensity
hydrographic data (21). (C) Oxygen isotopic composition of the shells of benthic foraminifera for the LGM suggest reconstructions, possibly by
that the modern density contrast in the upper 2 km was absent or reversed (21). using the elemental chemistry of

REVIEW
the shells (52) and paleosalinity estimates from of a detailed history of ocean circulation over the 32. J. C. Duplessy et al., Radiocarbon 31, 493 (1989).
measurements of chloride in pore waters (53); abrupt climate changes that punctuated the cooler 33. N. J. Shackleton et al., Nature 335, 708 (1988).
34. J. F. Adkins, E. A. Boyle, L. Keigwin, E. Cortijo, Nature
and (iii) better constrain water-mass properties climates of the past. Changes in the Atlantic 390, 154 (1997).
for accurate use of the 14C clock. Other ap- MOC play a critical role in many of the hy- 35. A. Mangini et al., Nature 392, 347 (1998).
proaches that show promise are the quantitative potheses put forth to explain these abrupt climate 36. L. F. Robinson et al., Science 310, 1469 (2005);
analysis of the grain size population in the transitions, and there is already compelling evi- published online 3 November 2005 (10.1126/
science.1114832).
terrigenous sediment fine fraction that provides dence to support this link, at least for the most 37. J. F. Adkins, E. A. Boyle, in Reconstructing Ocean History:
a proxy for near-bottom flow speed, which has recent of these abrupt climate change events A Window into the Future, F. Abrantes, A. C. Mix, Eds.
the advantage of having a direct physical con- (44, 60). The quantitative reconstruction of these (Kluwer Academic, New York, 1999), pp. 103–120.
nection to flow and an immediate response to any circulation changes remains a challenge that, if 38. P. Huybers, G. Gebbie, O. Marchal, J. Phys. Oceanogr.
37, 394 (2007).
changes in flow speed (54). New water-mass tackled successfully, will further our understand- 39. C. Wunsch, Quat. Sci. Rev. 22, 371 (2003).
tracers that are independent of nutrient cycling, ing of the relationship between ocean circulation 40. L. D. Keigwin, Paleoceanography 19, PA4012 (2004).
such as the Nd isotopic composition of metallic and climate, enabling us to better constrain the 41. M. P. Bacon, R. F. Anderson, J. Geophys. Res. 87, 2045
precipitates in deep-sea sediments, are also being scenarios for the future. (1982).
42. E. F. Yu, R. Francois, M. P. Bacon, Nature 379, 689
developed and successfully applied on a number
(1996).
of time scales [e.g., (55)], and magnetic proper- References and Notes 43. O. Marchal, R. Francois, T. F. Stocker, F. Joos,
ties have been used to reconstruct the flow path 1. A. C. Mix, E. Bard, R. Schneider, Quat. Sci. Rev. 20, 627 Paleoceanography 15, 625 (2000).
of bottom waters (56). Although challenges (2001). 44. J. F. McManus, R. Francois, J. M. Gherardi, L. D. Keigwin,
remain before we arrive at a well-constrained 2. J. C. Duplessy, J. Moyes, C. Pujol, Nature 286, 479 S. Brown-Leger, Nature 428, 834 (2004).
(1980). 45. I. R. Hall et al., Geophys. Res. Lett. 33, L16616 (2006).
quantitative estimate of the glacial circulation, 3. S. S. Streeter, N. J. Shackleton, Science 203, 168 (1979). 46. J. M. Gherardi et al., Earth Planet. Sci. Lett. 240, 710
much will be gained in the coming years by the 4. E. A. Boyle, L. D. Keigwin, Science 218, 784 (1982). (2005).
combined use of the tools currently at our 5. W. B. Curry, G. P. Lohmann, Quat. Res. 18, 218 (1982). 47. N. Kumar, R. Gwiazda, R. F. Anderson, P. N. Froelich,
disposal. It is worth recalling that the basic 6. H. Stommel, Tellus 13, 224 (1961). Nature 362, 45 (1993).
7. S. Manabe, R. J. Stouffer, J. Clim. 1, 841 (1988). 48. Z. Chase, R. F. Anderson, M. Q. Fleisher, P. W. Kubik,
structure of the modern Atlantic MOC was 8. A. Kitoh, S. Murakami, H. Koide, Geophys. Res. Lett. 28, Earth Planet. Sci. Lett. 204, 215 (2002).
largely described by the 1950s on the basis of a 2221 (2001). 49. M. Kucera et al., Quaternary Sci. Rev. 24, 951 (2005).
few measurements of water-mass properties and 9. C. D. Hewitt, R. J. Stouffer, A. J. Broccoli, J. F. B. Mitchell, 50. A. Paul, C. Schafer-Neth, Paleoceanography 18, 1058
the thermal wind relationship, whereas quantita- P. J. Valdes, Clim. Dyn. 20, 203 (2003). (2003).
10. B. L. Otto-Bliesner et al., J. Clim. 19, 2526 (2006). 51. P. Legrand, C. Wunsch, Paleoceanography 10, 1011
tive estimates of the modern MOC based on (1995).
11. S. I. Shin et al., Clim. Dyn. 20, 127 (2003).
inverse methods have only been possible in the 12. M. Vellinga, R. A. Wood, Clim. Change 54, 251 (2002). 52. H. Elderfield, J. Yu, P. Anand, T. Kiefer, B. Nyland, Earth
past two decades or so (57). 13. R. Zhang, T. L. Delworth, J. Clim. 18, 1853 (2005). Planet. Sci. Lett. 250, 633 (2006).
Different circulation scenarios are consistent 14. M. M. Hall, H. L. Bryden, Deep Sea Res. 29, 339 (1982). 53. J. F. Adkins, D. P. Schrag, Earth Planet. Sci. Lett. 216,
15. D. Roemmich, C. Wunsch, Deep Sea Res. 32, 619 (1985). 109 (2003).
with the currently available data for the LGM,
16. J. Lynch-Stieglitz, W. B. Curry, N. Slowey, Paleoceanography 54. I. N. McCave, I. R. Hall, Geochem. Geophys. Geosyst. 7,
but certain hypotheses regarding the glacial 14, 360 (1999). Q10N05 (2006).
circulation in the Atlantic already are testable. 17. C. Emiliani, J. Geol. 63, 538 (1955). 55. A. M. Piotrowski, S. L. Goldstein, S. R. Hemming,
In particular, general circulation models provide 18. J. Hirschi, J. Lynch-Stieglitz, Geochem. Geophys. Geosyst. R. G. Fairbanks, Science 307, 1933 (2005).
a set of circulation hypotheses that are physically 7, Q10N04 (2006). 56. C. Kissel et al., Earth Planet. Sci. Lett. 171, 489
19. J. Lynch-Stieglitz, Geochem. Geophys. Geosyst. 2, (1999).
consistent (given the assumptions used in con- 10.1029/2001GC000208 (2001). 57. A. Ganachaud, C. Wunsch, Nature 408, 453 (2000).
structing the models) with the ice sheets and CO2 20. J. Marotzke et al., J. Geophys. Res. 104, 29529 (1999). 58. A. N. LeGrande et al., Proc. Natl. Acad. Sci. U.S.A. 103,
concentration of the last ice age. We can already 21. J. Lynch-Stieglitz et al., Geochem. Geophys. Geosyst. 7, 837 (2006).
say with some confidence that the data can rule Q10N03 (2006). 59. A. Schmittner, A. Oschlies, X. Giraud, M. Eby,
22. G. Gebbie, P. Huybers, Geochem. Geophys. Geosyst. 7, H. L. Simmons, Global Biogeochem. Cycles 19, GB3004
out some of the more extreme scenarios produced Q11N07 (2006). (2005).
by the models. To compare the less extreme 23. T. Bickert, A. Mackensen, in The South Atlantic in the Late 60. P. U. Clark, N. G. Pisias, T. F. Stocker, A. J. Weaver,
model scenarios directly with the data, we need Quaternary: Reconstruction of Material Budget and Nature 415, 863 (2002).
to account for the multiple physical, chemical, Current Systems, G. Wefer, S. Mulitza, V. Ratmeyer, Eds. 61. M. E. Conkright et al., World Ocean Atlas 2001: Objective
(Springer-Verlag, Berlin, 2004), pp. 671–693. Analyses, Data Statistics, and Figures, CD-ROM
and biological controls on the paleoceanographic Documentation (National Oceanographic Data Center,
24. W. B. Curry, D. W. Oppo, Paleoceanography 20, PA1017
observations. A better representation in the mod- (2005). Silver Spring, MD, 2002).
els of the full complexity of these processes that 25. T. M. Marchitto, W. S. Broecker, Geochem. Geophys. 62. We thank the Scientific Committee on Ocean Research,
lead to the record left behind in the sediments and Geosyst. 7, Q12003 (2006). IMAGES, and NSF for their support of the Working Group
26. J.-C. Duplessy et al., Paleoceanography 3, 343 (1988). on Past Ocean Circulation and the workshop that was
the explicit prediction of the values of the
27. M. Sarnthein et al., Paleoceanography 9, 209 (1994). held on this topic in March 2005 at Georgia Tech. We
paleoceanographic proxies will allow for a more 28. J. Lynch-Stieglitz, in The Oceans and Marine Geochemistry, also thank the scientists who contributed their ideas to
direct comparison between model output and H. Elderfield, Ed., vol. 6 of Treatise on Geochemistry this review through their participation in that workshop
data [e.g., (58, 59)]. (Elsevier, Oxford, 2003), pp. 433–451. and the ORMEN/VAMOC Workshop on LGM ocean
Reconstructing ocean circulation during the 29. T. M. Marchitto, W. B. Curry, D. W. Oppo, Paleoceanography circulation in Amsterdam in October 2005. We thank
15, 299 (2000). T. Bickert for providing data for Fig. 1.
LGM is a critical first step, but there is already a 30. K. Matsumoto, J. Lynch-Stieglitz, Paleoceanography 14,
strong community focus on generating the time- 149 (1999).
series data that will allow for the reconstruction 31. W. S. Broecker et al., Radiocarbon 30, 261 (1988). 10.1126/science.1137127

Discover the
leading resource in
the life sciences ...
Encyclopedia of
Life
Sciences
www.els.net
Spanning the entire
spectrum of life
science research,
the Encyclopedia
of Life Sciences
features
thousands of specially
commisioned and peer-
reviewed articles, most of which are
accompanied by colour images and
tables.
• Available in print and online
• Over 3,500 original articles
• Contributions from 5,000 of the
world’s leading scientists
• Introductory, advanced and
keynote articles
• More than 9,000 illustrations and
figures
Original 20 Volume
print set
Hardcover
978-0-470-01617-6
£3150 / $5670 / €4899
NEW 6
Supplementary
print volumes
Hardcover
978-0-470-06141-1
£795 / $1435 / €1249, valid until 30 June
2007 - thereafter £995 / $1795 / €1549
26 Volume print set (20 Volumes + 6
Supplementary volumes)
Hardcover
10107
978-0-470-06651-5
£3550 / $6390 / €5499, valid until
30 June 2007 - thereafter
£4145 / $7465 / €6449
HOW TO ORDER NORTH, CENTRAL &

SOUTH AMERICA
EUROPE, MIDDLE EAST, John Wiley & Sons Inc
ASIA & AFRICA Tel: 877 762 2974
John Wiley & Sons Ltd Fax: 800 597 3299
Tel: +44 (0)1243 843294 E-mail: [email protected]
Fax: +44 (0)1243 843296 www.wiley.com
E-mail:
[email protected] GERMANY, SWITZERLAND
www.wiley.com & AUSTRIA
Wiley-VCH Verlag GmbH
Tel: +49 6201 606 400
Fax: +49 6201 606 184
E-mail: [email protected]
www.wiley-vch.de
BREVIA
growing beneath deciduous hosts (Fig. 1B).
Therefore, the fruiting season of these species
has changed in a habitat-dependent manner. If
Rapid and Recent Changes in Fungal these responses were due to microclimatic
differences beneath deciduous and coniferous
Fruiting Patterns trees, then there would likely be similar
differences in fruiting patterns of nonmycor-
rhizal forest-floor fungi. To examine this pos-
A. C. Gange,1* E. G. Gange,2 T. H. Sparks,3 L. Boddy4
sibility, we compared regression coefficients
of seven nonmycorrhizal leaf litter–decay
any studies have demonstrated recent both of which are greater than equivalent species and a further seven wood-decay fungi
M phenological responses to climate

change, but these largely involved
higher organisms, such as plants, insects, or
spring data previously reported for higher
organisms (5).
The alteration in fungal fruiting mirrors changes
that occurred in both forest types. In no case
did the regression coefficient differ (all P >
0.05); thus, microclimatic effects can be dis-
birds, and were restricted to events in spring (1). in British temperatures that have occurred since counted. These data suggest that changes in
Autumnal events have received far less atten- 1975 (6). To substantiate this, we examined re- the temporal allocation of nutrients to roots
tion, even though the end of the growing season lations between fruiting dates of each species have occurred in deciduous forests but not in
has seen significant delays (2). Fungi provide and monthly records of local temperature and coniferous woods, where there is no single
vital ecosystem services through decomposition, rainfall (4). Over the past 56 years, August large loss of leaf material. Nutrients are inter-
nutrient cycling, and soil aggregation, yet they temperatures have increased (F1,54 = 11.4, P < cepted by the mycorrhizal species and used
are missing from previous considerations of eco- 0.01), as has October rainfall (F1,54 = 5.8, P < for fruit body production (7).
system responses to global change (3). In this 0.05). The increase in late summer temper- Furthermore, climate warming seems to have
study, we analyzed a data set consisting of over atures and autumnal rains has caused early caused significant numbers of species to begin
52,000 individual fungal fruiting records, from season species to fruit earlier and late season fruiting in spring as well as autumn (fig. S1).
nearly 1400 localities, collected in southern species to continue fruiting later. Seventy-eight Given that active mycelial growth is required
England over the period 1950–2005. We ex- (91%) of the species showing an advanced before sporophore production, this is strong evi-
tracted information on a total of 315 autumnal fruiting date have a significant relation between dence that the mycelium of certain species must
fruiting species, each of which had been recorded first fruiting date and August temperature, be active in late winter and early spring as well
in more than 20 years (4). whereas 92 (88%) of the species showing as late summer and autumn, suggesting in-
The first fruiting date averaged across all spe- later last dates could be explained by positive creases in decay rates in forests.
cies has become significantly earlier, whereas relations between August temperature and Oc-
average last fruiting date has become signifi- tober rainfall.
cantly later (Fig. 1A). The increase in the over- We noticed that 47 (59%) of the deciduous 1. C. Parmesan, G. Yohe, Nature 421, 37 (2003).
all fruiting period is dramatic; in the 1950s, mycorrhizal species showed a delay in last 2. A. Menzel, P. Fabian, Nature 397, 659 (1999).
the average (±SE) was 33.2 ± 1.6 days, but fruiting date, whereas no coniferous mycor- 3. D. Schröter et al., Science 310, 1333 (2005); published
this has more than doubled to 74.8 ± 7.6 days rhizal species were delayed. To examine this online 27 October 2005 (10.1126/science.1115233).
4. Materials and methods are available as supporting
in the current decade. For the species that further, we studied the 11 mycorrhizal species material on Science Online.
show significantly earlier first fruiting dates that were recorded beneath both coniferous 5. T. L. Root et al., Nature 421, 57 (2003).
(n = 85), the average advancement is 8.6 ± and deciduous hosts. Average fruiting date in 6. A. H. Fitter, R. S. R. Fitter, Science 296, 1689 (2002).
0.6 days decade−1, whereas for species show- each year was calculated and regressed against 7. F. T. Last, J. Pelham, P. A. Mason, K. Ingleby, Nature 280,
168 (1979).
ing significantly later last fruiting dates (n = time (56 years). Eight of the species showed a 8. We are extremely grateful to all those who collected
105), the delay is 7.5 ± 0.5 days decade−1, significantly larger regression coefficient when fungi, especially I. Gange, the late J. Hindley, A. McKee,
W. Freemantle, R. Nicholls, and R. Chapman.
Regression coefficient
360 A 1.5 B * *
1 * * * * Supporting Online Material
*
Day of year
320 * www.sciencemag.org/cgi/content/full/316/5821/71/DC1
0.5 Materials and Methods
280 0 Fig. S1
References
240 -0.5
-1 13 November 2006; accepted 8 January 2007
200 A.c. A.r. C.t. H.c. L.a. L.l. P.i. R.a. R.o. R.x. T.t. 10.1126/science.1137489
1945 1965 1985 2005
deciduous coniferous
Fig. 1. (A) Average first fruiting date (lower line) and average last fruiting date (upper line) of 315
1
School of Biological Sciences, Royal Holloway, University
fungal species over 56 years. The underlying pattern is represented by lowess (locally weighted of London, Egham, Surrey TW20 0EX, UK. 2Belvedere, South-
ampton Road, Whaddon, Salisbury, Wilts SP5 3DZ, UK.
scatter plot smoother) lines. (B) Regression coefficients for mean fruiting date versus years for 11 3
Natural Environment Research Council Centre for Ecology
mycorrhizal fungal species when growing under coniferous or deciduous trees. A.c. represents and Hydrology, Monks Wood, Cambridgeshire PE28 2LS, UK.
Amanita citrina; A.r., A. rubescens; C.t., Cantherellus tubaeformis; H.c., Hebeloma crustuliniforme; L.a., 4
Cardiff School of Biosciences, Cardiff University, Main Build-
Laccaria amethystina; L.l., L. laccata; P.i., Paxillus involutus; R.a., Russula atropurpurea; R.o., R. ochroleuca; ing, Museum Avenue, Cardiff CF10 3TL, UK.
R.x., R. xerampelina; and T.t., Tricholoma terreum. Asterisks above bars indicate a significant difference *To whom correspondence should be addressed. E-mail:
in coefficients between the host types at P = 0.05. [email protected]

Looking for solid ground in the ever-changing
landscape of science & technology
policy and budget issues?
Join the nation’s top S&T experts at the 32nd Annual
AAAS Forum on Science & Technology Policy
3–4 May 2007 • Washington DC
International Trade Center in the
Ronald Reagan Building
The AAAS Forum on Science and Technology Policy provides a setting

for discussion and debate about the federal budget and other policy
issues facing the science, engineering, and higher education commu-
nities. Initiated in 1976 as the AAAS R&D Colloquium with about 100
participants, the Forum has emerged as the major public meeting in
the U.S. devoted to science and technology policy issues. It annually
draws upwards of 500 of the nation’s top S&T policy experts.
• Get a full analysis of the • Registrants will receive, at

President’s federal R&D the Forum, AAAS Report
funding proposals. XXXII: Research and Develop-
ment, FY 2008, a comprehen-
• Have an opportunity to sive analysis of the proposals
meet directly with key for the FY 2008 budget, pre-
S&T policymakers. pared by AAAS and a group
• Learn how the changes in of its affiliated scientific,
Congress are affecting S&T engineering, and higher
policy issues. education associations.
• Network with colleagues, For more complete details on

including top decisionmakers the program, hotel registration
in science and technology and on-line registration,
policy from all sectors. please visit the website:
www.aaas.org/forum.
• Learn about broader national
and international develop-
ments that will affect strate-
gic planning in universities,
industries, and government.
www.aaas.org/forum
RESEARCH ARTICLES
likely candidate for the protein from which these
other enzymes evolved.
We recently isolated the genes for chrysan-
Chimeras of Two Isoprenoid Synthases themyl diphosphate synthase (CPPase), an enzyme
with cyclopropanation activity, from Chrysan-
Catalyze All Four Coupling Reactions themum cinerariaefolium (chrysanthemum)
(14) and Artemisia tridentata ssp. spiciformis
in Isoprenoid Biosynthesis (snowfield sagebrush) (15). The encoded sage-

brush FPPase and CPPase proteins have simi-
lar sequences (75% identity and 96% similarity),
Hirekodathakallu V. Thulasiram,* Hans K. Erickson,† C. Dale Poulter‡ suggesting that they share a recent common
ancestor. Because FPPases are universally
The carbon skeletons of over 55,000 naturally occurring isoprenoid compounds are constructed distributed, whereas CPPases are confined to
from four fundamental coupling reactions: chain elongation, cyclopropanation, branching, and members of a closely related family of higher
cyclobutanation. Enzymes that catalyze chain elongation and cyclopropanation are well studied, plants, it is apparent that the cyclopropanation
whereas those that catalyze branching and cyclobutanation are unknown. We have catalyzed the activity evolved from the chain-elongation ac-
four reactions with chimeric proteins generated by replacing segments of a chain-elongation tivity of a parental FPPase. In contrast, the
enzyme with corresponding sequences from a cyclopropanation enzyme. Stereochemical and cyclopropanation enzymes squalene (13) and
mechanistic considerations suggest that the four coupling enzymes could have evolved from a phytoene (16) synthase have no discernable
common ancestor through relatively small changes in the catalytic site. sequence similarity with chain-elongation en-
zymes, suggesting an independent divergence of
soprenoid metabolism is the most chemical- the other four (1′-1, 1′-3, 2-1′-3, and c1′-1-2) are chain-elongation and cyclopropanation activities
I ly diverse of nature’s biosynthetic pathways.

Over 55,000 naturally occurring isoprenoid
molecules have been discovered, with a large
produced by rearrangement of 1′-2-3 structures
(4, 5). The basic building reaction in the path-
way is chain elongation, where the growing
at a much earlier time, probably at the very
beginning of cellular life.
On closer examination, we discovered that
number of new structures reported each year. isoprenoid chain of an isoprenoid allylic diphos- CPPase also synthesized lesser amounts of the
Isoprenoid compounds play crucial metabolic phate is added to the double bond in isopentenyl 1′-2 branched product lavandulyl diphosphate
and structural roles in cells, and the pathway is diphosphate (IPP) (Fig. 2). This reaction occurs (LPP) and the chain-elongation product GPP. In
found in all forms of life. Among the better- in all organisms. The 1′-4 linkage is by far the preliminary experiments with a series of chimer-
known classes of isoprenoids are sterols (hor- most common that is seen in isoprenoid com- ic proteins constructed by replacing amino acids
mones and membrane components), carotenoids pounds, and compounds with this attachment of sagebrush FPPase with corresponding sequen-
(visual pigments and photoprotective agents), between isoprene units are called head-to-tail or ces from sagebrush CPPase, we found a transition
prenylated proteins (membrane structure and regular terpenes. Other attachment patterns are in activities from chain elongation to branching
cell signaling), dolichols (glycoprotein synthesis designated as non–head-to-tail or irregular. The and finally to cyclopropanation (17). Studies
and bacterial cell wall synthesis), monoterpenes cyclopropanation reaction is the first pathway- with a full set of chimeric proteins demonstrate
(insect sex pheromones and fragrances), and specific step in the biosynthesis of sterols (6) that they catalyze all four of the fundamental
sesquiterpenes (plant defensive agents). The car- and carotenoids (7) to give metabolites widely isoprenoid-coupling reactions and that the abso-
bon skeletons of isoprenoid compounds are con- distributed in Eukarya, Archaea, and some lute stereochemistries of the chiral products are
structed from 3-methyl-1-butyl units, which are Bacteria. Branching is only found in a limited identical to their naturally evolved counterparts.
joined in one of nine different patterns (Fig. 1) number of plants (1, 8). The only documented Synthesis and expression of chimeric
(1–3). In four of these structures, C1′ of one unit compounds from cyclobutanation are mealybug enzymes. By means of procedures reported
(red) is joined to a single carbon of the other mating pheromones (2, 9). previously, a common set of specific restriction
unit (black) (1′-1, 1′-2, 1′-3, and 1′-4). In one The enzymes that catalyze chain elongation
case, C1′ is inserted between atoms in the other can be divided into two convergently evolved
unit (2-1′-3), and in three cases, C1′ is joined families based on the stereochemistry, E or Z, of
to the other unit in a cyclic structure (c1′-1-2, the double bond in the newly added isoprene
c1′-2-3, and c1′-2-3-2′). The example that does unit (10). Members of the E family typically
not involve C1′ is the 4′-4 pattern seen in mem- synthesize shorter-chain isoprenoid diphosphates
brane lipids from Archaea (3). found early in the pathway, whereas those in the
Except for the 4′-4 attachment, the isopre- Z family synthesize longer-chain diphosphates.
noid skeletons in Fig. 1 are formed early in the Farnesyl diphosphate synthase (FPPase) is the
isoprenoid pathway, before the modification re- prototypal representative of the E family. FPPase
actions that are required to synthesize specific catalyzes two reactions: the sequential conden-
natural products. Four of the basic isoprenoid sation of dimethylallyl diphosphate (DMAPP,
structures (1′-2, 1′-4, c1′-2-3, and c1′-2-3-2′) are C5) and geranyl diphosphate (GPP, C10) with
synthesized by joining simpler units, whereas IPP to give farnesyl diphosphate (FPP, C15).
Structural studies and phylogenetic comparisons
Department of Chemistry, University of Utah, 315 South indicate that the a-helical “isoprenoid fold” orig-
1400 East, Room 2020, Salt Lake City, UT 84112, USA. inally discovered in avian FPPase is found in all
*Present address: Division of Organic Chemistry, National of the E-family chain-elongation enzymes (11)
Chemical Laboratory, Pune 411008, India. and in more distant relatives that catalyze the Fig. 1. Patterns found in nature for connecting
†Present address: ImmunoGen Incorporated, 128 Sidney
Street, Cambridge, MA 02139, USA.
cyclopropanation reactions in sterol biosynthesis isoprene units. Colors indicate isoprene units (red
‡To whom correspondence should be addressed. E-mail: (12) and terpenoid cyclization reactions (13). An and black) and bonds joining the isoprene units
[email protected] ancestral FPPase or a closely related relative is a (green).

RESEARCH ARTICLES
sites at identical locations was introduced into were separated by thin-layer chromatography mers when the enzyme was incubated with IPP
the open reading frames (ORFs) for FPPase and (TLC) and analyzed by phosphor auto- and DMAPP (Fig. 4 and table S2) (18). A sim-
CPPase (fig. S1) (18). The kinetic constants for radiography (fig. S2). The screen for irregular ilar incubation with CPPase-M gave four C10
the modified (M) proteins, FPPase-M and monoterpene synthesis involved incubation products: chrysanthemol (COH) (15) from cyclo-
CPPase-M, were similar to those of the wild- with [14C]DMAPP. Incubation of FPPase-M with propanation, lavandulol (LOH) (17) from branch-
type (WT) enzymes (table S1). Eleven chi- [14C]IPP and DMAPP gave products that comi- ing, and geraniol and nerol (19) from chain
meras were constructed from FPPase-M and grated with C10 (GPP) and C15 (FPP) isoprenoid elongation. In the series C69F → C243F, irreg-
CPPase-M by fusing fragments of the two diphosphates. No products were seen when the ular monoterpenes were first detected as mi-
proteins at the common restriction sites. In six enzyme was incubated with [14C]DMAPP. In- nor products with the C98F chimera, which
of the constructs, amino acids in FPPase-M, cubation of CPPase-M with [14C]DMAPP gave gave LOH and an unexpected isomer, maconelliol
beginning at the N terminus, were replaced with products that comigrated with chrysanthemyl (MOH) (2), from a cyclobutanation reaction. Syn-
increasingly longer segments from CPPase-M. diphosphate (CPP). However, the enzyme also thesis of irregular monoterpenes became the
A complementary set of six constructs was pre- gave products characteristic of a C10 di- dominant reaction for the C243F chimera, where
pared in a similar manner by replacing amino phosphate when incubated with [14C]IPP and LOH was favored over COH by a ratio of ~3:1.
acids in CPPase-M, again beginning at the N DMAPP in the chain-elongation assay. TLC For CPPase-M, the major product was COH,
terminus, with the corresponding segments traces for the series of chimeras from C69F to which was only favored over LOH by a ratio of
from FPPase-M. The chimeras were named C243F reflect a gradual transition from the C10 ~2:1. In the F69C → F178C series, irregular
by designating the site of the junction and the and C15 chain-elongation products typically products (LOH and COH) were only detected
origin of the amino acids, either FPPase-M or synthesized by FPPase to the irregular products for the F69C chimera (table S2). The propor-
CPPase-M, on either side of the junction. For synthesized by CPPase. Between C69F and tion of chain-elongation products to irregular
example, the “C69F” chimera (where C indicates C178F, chain-elongation activity gradually shifted products increased as the concentration of IPP
CPPase and F indicates FPPase) has a junction from predominant synthesis of C15 FPP to increased relative to that of the allylic substrate,
site constructed at the common BseR I restriction exclusive synthesis of C10 GPP. Activity in the reflecting competition between the isomeric
sites in the ORFs for FPPase-M and CPPase-M [14C]DMAPP assay was readily detected for the substrates for binding.
where the first 69 amino acids (FPPase-M C219F chimera and became progressively greater Except for CPPase-M and the C243F and
numbering) are from CPPase-M and the re- for C243F and CPPase. The F219C protein F69C chimeras, reactions proceeded slowly
maining amino acids are from FPPase-M. The was inactive in both assays. Activity for irreg- when DMAPP was the sole substrate. In ad-
chimeric proteins were obtained for 11 of the ular products was low in all of the chimeras in dition to COH, LOH, and MOH, small amounts
12 constructs (only F243C failed to give pro- the F → C series. Trace amounts of materials of planococcol (POH) (9), an isomer of MOH,
tein) and were purified (>95%) by Ni2+-affinity were seen in all of the TLC traces, with the were detected in extracts from incubations with
chromatography. Removing the His6 tag in F178C chimera giving somewhat more C10 the C98F and C147F chimeras, indicating that
FPPase-M and CPPase-M did not alter their product than the others. these two enzymes also synthesized planococcyl
activity, and the histidine tags were not removed Product analysis. The TLC radioassay diphosphate (PPP) (Fig. 4 and table S3). Thus,
from the parental enzymes or chimeric enzymes did not resolve individual components within most of the proteins that we studied synthesized
used in this study. each group of C5, C10, and C15 diphosphates. irregular monoterpenes. The exceptions were
A screen for chain-elongation and “ir- Gas chromatographic (GC) and GC mass spec- FPPase-M and C178F, which only catalyzed
regular” products. FPPase-M, CPPase-M, and trometric analysis of alcohols obtained after hy- chain elongation, and F219C, which was inactive
11 chimeric proteins were screened for synthesis drolysis of the diphosphates with alkaline in all of the assays. Several of the enzymes syn-
of chain-elongation and irregular products (Fig. phosphatase showed that C10 and C15 chain- thesized at least one compound generated by the
3). The screen for chain elongation involved elongation alcohols were the exclusive products cyclopropanation, branching, and cyclobutanation
incubation of the proteins with [14C]IPP and from FPPase-M with selective, but not ex- reactions in addition to chain-elongation products.
either DMAPP or GPP. Products and substrates clusive, formation of the E double-bond iso- The WT A. tridentata ssp. spiciformis CPPase was
Fig. 2. Building reactions in the isoprenoid biosynthetic pathway. PPO, diphosphate; R, CH2(CH2CH= Fig. 3. Products from incubations with IPP and
C(CH3)CH2)nH, where n = 0, 1, 2, 3, and so forth. DMAPP. NPP, neryl diphosphate.

RESEARCH ARTICLES
slightly more selective for CPP than CPPase-M. coupling reactions have been identified and char- stereoisomers of these two important metabo-
The WT CPPase from C. cinerariaefolium was acterized. Metabolites from branching and cy- lites. Work with recombinant squalene synthase
slightly more selective for cyclopropanation clobutanation are narrowly distributed, and the indicates that the relative orientation of the two
relative to branching and chain elongation than biosynthetic enzymes and the corresponding molecules of FPP, which are condensed to form
the sagebrush enzymes. genes have not been identified. presqualene diphosphate, is identical to the
Stereochemistry. The absolute stereochem- CPPase and the FPPase/CPPase chimeras “endo” orientation predicted for DMAPP (4).
istries of the irregular products from the en- synthesize single enantiomers of CPP, LPP, The stereochemical similarities between the
zyme catalyzed reactions were established by maconelliyl diphosphate (MPP), and PPP. The compounds synthesized by the chimeric proteins
hydrolyzing the isoprenoid diphosphates to the absolute stereochemistries of the cyclopropane and those synthesized by their respective WT
corresponding alcohols and comparing their GC ring in CPP, the chiral center in LPP, and enzymes in nature are consistent with a scenario
retention times by coinjection with authentic sam- cyclobutane rings in MPP and PPP require that in which the naturally occurring enzymes evolved
ples on a chiral column (figs. S3 and S4). In each C1 of the dimethylallyl unit destined to alkylate from a common ancestor that provided the orig-
case, a single enantiomer [(1R,3R)-COH (19), the C2-C3 double bond of the other dimethylallyl inal template for the orientation of the bound
(R)-LOH (20), (R)-MOH (2), and (1R,3R)-POH unit is located on the Re face of the C2-C3 substrates. Genes for the cyclopropanation en-
(9)] was found. The absolute stereochemistries of double bond. If one assumes that formation of zymes evolved twice, presumably from an
the alcohols isolated from our enzyme-catalyzed PPP proceeds through a “least motion” reaction E-selective chain-elongation template: once at
reactions were identical to those of the corre- coordinate, the absolute configuration of the C3 the very beginning of cellular evolution for bio-
sponding alcohols isolated from natural sources. cyclobutane carbon dictates that the enzymes synthesis of squalene and phytoene, and subse-
Overview. The isoprenoid pathway uses bind the two molecules of DMAPP in an quently much later for biosynthesis of CPP.
only four different coupling reactions to build “endo” orientation (fig. S5). The rather conservative changes that we
the carbon skeletons of its metabolites from the Thus far, only a single set of enantiomers made in the catalytic site suggest that the elon-
fundamental five-carbon building blocks IPP has been reported for the naturally occurring ir- gation and irregular isoprenoid products are
and DMAPP. Metabolites built by chain elon- regular isoprenoid compounds formed by branch- formed by a similar chemical mechanism with
gation and cyclopropanation are broadly distrib- ing, cyclopropanation, and cyclobutanation branches leading to the individual products. A
uted in nature, and the enzymes that catalyze the reactions. The absolute stereochemistries of the comprehensive mechanism for the four cou-
alcohols obtained by hydrolysis of CPP, LPP, pling steps during biosynthesis of irregular
MPP, and PPP in our studies are identical to isoprenoid compounds, based on what is known
those of COH, MOH, and POH isolated from about the stereochemistries of chain elongation,
natural sources. Likewise, the topologically re- cyclopropanation, branching, and cyclobutanation
lated 1R,2R,3R enantiomers of presqualene di- and the mechanism of chain-elongation reac-
phosphate (21) and prephytoene diphosphate tion, is shown in Fig. 5. The chain-elongation
(22) are the only known naturally occurring reactions catalyzed by FPPase proceed by a
Fig. 4. Relative percentage of products from in-

cubations with FPPase-M, CPPase-M, and the C → F
series of chimeras. FPP, blue; GPP, gold; LPP,
green; CPP, red; MPP, pink; PPP, orange. Di-
phosphate products were hydrolyzed by treatment
with alkaline phosphatase, and the relative per-
centages of the corresponding alcohols were
determined by GC. (A) Incubation for 2 hours at
30°C with IPP (200 mM) and DMAPP (200 mM).
(B) Incubation for 12 hours at 30°C with DMAPP Fig. 5. A dissociative electrophilic alkylation mechanism for chain elongation, cyclopropanation,
(3 mM). branching, and cyclobutanation.

RESEARCH ARTICLES
dissociative electrophilic alkylation of the dou- zyme avian FPPase (12) has also been found in 10. C. D. Poulter, Phytochem. Rev. 5, 17 (2006).
ble bond in IPP by the allylic cations generated the cyclopropanation enzyme squalene synthase 11. L. C. Tarshis, M. Yan, C. D. Poulter, J. C. Sacchettini,
Biochemistry 33, 10871 (1994).
from DMAPP or GPP (23). By analogy, for (13) (sterol biosynthesis) and several different 12. J. Pandit et al., J. Biol. Chem. 275, 30610 (2000).
biosynthesis of irregular monoterpenes, we terpenoid cyclases (14) along with aspartate-rich 13. D. W. Christianson, Chem. Rev. 106, 3412 (2006).
suggest that a related dissociative electrophilic motifs involved in binding allylic diphosphate 14. S. B. Rivera et al., Proc. Natl. Acad. Sci. U.S.A. 98, 4373
alkylation of the double bond in DMAPP by the substrates, indicating that the enzymes evolved (2001).
15. A. Hemmerlin, S. B. Rivera, H. K. Erickson, C. D. Poulter,
dimethylallyl cation results in a protonated from a common ancestor. Phylogenetic correla- J. Biol. Chem. 278, 32132 (2003).
cyclopropane intermediate. This species can be tions suggest that the cyclopropanation enzyme 16. D. Iwata-Reuyl, S. K. Math, S. B. Desai, C. D. Poulter,
deprotonated to give CPP or rearrange to a phytoene synthase (carotenoid biosynthesis) also Biochemistry 42, 3359 (2003).
tertiary cation, which can in turn be deprotonated has an isoprenoid fold. Our discovery that chi- 17. H. K. Erickson, C. D. Poulter, J. Am. Chem. Soc. 125,
6886 (2003).
to give LPP. Alternatively, the tertiary cation can meric enzymes from FPPase and CPPase cata- 18. H. V. Thulasiram, C. D. Poulter, J. Am. Chem. Soc. 128,
cyclize to give a cyclobutylcarbinyl cation that lyze branching and cyclobutanation reactions 15819 (2006).
can then be deprotonated to give MPP or LPP. suggests that WT enzymes with these activities 19. K. Alexander, W. W. Epstein, J. Org. Chem. 40, 2576
Formation of any specific product would be con- also share this common ancestor. (1975).
20. M. Soucek, L. Dolejs, Collect. Czech. Chem. Commun. 24,
trolled by the ability of the enzyme to stabilize a
3802 (1959).
specific intermediate along the reaction coordi- 21. G. Popjak, J. Edmond, S.-M. Wong, J. Am. Chem. Soc. 95,
nate through dipolar and electrostatic interactions References and Notes
2713 (1973).
1. K. Gunawardena, S. B. Rivera, W. W. Epstein,
and to facilitate the selective removal of pro- 22. L. J. Altman, R. C. Kowerski, D. R. Laungani, J. Am. Chem.
Phytochemistry 59, 197 (2002).
tons. The stereochemistries of the products Soc. 100, 6174 (1978).
2. A. Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 101, 9601
23. J. M. Dolence, C. D. Poulter, in Comprehensive Natural
result from the conformations of the two bound (2004).
Products Chemistry, O. Meth-Cohn, Ed. (Elsevier, Oxford,
substrate molecules before the reaction. Only 3. C. H. Heathcock, B. L. Finkelstein, T. Aoki, C. D. Poulter,
UK, 1999), vol. 5, pp. 18473–18500.
Science 229, 862 (1985).
minor changes in the relative positions of the 4. M. B. Jarstfer, D. L. Zhang, C. D. Poulter, J. Am. Chem.
24. We thank A. Zhang for providing a sample of (R)-MOH.
substrates are required to accommodate the for- This work was supported by NIH grant GM 21328.
Soc. 124, 8834 (2002).
mation of the different products. 5. B. S. J. Blagg, M. B. Jarstfer, D. H. Rogers, C. D. Poulter,
Supporting Online Material
This scenario provides an attractive mecha- J. Am. Chem. Soc. 124, 8846 (2002).
www.sciencemag.org/cgi/content/full/316/5821/73/DC1
6. H. C. Rilling, W. W. Epstein, J. Am. Chem. Soc. 91, 1041
nism for the evolution of the isoprenoid path- (1969). Materials and Methods
way through gene duplication and random Figs. S1 to S5
7. L. J. Altman et al., J. Am. Chem. Soc. 94, 3257
Tables S1 to S3
mutagenesis of the duplicate genes to give new (1972).
Electron Impact (EI) Mass Spectra
proteins, one of which is constrained to retain 8. J. D. Berkowitz, J.-L. Giner, T. Andersson, J. Nat. Prod. 63,
Chemical Ionization (CI) Mass Spectra
267 (2000).
its original function, whereas the other is free to 9. B. A. Bierl-Leonhardt, D. S. Moreno, M. Schwarz, References
acquire a new activity. The isoprenoid fold first J. Fargerlund, J. R. Plimmer, Tetrahedron Lett. 22, 389 20 November 2006; accepted 16 February 2007
seen in the E-selective chain-elongation en- (1981). 10.1126/science.1137786
Schemas and Memory Consolidation considered the issue of what the animal itself
brings in the way of knowledge to a learning
situation, with the exception of studies of spatial
Dorothy Tse,1* Rosamund F. Langston,1* Masaki Kakeyama,2 Ingrid Bethus,1 and relational memory (7–9). This is partly
Patrick A. Spooner,1 Emma R. Wood,1 Menno P. Witter,3 Richard G. M. Morris1† because most experiments are conducted with
“experimentally naïve” animals, and also be-
Memory encoding occurs rapidly, but the consolidation of memory in the neocortex has long cause the creation of a mental schema is difficult
been held to be a more gradual process. We now report, however, that systems consolidation can to map precisely onto concrete neuroscience
occur extremely quickly if an associative “schema” into which new information is incorporated concepts such as anatomical connectivity or
has previously been created. In experiments using a hippocampal-dependent paired-associate task synaptic plasticity. The present experiments test
for rats, the memory of flavor-place associations became persistent over time as a putative the idea that the schema concept is directly
neocortical schema gradually developed. New traces, trained for only one trial, then became relevant to the neural mechanisms of systems
assimilated and rapidly hippocampal-independent. Schemas also played a causal role in the memory consolidation (10–12).
creation of lasting associative memory representations during one-trial learning. The concept Experiments on schema learning. We trained
of neocortical schemas may unite psychological accounts of knowledge structures with rats to learn several flavor-place associations
neurobiological theories of systems memory consolidation. concurrently, using different flavors of food
(flavor cues) and sand wells (place cues) located
he concepts of “mental schema” and cative skill of the speaker in logically conveying within a familiar testing environment called an
T “mental models” as frameworks of

knowledge are now well established (1, 2),
with implications for story recall, deductive
his or her message. In the absence of such mental
frameworks, we are unable to follow scientific
inferences in a talk and have the phenomeno-
“event arena” (13). The task was to learn which
1
Laboratory for Cognitive Neuroscience, Centre for Cogni-
tive and Neural Systems, and Centre for Neuroscience
inference, and education (3, 4). For example, logical experience of being functionally amnesic
Research, University of Edinburgh, 1 George Square,
the memory of grammatically correct but seman- for its content a surprisingly short time later. Edinburgh EH8 9JZ, Scotland, UK. 2Division of Environ-
tically unusual prose passages is substantially Curiously, this fundamental idea about mem- mental Health Sciences, Center for Disease Biology and
better when subjects have an activated and ory has had little impact in neuroscience. Selec- Integrative Medicine, Graduate School and Faculty of
relevant mental framework with which to under- tive activation of a specific region within the Medicine, University of Tokyo, Faculty of Medicine Building
stand them (5). An everyday experience for posterior parietal cortex occurs in human subjects 1, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
3
Centre for the Biology of Memory, Medical-Technical
working scientists is remembering complex new when, having been given relevant pictorial Research Centre, NO-7489 Trondheim, Norway.
information in an academic seminar. Our ability information earlier, they correctly interpret un- *These authors contributed equally to this work.
to do so depends as much on our possession of an usual textual information that would otherwise be †To whom correspondence should be addressed. E-mail:
appropriate mental schema as on the communi- incomprehensible (6). Animal studies have rarely [email protected]

RESEARCH ARTICLES
flavor was in which location such that, when cued similar way. Probe tests, other controls, and novel If the animals develop a neocortical associative
with a specific flavor in start boxes at the side of context training were scheduled at various stages schema for this task, and if this is activated when
the arena, the animals would be rewarded for before and after making sham or hippocampal the animals enter the apparatus, it might aid the
going to the correct location by receiving more of lesions (fig. S4). Using the same paired-associate encoding of new paired associates and their rapid
that same food (i.e., “cued recall”). They should layout as in experiment 1, we examined acquisi- assimilation into the schema. A single training
be able to recall that banana-flavored food is at tion of sand-well choice behavior during training. session of six trials was given (Fig. 2A, session 21)
one location, bacon-flavored food at another, and A “performance index” was calculated, and this in which paired associates (PAs) 1 and 6 were re-
so on (Fig. 1, A and B). Such paired-associate index improved monotonically across sessions placed by two new PAs, 7 and 8, hidden at two
learning is likely to be mediated by the hippo- (Fig. 2A). In nonrewarded probe trials, preferen- nearby locations; PAs 2 to 5 were trained normally.
campus initially (14–16), with long-term storage tial digging at the correct location rather than the Note that PAs 7 and 8 received only one rewarded
of paired-associate memory traces eventually con- other five locations increased from chance levels trial each. The inset of Fig. 2C shows how the new
solidated in the neocortex (17, 18). This makes at the outset of training to a highly significant PAs were located near those of the now-closed
this paradigm ideal for looking at the temporal preference for the cued location (Fig. 2B). To sand wells. A nonrewarded probe trial was given
dynamics of systems memory consolidation exclude the possibility that an olfactory cue in the 24 hours later to test memory for the new associ-
(10, 12, 19, 20), a process widely held to be correct sand well guided choice performance on ates. Preferential digging was observed at the cor-
quite slow. Additionally, the use of location as training days, we conducted a single session of rect cued location in the arena relative to the new
one member of each paired associate allowed six trials in which the daily protocol was noncued location (i.e., less digging at location 8 for
the animals to learn each association as either an unchanged, except that no cue flavors were those animals on a PA7 trial, and vice versa) and to
isolated declarative “fact,” in which spatial offered in the start box. Choice performance fell any of the original locations (PAs 2 to 5; Fig. 2D).
information is generally considered as no differ- to chance (Fig. 2A, session 18), returning to The rapid acquisition of new PAs in a single trial,
ent from other kinds of information (10), or as above chance on the next normal session. The and their retention over 24 hours, are indications
some kind of mapping of flavors to arena loca- possibility of cryptic olfactory guidance by cues that the prior learning of an associative schema
tions, resulting in the formation of a spatial or on the arena near the correct sand well was also may aid the encoding, storage, and/or consoli-
relational framework (7, 21). ruled out in a later session by physically rotating dation of new PAs. In contrast, animals trained on a
After habituation, the animals were started the arena through 90° after the third trial of a similar one-trial task, but with novel PAs each day,
from one start box of the arena (at north, south, session and back to its normal orientation after showed consistent forgetting over 90 min (13).
east, or west) on all six trials of a session. A the third trial of a second session on the next day. Time course of memory consolidation.
different start box was used for each session. A The sand wells and intramaze cues were re- Hippocampal or sham lesions were then made
trial began when the rat was given a cue flavor in located such that their places relative to the distal 24 hours later—a much shorter time after training
the start box. Upon entering the arena, the animal room cues remained the same. Arena rotation had of the new flavors (48 hours) than is usually
was confronted by six sand wells (Fig. 1, A and no effect (fig. S4, A3). With a different start box thought necessary for systems consolidation to
B) of which only one contained flavored food— used in each session, it would appear that the be completed (24–27), and shorter than the
the same flavor given as a cue in the start box animals can visually perceive their own location usual time scale of differential changes in the
(22). The animals visited and sometimes dug at relative to the intra- and extramaze landmarks patterns of glucose use or immediate early gene
incorrect sand wells, which did not contain food on and use allocentric memory representations to activation between hippocampus and neocortex
that particular trial, until they found the correct one. identify the correct goal location among the six after learning (19, 28). After recovery from sur-
On each trial, the animals would retrieve the first of available sand wells. gery, a series of nonrewarded probe tests (with
three buried food pellets, return to the start box to
eat it, and then run back to the correct sand well to Fig. 1. Paradigm for
collect and transport the second and third pellets. hippocampal-dependent
One hour later, the second trial began with a paired-associate (PA) learn-
different cue flavor in the start box and a different ing. (A) The large event
sand well baited. There were six trials per session, arena (1.6 m by 1.6 m)
with the next session run 48 hours later (23). contains a 7 × 7 grid of
We began by examining the impact of neuro- locations at which sand
toxic hippocampal lesions made before training wells can be made avail-
(experiment 1). After 13 sessions, sham-lesioned able and four surround-
animals were digging less frequently at incorrect ing start boxes. After
sand wells before going to the correct one, being given a cue flavor
whereas the hippocampal-lesioned animals did in a start box, the animals
not improve. A single nonrewarded probe trial recall the spatial location
was then scheduled, which started with the with which it is associ-
provision of a cue flavor in the start box. The ated, and run into the
sham-lesioned animals spent significantly more arena to that location to
secure more of that flavor
time digging at the cued location than at the
of food. (B) The spatial
other five incorrect sand wells, whereas the
arrangement of the six
hippocampal-lesioned animals were at chance PAs and the “schema”
(Fig. 1C; see tables S1 and S2 and figs. S1 to S3 this constitutes (F, flavor;
for the lesions and full experimental design). L, location). (C) Preferen-
The lesions were extensive, leaving minimal tial digging during a non-
residual tissue throughout the longitudinal axis rewarded probe test [probe trial 1 (PT1)] by sham-lesioned but not hippocampal (HPC)-lesioned animals
of the hippocampus (Fig. 1D). (ns = 6). Groups t = 5.25, df = 10, P < 0.001; sham versus chance, t = 5.01, df = 5, P < 0.005; HPC
To investigate the properties of paired-associate versus chance, not significant (n.s.). (D) A three-dimensional reconstruction of the volume of
learning and its consolidation in more detail hippocampus lesioned in a representative rat (red), together with typical overlying cortical damage
(experiment 2), we trained normal animals in a (yellow). The gray region represents the transparent volume of the rat brain.

RESEARCH ARTICLES
interpolated training days using the original
flavor-place pairs) was given to examine memory
for the original schema and the two new PAs.
These consisted of separate tests of the original
PAs 2 to 5 and new PAs 7 and 8, each repeated
once across a series of four sessions to enable
both PA7 and PA8 to be tested in all animals. The
hippocampal-lesioned group not only could
successfully recall the original PAs learned over
the previous month (Fig. 2D) but also, remark-
ably, could remember the newly acquired pairs
PA7 and PA8. Because the lesions were near-
complete (~90%; see Fig. 1D and fig. S2B), these
two findings imply that (i) the memory traces for
these PAs must be stored outside the hippocam-
pus, probably in the neocortex; and (ii) con-
solidation of new associates whose acquisition is
mediated by the hippocampus takes place within
48 hours.
To be more confident of these claims, it was
essential to establish that the learning of further
new PAs still required the integrity of the hip-
pocampus in these same animals. Accordingly,
immediately after this series of postoperative
probe tests, we conducted a single six-trial train-
ing session with PAs 2 to 5 of the original schema,
but with PAs 7 and 8 now replaced by sand wells
containing two new flavors in nearby locations in
the arena (PAs 9 and 10; Fig. 2E). The probe test
conducted 24 hours later showed that sham-
lesioned animals could readily learn and recall
these new pairs, whereas the hippocampal-
lesioned group could not. Thus, the one-trial
acquisition of new PAs in this paradigm in
experienced animals was still blocked by hippo-
campal lesions. Hence, it is unlikely that any
relearning took place after the hippocampal
lesions during the earlier series of four probe
tests that had examined remote memory (the
interpolated training was restricted to the well-
trained PAs 2 to 5). The effective cued recall of
the new PAs 7 and 8 introduced before the lesion Fig. 2. Acquisition of an associative schema and its role in new learning and consolidation. (A)
must therefore reflect rapid, successful systems Acquisition of PAs. The animals (n = 18) made fewer choice errors over training (F = 18.24, df =
consolidation. 5.7/97.5, P < 0.001; Greenhouse-Geisser correction, including degrees of freedom) such that the
Although the animals appear to have acquired performance index, computed as 100 – [100 × (errors/5)], was significantly above chance from
an associative schema reflecting the mapping of session 10 onward (ts > 5.08, df = 1/17, Ps < 0.001). Removing cue flavors from the start box on
flavors to places in the arena, an alternative might session 18 resulted in performance dropping to chance and then returning to 70% correct on a
be a response-based “win-stay, lose-shift” infer- succeeding normal session (session 19). (B) Cued-recall probe trials. Nonrewarded probe tests
ence strategy in the manner of a learning set (29). revealed a graded learning of the original PAs (cued flavor = solid bars) across sessions 2, 9, and
It is not entirely clear how such a procedural 16 (F = 16.24, df = 1.54/26.22, P < 0.001; above chance in PTs 2 and 3; ts = 3.94 and 6.17, df =
strategy could be applied in this context, with six 17, P < 0.005 and P < 0.001, respectively). (C) Effective recall in PT4 of the location of the cued
choice locations and only one trial per day to new PA (solid bar), coupled with avoidance of the noncued new PA (gray bar) and the remaining
each cued location. However, as procedural strat- original associates (open bar) 24 hours after a single session of training with only one trial of each
egies are generally context-independent, this new PA (repeated-measures F = 65.28, df = 1.7/29.1, P < 0.001; cued location above chance, t =
10.29, df = 17, P < 0.001; noncued versus original, n.s.). (D) Postoperative retention. Both sham-
account would predict that the learning of an
lesioned (n = 8) and HPC-lesioned (n = 10) animals could effectively remember both original PAs
entirely new set of six PAs in a new context (PTs 5 and 7) and new PAs introduced for a single trial 2 days before surgery (PTs 6 and 8). Both
would occur very quickly. In contrast, the schema groups dug at the sand wells of the original associates (flavors 2 to 5) significantly more than
hypothesis requires that a new schema be chance (HPC t =3.60, df = 9, P < 0.01; sham t = 12.89, df = 7, P < 0.001; sham versus HPC group,
gradually learned. The same animals of experi- t = 2.86, df = 16, P < 0.05). Both groups also dug equally at the cued locations of the new
ment 2 were first trained on a new set of PAs in associates relative to the noncued locations (Group × Location F < 1, n.s.), and at these cued
the same event arena (fig. S7) and then in a novel locations better than chance (ts > 8.07, df = 9 and 7, P < 0.001). (E) Postoperative new training.
event arena in a different room with new intra- Hippocampal lesions prevented the learning of new PAs (PAs 9 and 10; Group × Location F =
and extramaze landmarks, new flavors, and a 60.23, df = 1.64/26.17, P < 0.001). Digging at the cued new location in PT9 was significantly
distinct spatial geometry to the new set of sand above chance only in the sham group (t = 17.07, df = 7, P < 0.001) and significantly lower in the
wells (Fig. 3, A and B). Acquisition again took HPC group than in the sham group (t = 13.78, df = 16, P < 0.001).

RESEARCH ARTICLES
place gradually, such that the learning curve of of the normal animals in the first event arena. The test performance early in training followed the
the now experienced sham-lesioned animals hippocampal-lesioned animals did not learn the same gradual pattern in the sham group, resulting
did not differ from the original rate of learning new spatial schema despite repeated trials. Probe in effective probe test performance only by ses-
sion 67 (Fig. 3C). These findings argue against a
response-based strategy, such as a learning set,
because learning was no faster in the new room
with new flavor-place geometry.
Completion of training in the second room
offered the opportunity of returning the ani-
mals to the first arena to examine their now
remote memory of the original set of PAs first
learned 4 to 5 months earlier. Remarkably, the
hippocampal-lesioned animals were above
chance in cued spatial recall (session 68, Fig.
3D) and even showed a nonsignificant trend
toward better performance than did sham-
lesioned controls in a probe test as early as
session 2. The sham-lesioned animals may have
sustained some associative interference arising
from their successful training on other sand-well
arrangements in this and the other contexts, but
after as few as six sessions of retraining, both
groups showed effective cued recall of the
original PAs (Fig. 3E). Thus, the failure to learn
new PAs in a new context after a hippocampal
lesion did not affect the ability to remember, after
several months, information acquired before the
lesion—a pattern exactly like that shown by
patient E.P. in his knowledge of current and past
hometown topography (30).
Fig. 4. Identifying the interval between training

and hippocampal lesions for consolidation. A
striking temporal gradient of retrograde amnesia
is observed in this paradigm. HPC lesions made 3
hours after training (n = 7) on the novel flavor
tested 14 days later prevented consolidation,
whereas consolidation was complete when HPC
Fig. 3. Gradual acquisition of new PAs in a new context by experienced animals. (A) Acquisition of PAs. lesions (n = 6) were made after 48 hours (Group ×
The now experienced sham group (n = 8) learned a new set of six PAs in the second event arena at a Delay F = 15.77, df = 1/13, P < 0.005). The HPC
comparable rate to that shown by normal animals in the first event arena (Group × Session F = 1.97, df = and control 48-hour groups did not differ (t < 1).
6.9/116.9, 0.10 > P > 0.05, treating Group as a between-subjects factor). Relative to the sham-lesioned The performance of the HPC 48-hour group was
group, the HPC-lesioned group (n = 10) failed to learn (Group F = 128.63, df = 1/15, P < 0.001; Group × significantly higher than that of the HPC 3-hour
Session F = 7.42, df = 5.9/89.3, P < 0.001). (B) Spatial arrangement of the new PAs (PAs 11 to 16) in the group (t = 4.82, df = 11, P < 0.001), but the
new event arena. (C) Cued-recall probe trial. Proportion of digging at the cued location relative to the corresponding two control groups (ns = 9) did not
noncued locations by sham- and HPC-lesioned animals (PT15, session 67). The sham group was above differ (t < 1). The control groups were above chance
chance (t = 2.38, df = 7, P < 0.05); the HPC group was not (t < 1). However, the difference between at both training-lesion intervals (ts > 5.1, df = 8,
groups showed only a trend toward significance (t = 1.83, df = 15, 0.10 > P > 0.05). (D) Return to the P < 0.001); the HPC 3-hour group did not differ
original event arena and flavors (flavors 1 to 6). Inset indicates transition to the original schema acquired from chance (t < 1), whereas the HPC 48-hour
before surgery. The HPC group is above chance at the outset (t = 3.9, P < 0.005; session 68), but neither group was above chance (t = 4.90, df = 5, P <
Group nor Group × Session effects were significant for the performance index (Ps > 0.05). After six 0.005). Separate analyses of the postsurgery mem-
sessions of retraining, the sham group caught up, and both groups were well above chance (ts = 8.7 and ory for the original PAs learned over 14 sessions
8.9, Ps < 0.001). (E) Performance in the probe test (PT16) indicated that both HPC and sham groups were showed above-chance performance for both the
consistently above chance in preferentially digging at the cued location (t = 4.37, df = 8, P < 0.005; t = HPC and sham groups (HPC t = 5.80, df = 12, P <
3.19, df = 7, P < 0.025, respectively) and did not differ from each other (t < 1, n.s.). 0.001; sham t = 9.85, df = 17, P < 0.001).

RESEARCH ARTICLES
If systems consolidation within the neocortex introduction of PAs 9 and 10. Cued recall was changed every two sessions (Fig. 5B). The sched-
can take place in as little as 48 hours, it becomes examined for the new associates shortly before uled inconsistency was therefore in the relational
of interest to find out the minimal time required surgery and was found to be effective for all pairing of the items rather than the identity of the
for it to occur. Some theoretical models suppose animals. After surgery, cued recall for the new flavors or the locations of the sand wells. More-
that a memory trace stored in the hippocampus, one-trial PAs was at chance for those animals over, a change only every two sessions did not
serving as an “index” or “pointer” to cortically subject to hippocampal lesions 3 hours after preclude the animals attempting to learn these PAs
encoded information, must last sufficiently long acquisition, but—replicating the results of exper- across sessions, but would have precluded the
to guide the slower systems-level consolidation iment 2—it was effective when lesions were creation of a context-specific schema. Choice per-
process that is thought to take place in sleep, made 48 hours after training (Fig. 4). This is a formance gradually improved in the consistent
requires sharp-wave activity, and has previously strikingly steep upward temporal gradient of schema room but not in the inconsistent room
been shown to involve hippocampal-neocortical remote memory. (Fig. 5A); nonrewarded probe tests also es-
interactions over time (31–35). The prediction is Causal role for schemas in learning. The tablished that the animals dug preferentially in
that hippocampal lesions made 3 hours after final issue to consider is whether an activated the cued location in the arena of a start-box flavor
training to animals that do not sleep during this schema is causally necessary for rapid memory in the consistent but not the inconsistent context
short training-surgery interval should prevent consolidation (5). An alternative account of these (Fig. 5C). This difference between the two con-
neocortical consolidation. In experiment 3 (using experiments could be that the animals find it texts is not in itself surprising and would occur
a new set of 18 rats that acquired the basic increasingly easier over the course of training to even if the animals were still trying to learn in-
schema of PAs 1 to 6 over 14 sessions as before), encode, store, and/or consolidate individual PAs dividual PAs in the inconsistent room. However,
we compared the impact of hippocampal lesions as a result of increasing familiarity with the con- this differential rate of learning sets the stage for a
given 3 or 48 hours after the training of two new text of learning, with the “schema” concept being last and crucial test of the schema concept.
PAs in single trials (PAs 7 and 8). This ex- superfluous. To contrast these alternatives, we This test involved the learning of new PAs. If
periment used a “reverse” day-night cycle (with trained normal animals in two event arenas con- animals learn PAs as isolated “facts,” and if they
all testing during the animal’s night) to minimize, currently (experiment 4). In one room, they were do so ever more quickly because of context fa-
in the case of the 3-hour interval, the likelihood of trained on a “consistent” schema in which flavors miliarity as training in this protocol proceeds, the
sleep episodes between the end of training and 1 to 6 were always placed consistently at loca- rate of learning in the two contexts should be the
the time of the lesion. A partial within-subjects tions 1 to 6, respectively (schema 1 = PAs 1 to 6; same. However, if the animals bring something
design was also used (fig. S8), with some animals Fig. 5B). In the other room, the animals were like “activated schema” to bear on the process of
having hippocampal lesions at appropriate time trained on “inconsistent” schema in which a learning, a difference between the two contexts
points soon after novel PAs 7 and 8, and others single set of six locations (locations 11 to 16) and might be observed. The “consistent schema”
that were only anesthetized in this first phase a set of six flavors (flavors 11 to 16) were used, would only be activated in its appropriate con-
given hippocampal or sham lesions after the later but the mapping of flavors to locations was text. Procedurally, the comparison in the rate of
Fig. 5. A consistent activated

schema promotes effective memory.
(A) Differential acquisition of con-
sistent and inconsistent schemata.
Effective acquisition by normal rats
(n = 9) occurred when mapping of
flavors to places remained con-
sistent, with six, four, two, and then
single sessions (sessions 1 to 40;
white background). Above-chance
performance was consistent from
session 15 onward (P < 0.025 for
each comparison with chance). The
same animals failed to learn a series
of inconsistent schemas in the
second event arena (selected days
are above chance, e.g., session 27,
but performance never rose above
60% correct; gray background). (B)
With the consistent schema, the
mapping of flavors to places is con-
sistent across sessions; inconsistent
schema used a common set of six
flavors and locations that were
associated for two sessions but then
changed every third session (see
N + 2, shaded gray). (C) Preferential
digging in the probe trials at the
cued locations for the consistent
schema (PT1: t = 10.9, df = 8, P < 0.001) but not for the inconsistent schema schema was not (PTs 4 and 6; Group × Location F = 13.92, df = 1.64/26.30, P <
(PT2: t < 1). (D) New PA probes. Performance 24 hours after exposure to the two 0.001). Approach latencies from the start box to the correct sand well during
new cue flavors and their locations when the animals would be encoding these probe trials were equivalent in the consistent (20.9 ± 1.9 s) and
information using a consistent activated schema (PTs 3 and 5) was consistently inconsistent (20.0 ± 2.5 s) contexts, indicating comparable motivation to
good to the cued new location, whereas performance after use of an inconsistent perform each task.

RESEARCH ARTICLES
learning new information had to be done in a uncontrolled olfactory cues. Similarly, the use of (30). It is unclear why effective remote spatial
manner that ensured an identical behavioral two new PAs exploring associative assimilation memory is found here but not in the water maze
protocol in the consistent and inconsistent rooms. into a schema, rather than a single PA, ensured that (48). One possibility is that the water maze is
In this phase, beginning at session 29, the ani- the effective recall in probe trials was not an more “recall-like” in character (10), requiring an
mals were therefore trained on four successive artifact of stimulus novelty. animal to generate its own reminder cues. The PA
sequences of three training sessions beginning as Discussion. These findings have implications paradigm used here could allow apparent cued
follows: session 29, further consistent-context for a number of key issues in the neurobiology of recall to be mediated in part by cued recognition
training of flavors 1 to 6; session 30, two new learning and memory. First, they indicate that based on proximal intramaze cues.
PAs trained in a session consisting of only two the rate at which systems consolidation occurs Third, the failure of animals with near-
trials (PAs 7 and 8); session 31, a nonrewarded in the neocortex can be influenced by what is complete hippocampal lesions to acquire PAs
probe test for these novel associates. This three- already known. In contrast, in the complementary over many trials of training (experiments 1 and 2)
session sequence was then repeated in the incon- learning systems approach (36, 37), the hippo- calls into question the capacity for effective
sistent context (sessions 32 to 34) using flavors campus is said to be “specialized for rapidly “semantic-like” learning in the absence of func-
11 to 16, then PAs 17 and 18 followed by a non- memorizing specific events” (37) and the neo- tional hippocampal tissue. This idea emerged
rewarded probe test; and again in the consistent cortex for “slowly learning the statistical regu- particularly in studies of developmental amnesia
and then the inconsistent context with PAs 9 and larities of the environment.” Consolidation of (49), but it has proved difficult to distinguish
10 and PAs 19 and 20, respectively. The sequence memory traces in the neocortex is held to be a whether the intriguing dissociations between
ended with PT6 on session 40 (Fig. 5). The use of largely time-dependent process determined by impaired episodic and intact semantic memory
only two rewarded trials instead of the usual six the specific patterns of information representa- in such patients are due to intact neocortical
trials per day on session 2 of this three-session tion, anatomical connectivity, and synaptic plas- learning of semantic information (50), to func-
sequence ensured that both the behavioral pro- ticity expression rules that it can support. Broadly tional reorganization in the developing brain, or
cedure and the memory-encoding demands on the speaking, this is a fair characterization of a large to islands of residual hippocampal function in
animals were identical in the two training contexts body of data (27), but it does not quite capture the these amnesic patients. When the medial tempo-
on session 2. Figure 5D shows successful acqui- potential that the neocortex has for rapid ral lesions are large, as in patient E.P., essentially
sition and 24-hour retention of these new PAs only consolidation when newly acquired information no declarative fact learning occurs (51). Our
when encoding occurred in the consistent-schema is compatible with previously acquired knowl- findings suggest that, in animals in which it is
context. The apparent motivation of the animals to edge. Given our observation that the neocortex possible to make selective 90% lesions of the
perform these two learning tasks was equivalent, can sometimes consolidate very rapidly, it fol- hippocampus as adults, the acquisition of new
as indexed by equivalent approach latencies to the lows that it must also be able to encode asso- flavor-place PAs is also consistently blocked and
target sand well in both the consistent and incon- ciative memory traces very rapidly—perhaps not rescued by multiple training trials. The
sistent contexts (Fig. 5D). even “on-line” within sensory-perceptual sys- generality of this observation beyond the spatial
These findings indicate that animals—no less tems. The widely held supposition that the neo- domain should be followed up in young animals,
than people—can bring activated mental schemas cortex is a slow learner therefore needs to be including primates, in order to model the situa-
to bear in a PA learning task and thereby encode, reappraised. The distinct temporal dynamics of tion in developmental amnesia more closely.
assimilate, and rapidly consolidate relevant new these memory processes may contribute to the That the acquisition of a schema took about a
information after a single trial. The capacity of usual finding that the cortex does learn more month points to the possibility of it involving
animals to make deductive inferences on the basis slowly than subcortical structures—a generality some kind of neuroanatomical growth process in
of their “mental models” of the world is, of that extends to conditional-associative motor the neocortex that creates an associative “space”
course, far more limited than that of humans (4), learning (38)—but that this may not always occur. in which new PAs can be rapidly stored without
but the principle that associative schemas can be A second finding is that the storage and recall interference—analogous to “phase sequences”
useful in memory is not unique to humans. of allocentric spatial memory can occur outside (52). Intercortical synaptic connections may be
In experiment 1, animals used hippocampal- the hippocampus in the rat, even for information created or unmasked within a functional network
dependent learning to acquire several PAs con- that has been acquired in a single trial as a that has only silent or baseline synaptic strengths.
currently, of which one member of each pair was consequence of hippocampal-dependent process- These could then be rapidly potentiated by
a spatial location in a familiar environment. This ing. This conflicts with both the cognitive-map relevant information when the network is in an
enabled the animals to treat these several as- theory and the multiple-trace theory of memory “active” state (an activated schema). The initial
sociates as a connected spatial set, rather than as consolidation (7, 39, 40). Spatial memory has growth process would necessarily take a period
individual “facts,” and so build up a framework been shown previously in rats with hippocampal of days or weeks—the very time period that has
in which similar new information could be lesions, but the information was either acquired hitherto been thought to mediate systems con-
stored. The construction of this “schema” took postoperatively and inflexibly over very extended solidation and to occur only after learning (20).
about a month—approximately the same period training (41, 42) or “semanticized” over many Thus, an intriguing speculation to emerge from
that several studies of retrograde amnesia have months before the lesion (43). The long-sought the present data, with conceptual similarities to
suggested is always required after learning for upward gradient of remote spatial memory in rats the principles of synaptic tagging and capture
effective systems consolidation to occur. We when varying intervals of time are systematically (53, 54), is that an associative space into which
observed, however, that if the several weeks of scheduled before making hippocampal lesions new information can be assimilated can be con-
schema building was completed before new learn- (44–47) is now definitively shown using a cued- structed before the exposure to that information.
ing, the assimilation and consolidation of novel recall protocol for information acquired in one However, this construction of associative inter-
information within these neocortical schemata trial. The temporal gradient is much steeper than connections can be noncommittal or “experience-
could be very rapid (experiments 2 and 3). We might have been expected on the basis of prior expectant” in character (55).
also established that the possession of an activated work using a within-subjects design for con- The findings bring to neuroscience a set of
schema is causally important in the acquisition of textual fear conditioning (26). Moreover, the ideas hitherto largely discussed in the context of
new information (experiment 4). The use of rig- effective remote spatial memory in hippocampal- psychological studies of human memory. The
orous control protocols (e.g., the noncued memory lesioned animals upon their return to the first concept of “activated schemas” has been dis-
test, arena rotation) established that performance is event arena, learned as young animals, is strik- cussed only in relation to humans (3), as it im-
mediated by PA memory rather than by cryptic ingly similar to that displayed by patient E.P. plies a conscious awareness that rats are unlikely

to possess. However, even if they are implicit, 14. M. Bunsey, H. Eichenbaum, Nature 379, 255 (1996). 40. M. Moscovitch, L. Nadel, G. Winocur, A. Gilboa,
schemas are an economical way to characterize 15. R. P. Kesner, M. R. Hunsaker, P. E. Gilbert, Behav. Neurosci. R. S. Rosenbaum, Curr. Opin. Neurobiol. 16, 179 (2006).
119, 781 (2005). 41. R. G. M. Morris, F. Schenk, F. Tweedie, L. E. Jarrard,
the gradual acquisition of an organized frame- 16. S. Wirth et al., Science 300, 1578 (2003). Eur. J. Neurosci. 2, 1016 (1990).
work of associative “semantic-like” information 17. K. Sakai, Y. Miyashita, Nature 354, 152 (1991). 42. H. Eichenbaum, C. Stewart, R. G. M. Morris, J. Neurosci.
from “episodic-like” events that, once acquired, 18. Y. Miyashita, Science 306, 435 (2004). 10, 3531 (1990).
allows relevant new information to be assimilated 19. B. Bontempi, C. Laurent-Demir, C. Destrade, R. Jaffard, 43. G. Winocur, M. Moscovitch, S. Fogel, R. S. Rosenbaum,
Nature 400, 671 (1999). M. Sekeres, Nat. Neurosci. 8, 273 (2005).
and stored rapidly. Given that animals have daily 20. P. W. Frankland, B. Bontempi, Nat. Rev. Neurosci. 6, 119 44. J. J. Bolhuis, C. A. Stewart, E. M. Forrest, Q. J. Exp.
activities such as finding food and water, it is (2005). Psychol. B 47, 129 (1994).
important for them to retain an organized body of 21. H. Eichenbaum, P. Dudchenko, E. Wood, M. Shapiro, 45. R. J. Sutherland et al., Hippocampus 11, 27 (2001).
knowledge about where these may be found and H. Tanila, Neuron 23, 209 (1999). 46. R. E. Clark, N. J. Broadbent, L. R. Squire, Hippocampus
22. The food used as one member of each PA (diet pellets 15, 260 (2005).
to be able to update such a framework rapidly, manufactured in a range of different flavors) was hidden in 47. S. J. Martin, L. de Hoz, R. G. M. Morris, Neuropsychologia
within one trial. This inferential flexibility of a sand mixture that had been adulterated by ground-up 43, 609 (2005).
rodent cognition is now established in several pellets consisting of 1% of each of the six flavors used on 48. R. G. M. Morris, P. Garrud, J. N. Rawlins, J. O’Keefe,
domains (9). any daily session (6% total). This and other procedures Nature 297, 681 (1982).
masked any olfactory cue that might have guided the 49. F. Vargha-Khadem et al., Science 277, 376 (1997).
animals to the correct sand well. The animals were shown to 50. M. Mishkin, W. A. Suzuki, D. G. Gadian, F. Vargha-Khadem,
References and Notes use recall of spatial location exclusively in making their Philos. Trans. R. Soc. London Ser. B 352, 1461 (1997).
1. F. C. Bartlett, Remembering (Cambridge Univ. Press, sand-well choices. No food pellets were present in the 51. P. J. Bayley, L. R. Squire, J. Neurosci. 22, 5741 (2002).
Cambridge, 1932). sand mixture during nonrewarded probe tests. 52. D. O. Hebb, The Organization of Behaviour (Wiley, New
2. K. Craik, The Nature of Explanation (Cambridge Univ. 23. See supporting material on Science Online. York, 1949).
Press, Cambridge, 1943). 24. S. M. Zola-Morgan, L. R. Squire, Science 250, 288 (1990). 53. U. Frey, R. G. M. Morris, Nature 385, 533 (1997).
3. J. D. Bransford, Human Cognition: Learning, Understanding 25. J. J. Kim, M. S. Fanselow, Science 256, 675 (1992). 54. A. Govindarajan, R. J. Kelleher, S. Tonegawa, Nat. Rev.
and Remembering (Wadsworth, Belmont, CA, 1979). 26. S. G. Anagnostaras, S. Maren, M. S. Fanselow, J. Neurosci. Neurosci. 7, 575 (2006).
4. P. N. Johnson-Laird, Mental Models: Towards a Cognitive 19, 1106 (1999). 55. W. T. Greenough, J. E. Black, in Developmental
Science of Language, Inference, and Consciousness 27. P. J. Bayley, J. J. Gold, R. O. Hopkins, L. R. Squire, Neuron Behavioral Neuroscience, M. R. Gunnar, C. A. Nelson,
(Cambridge Univ. Press, Cambridge, 1983). 46, 799 (2005). Eds., vol. 24 of Minnesota Symposia on Child Psychology
5. J. D. Bransford, M. K. Johnson, J. Verb. Learn. Verb. Behav. 28. T. Maviel, T. P. Durkin, F. Menzaghi, B. Bontempi, Science Erlbaum, Hillsdale, NJ, 1992); pp. 155–200.
11, 717 (1972). 305, 96 (2004). 56. Supported by a UK Medical Research Council program
6. E. A. Maguire, C. D. Frith, R. G. M. Morris, Brain 122, 29. H. F. Harlow, Psychol. Rev. 56, 51 (1949). grant (R.G.M.M.), a UK Biotechnology and Biological
1839 (1999). 30. E. Teng, L. R. Squire, Nature 400, 675 (1999). Sciences Research Council studentship award (R.F.L.), and
7. J. O’Keefe, L. Nadel, The Hippocampus as a Cognitive 31. T. J. Teyler, P. DiScenna, Behav. Neurosci. 100, 147 (1986). a Fondation pour la Recherche Médicale fellowship (I.B.).
Map (Clarendon, Oxford, 1978). 32. G. Buzsaki, Neuroscience 31, 551 (1989). We thank J. Tulloch for assistance with the histology.
8. B. O. McGonigle, M. Chalmers, Nature 267, 694 (1977). 33. A. G. Siapas, M. A. Wilson, Neuron 21, 1123 (1998).
9. H. Eichenbaum, Neuron 44, 109 (2004). 34. B. L. McNaughton et al., in Sleep and Synaptic Plasticity, Supporting Online Material
10. L. R. Squire, Psychol. Rev. 99, 195 (1992). C. Smith, P. Maquet, Eds. (Oxford Univ. Press, New York, www.sciencemag.org/cgi/content/full/316/5821/76/DC1
11. Y. Dudai, R. G. M. Morris, in Brain, Perception and 2003), pp. 225–246. Materials and Methods
Memory: Advances in Cognitive Sciences, J. Bolhuis, Ed. 35. R. G. Morris, Eur. J. Neurosci. 23, 2829 (2006). Figs. S1 to S8
(Oxford Univ. Press, Oxford, 2001), pp. 147–162. 36. R. C. O’Reilly, J. W. Rudy, Psychol. Rev. 108, 311 (2001). Tables S1 and S2
12. J. L. McClelland, B. L. McNaughton, R. C. O’Reilly, 37. K. A. Norman, R. C. O’Reilly, Psychol. Rev. 110, 611 (2003). References
Psychol. Rev. 102, 419 (1995). 38. A. Pasupathy, E. K. Miller, Nature 433, 873 (2005).
13. M. Day, R. F. Langston, R. G. M. Morris, Nature 424, 205 39. R. S. Rosenbaum, G. Winocur, M. Moscovitch, 5 October 2006; accepted 23 February 2007
(2003). Behav. Brain Res. 127, 183 (2001). 10.1126/science.1135935
REPORTS
with the same dislocation character (i.e., char-
Nonstoichiometric Dislocation acteristic displacement vectors called Burgers
vectors, b). The possibility of nonstoichiometric
Cores in a-Alumina cores also arises but has usually been rejected
because it suggests the possibility of charged
dislocations (1, 2) and the presence of long-
N. Shibata,1* M. F. Chisholm,2 A. Nakamura,3 S. J. Pennycook,2 T. Yamamoto,4 Y. Ikuhara1 range Coulomb fields with a high associated
electrostatic energy. This has been suggested to
Little is known about dislocation core structures in oxides, despite their central importance in be the reason why the close-packed {111} crys-
controlling electrical, optical, and mechanical properties. It has often been assumed, on the tal plane in alkali halides cannot be an easy slip
basis of charge considerations, that a nonstoichiometric core structure could not exist. We report system (2, 3). Detailed knowledge of dislocation
atomic-resolution images that directly resolve the cation and anion sublattices in alumina core structures and compositions is critical to
(a-Al2O3). A dissociated basal edge dislocation is seen to consist of two cores; an aluminum column understand dislocations in ionic crystals.
terminates one partial, and an oxygen column terminates the second partial. Each partial core
is locally nonstoichiometric due to the excess of aluminum or oxygen at the core. The implication 1
for mechanical properties is that the mobile high-temperature dislocation core structure consists Institute of Engineering Innovation, University of Tokyo, 2-11-
16, Yayoi, Bunkyo, Tokyo 113-8656, Japan. 2Materials Science
of two closely spaced partial dislocations. For basal slip to occur, synchronized motion of the and Technology Division, Oak Ridge National Laboratory, Oak
partials on adjacent planes is required. Ridge, TN 37831–6030, USA. 3Department of Intelligent
Materials Engineering, Osaka City University, 3-3-138,
Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan. 4Depart-
he core structures of dislocations are tals, dislocation core termination can be deter-
T critical to the electronic, optical, and me-

chanical properties of a wide range of
materials. For most simple monometallic crys-
mined; however, in complex crystals such as
oxides, either cation or anion columns (or both)
can be the terminating atomic columns even
ment of Advanced Materials Science, University of Tokyo, 5-1-5,
Kashiwanoha, Kashiwa, Chiba 277-8561, Japan.
[email protected]

REPORTS
The inherent structural complication of alpha mal octahedral coordination of the oxygen to consists of 12 alternating cation and anion basal
alumina (a-Al2O3) and, indeed, other complex aluminum sites, Kronberg proposed the syn- layers. The cation layers are slightly puckered
compounds has led to conflicting models for chroshear mechanism, whereby two shears op- along the 〈0001〉 direction. Bilde-Sørensen et al.
dislocation glide (4–6). Alumina is widely used erate in different directions on adjacent atomic proposed that basal slip occurs between these
in many industrial areas because of its su- planes. This mechanism has been shown to shifted Al sites (5).
perior mechanical, thermal, and chemical sta- operate in the Laves phase compound HfCr2 The core structure of a basal edge dis-
bility at high temperatures. Slip on the {0001} (8). Later, Bilde-Sørensen et al. (5) argued that location in a-Al2O3 is observed by scanning
basal plane is reported to be the dominant the slip between Al and O planes would require transmission electron microscopy (STEM).
deformation system at elevated temperatures charge transport. Alternatively, they proposed Now, sub-angstrom resolution can be achieved
(7), and thus important for understanding the that dislocation slip would occur along the with this technique (9). Figure 1, B and C,
high-temperature mechanical behavior. Kronberg midplane on the puckered Al (cation) layer. show simultaneous high-angle annular dark-
(4) first proposed a basal dislocation slip mod- They argued that this choice of the slip plane field (HAADF) and bright-field STEM im-
el based on structurally related hexagonal allows the moving dislocations to carry no net ages of a-Al2O3 viewed along the 〈1100〉
metals. Slip was assumed to occur between Al charge.
and O basal-plane layers. To maintain the nor- Figure 1A shows a ball-and-stick model of
a-Al2O3 in the 〈1100〉 projection. From this
viewing direction, Al- and O-atom sites are ar-
ranged as distinct columns. The stacking se-
quence of a-Al2O3 along the 〈0001〉 direction
Fig. 3. Schematics of basal edge dislocation

core structures observed at low temperatures
and the mobile core at high temperatures. (A)
Fig. 1. Crystal structure and simultaneous Schematic illustration of basal edge dislocation
atomic-resolution high-angle annular dark-field core structure observed in this study. The per-
(HAADF) and bright-field (BF) STEM images of fect basal edge dislocation dissociates into two
a-Al2O3. (A) Unit structure of a-Al2O3 viewed from Fig. 2. Atomic-resolution bright-field STEM partial dislocations perpendicular to the slip
the 〈1 100〉 direction. The structure of a-Al2O3 image of a basal edge dislocation core in plane with the formation of a {1120} stacking
consists of alternating Al and O planes along the a-Al2O3. (A) Typical core structure of a basal fault in between. (B) Schematic illustration of
〈0001〉 direction, whose stacking sequence is dislocation observed from the 〈1100〉 direction. mobile basal edge dislocation core model at
··aaAcbaBcaaCcbaAcaaBcbaCcaa··. From the 〈1100〉 The dislocation core is dissociated with two high temperatures. Two partials move very close
projection, we can distinguish individual Al partials (⅓〈1010〉 and ⅓〈0110〉) connected by a together without forming a {1120} stacking
and O columns. (B and C) Simultaneous HAADF {1120} stacking fault. (B and C) Bright-field STEM fault in between. (C) Proposed atomic structure
(B) and bright-field (C) STEM images of images of the upper and lower partial dislocation model (〈1100〉 projection) of mobile perfect
a-Al2O3 viewed from the 〈1100〉 direction. Com- cores, respectively. The upper partial core is basal edge dislocation at high temperatures,
parison of the images shows that the bright spots terminated by an Al column, whereas the lower based on the present low-temperature obser-
in the bright-field image directly correspond to partial core is terminated by an O column. Both vations. Blue and red segments in the figure
the positions of atomic columns under the core terminations lie in between the Al and O correspond to the O and Al atomic columns,
conditions used to obtain the images (11). As atomic planes. Each partial core is non- respectively. In this model, the two dissociated
seen in the images, bright-field STEM can stoichiometric due to the excess of Al or O, but partials observed at low temperature are lo-
distinguish Al and O atomic columns in this the total basal dislocation chemically preserves cated on adjacent basal planes in the mobile
projection. Al2O3 stoichiometry. high-temperature dislocation.

REPORTS
direction. The Z-contrast image obtained Z-contrast image. It is apparent that at room metric, the total dislocation preserves Al2O3
with the HAADF detector is an incoherent temperature, this dislocation is not a single stoichiometry.
image (10); it is essentially a map of the scat- perfect dislocation (b = ⅓〈1120〉), but has an Because the dissociation occurs in the di-
tering power of the specimen. There is a direct extended structure with partials (b = ⅓〈1010〉 rection normal to the {0001} slip plane, as in
correspondence between the features in the and b = ⅓〈0110〉) connected by a {1120} Fig. 3A, glide of the dissociated dislocations
specimen and their image. In contrast, the stacking fault, consistent with the previous on the {0001} planes would require contin-
phase-contrast image obtained with a small reports of dislocation observations after basal slip uous structural rearrangements as the stack-
bright-field detector has coherent image char- deformation (12–14). The dissociation is con- ing fault between the partials is moved. In
acteristics, which is comparable to the paral- sidered to be driven by the reduction of strain this case, dislocation motion would require
lel beam high-resolution TEM. The contrast energy and suppressed by increasing stacking- atomic diffusion and would be very sluggish
is influenced by the focus of the objective fault energy. Dissociation occurs on the {1120} at low temperatures. It is thus expected that
lens and specimen thickness, orientation, and plane by self-climb (14). No evidence for dis- the dislocations that are mobile at high tem-
scattering power. This makes phase-contrast sociation on the {0001} basal plane is ob- peratures are not dissociated with a {1120}
images of unknown structures difficult to di- served. Our low-temperature observations show stacking fault (13, 14), as schematically shown
rectly interpret. However, this sensitivity can that the cores are separated in the 〈1120〉 in Fig. 3B. That is, the mobile dislocation core
be exploited to provide much greater con- direction by only 0.24 nm. This observation is at high temperature should consist of two close-
trast variations than can be obtained from consistent with stacking-fault energy calcula- ly spaced partial dislocations. At the most basic
the Z-contrast images of low–atomic number tions that show that the {0001} fault has higher level, dislocation glide occurs by the succes-
elements (such as Al and O). The simulta- energy than the {1120} fault at low temper- sive slip of terminating atomic columns on the
neously recorded Z- and phase-contrast images atures (15). stationary atomic planes. In a-Al2O3, the rel-
(Fig. 1, B and C) of the 〈1100〉 projection of The bright-field STEM images of each ative motions of {0001} O layers and the
a-Al2O3 reveal parallel kinked lines of spots dissociated partial core are shown in Fig. 2, B sandwiched Al layer are considered to be
that reflect the arrangement of alternating O and C. The arrows in the images indicate the involved in the basal dislocation glide. The
and Al columns along the 〈0001〉 direction. respective position of an extra half-plane ter- difference in core termination results in two
Under the conditions used to obtain these two mination of the two partials. The images re- slightly different slip processes. If the dislo-
images, the bright features of the Z-contrast veal that the upper core is terminated between cation is O terminated, the moving O plane
image are seen to correspond to the bright vertices of the kinked line of atoms at an Al- slips over an Al plane. If, instead, the dis-
features in the bright-field image. These bright column position, whereas the lower core is location is Al terminated, the moving Al plane
features identify the positions of the two O terminated at a vertex of the kinked line of slips over an O plane. On the basis of our low-
columns and the puckered Al column in each atoms at an O-column position. These obser- temperature observations, we propose that the
segment of the kinked lines seen in this pro- vations clearly show both dislocation core ter- slip planes of moving partials are located not
jection (11). minations and, thus, indicate that the slip on the same atomic plane, but on adjacent Al
Figure 2A shows a typical bright-field planes of the dislocations are located between and O {0001} planes. Figure 3C shows the
STEM image of a basal dislocation core in the Al and O atomic planes. Moreover, the possible perfect basal edge dislocation core
a-Al2O3. The line direction of the dislocation partials terminated by Al or O column in- structure mobile at high temperatures. We as-
is parallel to the observing direction, so that dicate that the partial cores are locally not sume that the two dissociated partials observed
the core structure is set at an “edge-on” condi- stoichiometric. Contrary to the common as- at low temperature are located on adjacent
tion. In this condition, bright spots in the sumption, nonstoichiometric core structures basal planes in the mobile high-temperature
image correspond to the positions of atomic actually exist in an ionic crystal. Although dislocation. Although other reconstructions
columns as determined from the simultaneous each partial dislocation core is nonstoichio- are possible, our observations show that these
Al- and O-terminated partials exist, and when
the dislocation glide stops, this model can
dissociate by climb to form partial dislocation
core structures consistent with our experi-
mental observation. Thus, we propose that
the atomic-scale basal slip processes are based
on the simple core model shown in Fig. 3C.
Because the schematic is viewed in a 〈1100〉
projection, the vector component normal to
the schematic is not shown. However, it should
be stated that the two partials have equal and
opposite vector components normal to the
schematic.
Fig. 4. Schematics of the mobile high-temperature basal edge dislocation core structure in cross Figure 4, A to C, shows the slip sequence
section (〈1100〉 projection). Blue and red segments in the figure correspond to the O and Al
of the core model shown in Fig. 3C. The par-
atomic columns, respectively. (A) The dislocation, consisting of adjacent partial dislocations with
tial dislocations (b = ⅓〈1010〉 and b = ⅓〈0110〉)
O and Al termination. The slip planes of the two partials are on adjacent atomic planes (slip
planes 1 and 2). “s” indicates the initial position of the dislocation. In these schematics, only the combine to produce a unit dislocation (b =
vector components perpendicular to the viewing direction are shown, but the two partials actually ⅓〈1120〉). Again, only the slip components per-
have equal and opposite vector components perpendicular to the schematic. (B) The first partial pendicular to the 〈1100〉 projection are shown
(O-terminated) slipping on a fixed Al plane. (C) The passage of the second partial (Al-terminated) in this schematic. The successive slip of the
slipping on an O plane. It is expected that (B) and (C) occur simultaneously, and during the two partials on adjacent planes (slip planes
process, slight relaxation in the O plane in between the slip planes 1 and 2 is required to preserve 1 and 2) recovers the perfect stacking sequence
the perfect stacking sequence in O layers. The plain-view schematics of the present basal on the Al sublattice (11). However, in sapphire,
dislocation motion show that the proposed dislocation motion preserves a perfect stacking there is a slight deviation from the perfect
sequence in Al layers (11). close-packed structure seen in the oxygen

REPORTS
sublattice that is correlated with the stacking of location. The structure of these partials is References and Notes
adjacent Al layers (16). This slight deviation in based on our low-temperature observations, 1. J. P. Hirth, J. Lothe, Theory of Dislocations (Krieger,
Malabar, FL, ed. 2, 1992).
oxygen positions can be schematically seen in in which each partial core is terminated by Al 2. R. W. Whitworth, Adv. Phys. 24, 203 (1975).
the projected structure model of Fig. 1A. If and O columns, respectively, but the total 3. J. J. Gilman, Acta Metall. 7, 608 (1959).
these deviations were a spatially fixed part of dislocation preserves Al2O3 stoichiometry. 4. M. L. Kronberg, Acta Metall. 5, 507 (1957).
the structure, the motion of a single partial Bilde-Sørensen et al. (5) also proposed a stoi- 5. J. B. Bilde-Sørensen et al., Acta Mater. 44, 2145 (1996).
6. D. E. Luzzi, G. Rao, T. A. Dobbins, D. P. Pope, Acta Mater.
would result in a stacking fault in the oxygen chiometric core model, but the slip plane 46, 2913 (1998).
sublattice. In the present slip model, the O (located at the midplane on the puckered Al 7. K. P. D. Lagerlöf, A. H. Heuer, J. Castaing, J. P. Rivière,
plane in between slip planes 1 and 2 is sheared layer) is not consistent with our images. Our T. E. Mitchell, J. Am. Ceram. Soc. 77, 385 (1994).
by only one partial, and thus a stacking fault images clearly show that the termination of 8. M. F. Chisholm, S. Kumar, P. Hazzledine, Science 307,
701 (2005).
on the O sublattice would be produced if we both partials is located in between the Al and
9. P. D. Nellist et al., Science 305, 1741 (2004).
assume rigid O layers. However, the devia- O layers at low temperature. Our results rep- 10. S. J. Pennycook, D. E. Jesson, Phys. Rev. Lett. 64, 938
tions are related to the positions of the Al resent a definitive starting point for realistic (1990).
atoms, neighboring O, atoms and vacant sites atomic-level modeling of slip processes, dis- 11. Materials and Methods are available as supporting
sandwiched between the O layers. Because location generation, and their effects on the material on Science Online.
12. T. E. Mitchell, B. J. Pletka, D. S. Phillips, A. H. Heuer,
the perfect Al stacking is preserved after the mechanical properties of a-Al2O3. Also, the Philos. Mag. 34, 441 (1976).
dislocation motion, the O sublattice can also present results will provide a crucial check for 13. A. Nakamura, T. Yamamoto, Y. Ikuhara, Acta Mater. 50,
preserve a perfect stacking sequence by an future theoretical calculations of dislocations 101 (2002).
appropriate small modification in the O atom in complex oxides. 14. K. P. D. Lagerlöf et al., Acta Metall. 32, 97 (1984).
15. P. R. Kenway, Philos. Mag. B 68, 171 (1993).
positions. It is important to note that these two We have provided experimental evidence 16. S. Geschwind, J. P. Remeika, Phys. Rev. 122, 757
partial motions are not independent in our that locally nonstoichiometric structures are (1961).
proposed model. Both partials move simulta- allowed in crystals with strong ionic char- 17. We thank K. P. D. Lagerlöf for valuable discussions.
neously on adjacent {0001} basal planes to acter. Simultaneous HAADF and bright-field This research was supported by the Japan Society for the
Promotion of Science and the Division of Materials
complete the perfect dislocation slip. A total STEM imaging with aberration correction is a Sciences and Engineering, Office of Basic Energy
basal dislocation is thus considered to possess powerful tool for observing such localized Sciences, U.S. Department of Energy.
two atomic slip planes. This core structure is defect structures, even in very complex crys-
expected to dissociate after the dislocation tals. The possibility for atomic-scale charac- www.sciencemag.org/cgi/content/full/316/5821/82/DC1
stops moving and form two partials, con- terization of dislocation core structures will Materials and Methods
sistent with our observations in Fig. 2. assist our understanding of dislocation activ- Figs. S1 and S2
We propose that basal slip in a-Al2O3 is ity and its effects on the electrical, optical, References
controlled by the partial dislocations that and mechanical properties of complex, mul- 11 October 2006; accepted 14 February 2007
dissociate from the perfect ⅓〈1120〉 dis- ticomponent materials. 10.1126/science.1136155
Acid Catalysis in Basic Solution: gen bonding networks near the active sites of
proteins can lead to well-tuned pKa matching
(2) and can result in pKa shifts of up to eight
A Supramolecular Host Promotes units, as shown in bacteriorhodopsin (3). Sim-
ilarly, purely electrostatic interactions can
Orthoformate Hydrolysis greatly favor charged states and have been re-
sponsible for pKa shifts of up to five units
for acetoacetate decarboxylase (4). Attempts
Michael D. Pluth, Robert G. Bergman,* Kenneth N. Raymond* have been made to isolate the contributions
of electrostatic versus covalent interactions to
Although many enzymes can promote chemical reactions by tuning substrate properties purely such pKa shifts; however, this remains a
through the electrostatic environment of a docking cavity, this strategy has proven challenging to difficult challenge experimentally. This chal-
mimic in synthetic host-guest systems. Here, we report a highly charged, water-soluble, metal- lenge emphasizes the importance of synthesiz-
ligand assembly with a hydrophobic interior cavity that thermodynamically stabilizes protonated ing host molecules that, like enzyme cavities,
substrates and consequently catalyzes the normally acidic hydrolysis of orthoformates in basic can enhance binding of small molecular guests
solution, with rate accelerations of up to 890-fold. The catalysis reaction obeys Michaelis-Menten and, in a few cases, catalyze chemical reactions
kinetics and exhibits competitive inhibition, and the substrate scope displays size selectivity, (5–9).
consistent with the constrained binding environment of the molecular host. Supramolecular assemblies with available
functional groups have been used to generate
ynthetic chemists have long endeavored and require certain properties of the substrate solution-state pKa shifts of up to two pKa units
S to design host molecules capable of se-

lectively binding slow-reacting substrates
and catalyzing their chemical reactions. Where-
to insure catalysis, enzymes are often able to
modify basic properties of the bound sub-
strate such as pKa (where Ka is the acid disso-
(10–13) and to catalyze chemical reactions
(14, 15). Synthetic hosts often rely on hydrogen
bonding or ion-dipole interactions for guest
as synthetic catalysts are often site-specific ciation constant) in order to enhance reactivity. inclusion, and numerous studies have inves-
Two common motifs used by nature to ac- tigated the effects of charge on guest binding
tivate otherwise unreactive compounds are affinities in supramolecular host-guest sys-
Department of Chemistry, University of California, Berke-
ley, Berkeley, CA 94720, USA. the precise arrangement of hydrogen-bonding tems (16, 17). We report here a synthetic supra-
networks and electrostatic interactions be- molecular host assembly that relies exclusively
[email protected] (R.G.B.); raymond@socrates. tween the substrate and adjacent residues of on electrostatic and hydrophobic interactions
berkeley.edu (K.N.R.) the protein (1). Precise arrangement of hydro- for thermodynamic stabilization of protonated

REPORTS
substrates. As nature has exploited pKa shifts spectrum showed a 1:1:1 triplet with spin- inference the encapsulated amines must be
to activate otherwise unreactive substrates spin H-P coupling constant (1JDP) = 75 Hz. In protonated as well, even at high pH.
toward catalysis, this stabilization is exploited H2O, the undecoupled 31P NMR spectrum For the amines encapsulated in 1, the
to promote acid-catalyzed hydrolyses in strongly showed a doublet (1JHP = 490 Hz) correspond- magnitude of the effective shift in basicity was
basic solution. ing to a one-bond P-H coupling that definitively investigated by monitoring 1:1 host guest com-
During the past decade, the Raymond group establishes binding of a proton to phosphorus. plexes as a function of pH. In order to confirm
has reported the formation and guest-hosting prop- Because similarly substituted amines and phos- that the encapsulated amines were exchanging
erties of supramolecular assemblies of the stoi- phines exhibit analogous base strengths, by with the amines in free solution and that 1 was
chiometry M4L6 [M is GaIII (1), AlIII, InIII, FeIII,
TiIV, or GeIV; L is N,N′-bis(2,3-dihydroxybenzoyl)-
1,5-diaminonaphthalene] (18, 19). These compo- Fig. 1. (Left) A sche-
nents self-assemble in solution to form tetrahedral matic representation of
clusters with chiral metal ions at the vertices and the host M4L6 assembly.
bridging ligands spanning each edge (Fig. 1). Only one ligand is shown
The strong mechanical coupling of the ligands for clarity. (Right) A mod-
el of [2-H+ ⊂ 1]11–; hy-
transfers chirality from one metal vertex to the
drogen atoms on the
others, thereby leading exclusively to DDDD or
host assembly are omit-
LLLL configurations with respect to the vertices. ted for clarity.
These enantiomers are stable, noninterconvert-
ing, and resolvable (20). The metal-ligand as-
sembly 1 is able to encapsulate a wide variety of
small monocationic guests in a 300 to 500 Å3
cavity protected from the bulk solution. The
naphthalene walls render the interior hydro-
phobic, whereas the tetra-anionic ligands in
combination with the trivalent metal centers
confer a 12– overall charge to the assembly. As
a host, 1 stoichiometrically mediates (21, 22)
as well as catalyzes (5, 23) several important
organic and organometallic reactions. In addi-
tion, it stabilizes reactive guests, such as the
tropylium cation (24), phosphine-acetone ad-
ducts (25), and iminium cations (26), all of
which rapidly decompose in water and are only
stable under anhydrous or extremely acidic
conditions.
The binding strength of monocationic guests
prompted our investigation into the ability of 1 to
thermodynamically drive the monoprotonation
of guest molecules within the cavity. Neutral
guests could then be either stoichiometrically or
transiently protonated to promote acid-catalyzed
reaction on encapsulation. To test our hypothesis,
we added a variety of amines and phosphines to
solutions of 1 in D2O. Upon addition of N,N,N′,N′-
tetramethyl-1,4-diaminobutane (2) or N,N,N′,N′-
tetraethyl-1,2-diaminoethane (3), upfield nuclear
magnetic resonance (NMR) resonances char-
acteristic of encapsulation were observed, cor-
responding to a 1:1 host-guest complex. Similarly,
two-dimensional 1H nuclear overhauser effect
spectroscopy (NOESY) (fig. S1) shows strong
through-space correlation between the naphtha-
lene protons of the assembly and the encap-
sulated guest (27).
In order to confirm that these weakly basic
compounds were being encapsulated in their
conjugate acid forms, we added an isostructural
phosphine, 1,2-bis(dimethylphosphino)methane
(4) to 1 and probed by using 31P NMR spec- Fig. 2. (A) Reaction and substrate scope for orthoformate hydrolysis in the presence of catalytic
troscopy. As with both amines, new upfield res- 1. Bu, butyl; Me, methyl; Pr, propyl. (B to D) All spectra taken with 50 equivalents (equiv.) of
onances corresponding to [4-H+⊂ 1]11– (⊂ denotes triethyl orthoformate with respect to 1 at pD = 11.0, 100 mM K2CO3, 22°C, in D2O. (B) Initial
encapsulation) were observed in both the 1H spectrum. (C) Spectrum after 60 min. (D) Spectrum of 1 with 2 equiv. NEt4+ after 60 min.
NMR and the 31P NMR spectra. In D2O, the ▼
Molecule 1 represented by ■; HC(OEt)3, ; NEt4+, ● for exterior and ○ for interior, and
proton-decoupled phosphorus (31P{1H}) NMR product HCO2H, .▲
REPORTS
not acting as a kinetic trap, we measured the found to be 5.2(5) kcal mol–1 and 4.8(4) kcal or basic solution (35). However, we found that
guest self-exchange rates of the encapsulated mol–1, respectively. Heating the host-guest com- in the presence of a catalytic amount of 1 in
amines (28) by using the selective inversion re- plexes to 75°C for 24 hours and returning the basic solution, triethyl orthoformate is quickly
covery (SIR) method (29) and found the amines sample to room temperature did not change the hydrolyzed (t1/2 ~ 12 min, pH = 11.0, 22°C) to
to be exchanging on the NMR time scale [for 2, ratio of encapsulated to free guest, confirming the corresponding formate ester, HC(O)(OR),
k320K = 0.24(3) s−1; for 3, k320K = 0.13(2) s−1] that the thermodynamic equilibrium had been and finally to formate, HCO2– (36). We moni-
(30). We carried out the SIR experiments at five reached. Although the pKa of 3-H+ is 10.8 in tored the reaction by 1H NMR spectroscopy and
different temperatures from 300 K to 340 K to free solution, stabilization of the protonated form observed that the resonances of host 1 shifted
extract the activation parameters (fig. S3). The by 1, which can be calculated as the product of upon substrate addition, suggesting that 1 is in-
activation parameters for guest exchange for 2 the pKa and the binding constant of the pro- timately involved in the reaction. The substrate
were DG‡298 (standard Gibbs energy of activa- tonated amine, shifts the effective basicity to 14.3 C-H resonance broadens to v1/2 = 14.3 Hz com-
tion) = 19(2) kcal mol–1, DH‡ (standard enthalpy (32). This dramatic shift highlights the substantial pared with the nonencapsulated v1/2 = 3.2 Hz,
of activation) = 10.8(9) kcal mol–1, and DS‡ stabilization of the protonated species over the which is suggestive of fast guest exchange.
(standard entropy of activation) = –28(4) neutral species upon encapsulation in the highly Increasing the concentration of 1 to 80 mM
entropy units (e.u.) and for 3 were DG‡298 = charged cavity (33). makes the encapsulated substrate observable
19.9(8) kcal mol–1, DH‡ = 16.7(6) kcal mol–1, We next sought to apply this host-induced (fig. S3). With a limited volume in the cavity
and DS‡ = –10.9(6) e.u. These values are con- shift in effective basicity to promote reaction of 1, substantial size selectivity was observed
sistent with those for the self-exchange activa- chemistry. We focused on the hydrolysis of or- in the orthoformate hydrolysis, with ortho-
tion parameters of tetraalkylammonium cations thoformates, HC(OR)3 (where R is an alkyl or formates smaller than tripentyl orthoformate
encapsulated in 1, suggesting that the same ex- aryl group), a class of molecules responsible for being readily hydrolyzed with 1 mole percent
change mechanism is present (31). Upon moni- much of the formulation of the Brønsted theory of 1 (Fig. 2).
toring 1:1 host-guest solutions of [2-H+ ⊂ 1]11– of acids almost a century ago (34). Although To further establish that the interior cavity of
and [3-H+ ⊂ 1]11– at different pHs, the free en- orthoformates are readily hydrolyzed in acidic 1 was catalyzing the hydrolysis, we explored the
ergies of binding (–DG°) for the amines were solution, they are exceedingly stable in neutral propensity of a strongly binding guest, NEt4+
(where Et is ethyl) [–DG° = 6.20(8) kcal mol–1],
to inhibit substrate binding. As expected, addi-
tion of NEt4+ to the solution completely in-
hibited the hydrolysis of orthoformates. In the
presence of NEt4+, the orthoformate methine
resonances sharpened to v1/2 = 3.4 Hz, confirm-
ing lack of encapsulation.
We probed the reaction mechanism by
using triethyl orthoformate as the substrate at
pH = 11.0 and 50°C. First-order substrate
consumption was observed under stoichiomet-
ric conditions (fig. S4). Working under satu-
ration conditions (see below), kinetic studies
revealed that the reaction is also first-order in
proton concentration and first-order in the
concentration of 1 while being 0th-order in
substrate (fig. S4). When combined, these
mechanistic studies establish that the rate law
for this catalytic hydrolysis of orthoformates
by host 1 obeys the overall termolecular rate
law: rate = k[H+][Substrate][1] but under sat-
uration conditions reduces to rate = k′ [H+][1].
We conclude that the neutral substrate
enters 1 to form a host-guest complex, leading
Fig. 3. Mechanism for catalytic orthoformate hydrolysis in the presence of catalytic 1.
to the observed substrate saturation. We con-
sidered the possibility that saturation is due to
Fig. 4. Eadie-Hofstee plot showing complete protonation of substrate outside of
competitive inhibition of the hydrolysis the assembly; however, it would not be pos-
of HC(OEt)3 by NPr4+ in H2O, pH = 11.0, sible to attain saturation at pH = 11, because
50°C, and 4.0 mM 1. protonated orthoformates have estimated pKa
values of about –5 (30). Similarly, we con-
sidered that protonation of the interior of the
assembly was the first step in the mechanism;
however, this mechanism would require a
binding constant of H+ in the assembly to be
greater than 1010, which is not attainable. In
the next step of the cycle, the encapsulated
substrate is protonated, presumably by depro-
tonation of water, and undergoes two succes-
sive hydrolysis steps in the cavity, liberating
two equivalents of the corresponding alcohol.

REPORTS
Lastly, the protonated formate ester is ejected to NEt4+ facilitates the competitive binding 15. H. H. Zepik, S. A. Benner, J. Org. Chem. 64, 8080
from 1 and further hydrolyzed by base in so- experiments by allowing for the weakly bind- (1999).
16. S. Shinkai, K. Araki, O. Manabe, J. Chem. Soc. Chem.
lution (Fig. 3) (37). ing substrate, HC(OEt)3, to more readily com- Commun. 3, 187 (1988).
The reaction mechanism in Fig. 3 shows pete for the binding cavity of 1. By varying 17. C. H. Haas, S. M. Biros, J. Rebek Jr., J. Chem. Soc. Chem.
direct parallels to enzymatic pathways that the concentration of substrate for each amount Commun. 48, 6044 (2005).
obey Michaelis-Menten kinetics because of an of inhibitor, we compared the saturation curves 18. D. L. Caulder, K. N. Raymond, J. Chem. Soc. Dalton Trans.
8, 1185 (1999).
initial pre-equilibrium followed by a first-order with use of an Eadie-Hofstee plot (Fig. 4) 19. D. L. Caulder, K. N. Raymond, Acc. Chem. Res. 32, 975
rate-limiting step. Lineweaver-Burk analysis (38, 39). The saturation curves intersect on (1999).
(fig. S5) using the substrate saturation curves the y axis, signifying that at infinite substrate 20. A. J. Terpin, M. Ziegler, D. W. Johnson, K. N. Raymond,
affords the corresponding Michaelis-Menten concentration the maximum reaction velocity Angew. Chem. Int. Ed. 40, 157 (2001).
21. D. H. Leung, D. Fiedler, R. G. Bergman, K. N. Raymond,
kinetic parameters of the reaction. Represen- is independent of the amount of inhibitor,
Angew. Chem. Int. Ed. 43, 963 (2004).
tative Michaelis-Menten parameters for tri- confirming competitive inhibition. If NPr4+ 22. D. H. Leung, R. G. Bergman, K. N. Raymond, J. Am.
ethyl orthoformate (Vmax = 1.79 × 10−5 M s−1, were competing for a different site than the Chem. Soc. 128, 9781 (2006).
KM = 21.5 mM, and kcat = 8.06 × 10−3 s−1, active site of 1 responsible for the catalytic 23. D. Fiedler, H. van Halbeek, R. G. Bergman, K. N. Raymond,
where Vmax is the maximum velocity of the hydrolysis, such as an exterior ion-pairing J. Am. Chem. Soc. 128, 10240 (2006).
24. J. L. Brumaghim, M. Michels, D. Pagliero, K. N. Raymond,
reaction, KM is the Michaelis constant, and site, then the saturation curves in the Eadie- Eur. J. Org. Chem. 22, 5115 (2004).
kcat is the turnover rate of the bound substrate) Hofstee plot would be parallel. Back calcula- 25. J. L. Brumaghim, M. Michels, K. N. Raymond, Eur. J.
and triisopropyl orthoformate (Vmax = 9.22 × tion of the binding constant of the NPr4+ Org. Chem. 24, 4552 (2004).
10−6 M s−1, KM = 7.69 mM, and kcat = 3.86 × inhibitor affords –DG° = 2.8(1) kcal mol–1, 26. V. M. Dong, D. Fiedler, B. Carl, R. G. Bergman,
K. N. Raymond, J. Am. Chem. Soc. 128, 14464
10−3 s−1) show substantial rate acceleration which is consistent with the known affinity of (2006).
over the background reaction. When compared this guest. 27. The 23Na NMR spectrum of encapsulated 2 in the
to the background hydrolysis reactions under Using synthetic hosts to modify the chem- presence of Na12Ga4L6 showed only a singlet at 1.0 parts
the same reaction conditions (triethyl ortho- ical properties of encapsulated substrates was per million, corresponding to free solvated sodium ions
formate kuncat = 1.44 × 10−5 s−1 and triiso- used to greatly enhance the reactivity of ortho- and suggesting that a Na–N,N,N′,N′-tetramethyl-1,2-
diaminoethane adduct was not the encapsulated guest.
propyl orthoformate kuncat = 4.34 × 10−6 s−1), formates and promote the acid catalyzed hy- 28. The exchange rates were measured at pD = 13.0 and 500
the rate accelerations (kcat/kuncat) for triethyl or- drolysis in basic solution. Similar strategies mM KCl to ensure uniform ionic strength.
thoformate and triisopropyl orthoformate are 560 could be used to hydrolyze other acid-sensitive 29. A. D. Bain, J. A. Cramer, J. Magn. Reson. A 118, 21
(1996).
and 890, respectively. Further analysis of the molecules in which the charged transition state
30. The error notation used throughout this paper, for
Michaelis-Menten kinetic parameters yielded of the reaction can be stabilized by a molecular example, k = 0.24(3), means that for the value of k =
additional information about the catalytic reac- host. The size selectivity in synthetic molecular 0.24 there is a standard uncertainty of 0.03.
tion. Assuming a fast pre-equilibrium with re- hosts is a property often used by nature but 31. A. V. Davis et al., J. Am. Chem. Soc. 128, 1324 (2006).
spect to kcat, KM is essentially the dissociation rarely incorporated into standard homogeneous 32. Formally, this is a shift in the pKa of the amine, but
because we are unable to observe the neutral amine
constant of the encapsulated neutral substrate. In or heterogeneous catalysis. This type of selec- guest inside of 1, this is more accurately referred to as an
order to compare how efficiently 1 catalyzes the tivity could be used to differentiate reactive sites effective shift in basicity.
hydrolysis of different substrates, the specificity of a substrate which would otherwise exhibit 33. Examination of crystal structures of 1 with various guests
factor (kcat/KM) can be examined. This parameter equivalent reactivity toward standard organic, shows that the catechol oxygens are not accessible to a
bound guest, thus removing the possibility that hydrogen
corresponds to the second-order proportional- organometallic, or inorganic catalysts. Such bonding between 1 and the guest is taking place.
ity constant for the rate of conversion of pre- strategies would be synthetically useful for com- Furthermore, examination of NOEs between 1 and the
formed enzyme-substrate complex, in this case mon organic protecting groups such as acetals or guest reveals strong through-space interactions between
[orthoformate ⊂ 1]12–, to product, thus provid- ketals and could also be applied to more bi- the guest and naphthyl protons but not between the
guest and catechol protons (fig. S1).
ing a measure of the effectiveness with which ologically relevant substrates such as amides or 34. J. N. Brønsted, W. F. K. Wynne-Jones, Trans. Faraday Soc.
two substrates can compete for the same site. phosphate esters, furthering the analogy to en- 25, 59 (1929).
Triethyl orthoformate and triisopropyl orthofor- zymatic systems. 35. E. H. Cordes, H. G. Bull, Chem. Rev. 74, 581 (1974).
mate have specificity constants of 0.37 M−1 s−1 36. At high pH, the formate ester product is quickly
hydrolyzed by OH– to formate ion (HCO2–). However, at
and 0.50 M−1 s−1, respectively, showing that References and Notes
1. N.-C. Ha, M.-S. Kim, W. Lee, K. Y. Choi, B.-H. Oh, J. Biol. lower pH, the formate ester product is stable and is the
triisopropyl orthoformate is more efficiently hy- Chem. 275, 41100 (2000). exclusive product.
drolyzed by 1. 2. W. W. Cleland, P. A. Frey, J. A. Cerlt, J. Biol. Chem. 273, 37. After the initial catalyzed step inside of 1, any of the
Also characteristic of enzymes that obey 25529 (1998). intermediates can be hydrolyzed by either acid or base;
Michaelis-Menten kinetics is that suitable in- 3. S. Szaraz, D. Oesterhelt, P. Ormos, Biophys. J. 67, 1706 however, we have not observed any intermediates
(1994). free in solution.
hibitors can compete with the substrate for the 4. F. H. Westheimer, Tetrahedron 51, 3 (1995). 38. G. S. Eadie, J. Biol. Chem. 146, 85 (1942).
enzyme active site, thus leading to inhibition. 5. D. Fiedler, R. G. Bergman, K. N. Raymond, Angew. Chem. 39. B. H. J. Hofstee, Science 116, 329 (1952).
The binding of an inhibitor to the enzyme Int. Ed. 43, 6748 (2004). 40. We gratefully acknowledge financial support from the
active site prevents the substrate from entering 6. M. Yoshizawa, M. Tamura, M. Fujita, Science 312, 251 Director of the Office of Energy Research, Office of Basic
(2006). Energy Sciences, Chemical Sciences Division (U.S.
and impedes the reaction. If the inhibitor binds Department of Energy) under contract DE-AC02-
7. J. Kang, J. Rebek Jr., Nature 385, 50 (1997).
reversibly to the enzyme active site, then the 8. K. R. Rao, Y. V. D. Nageswar, N. S. Krishnaveni, 05CH11231 and an NSF predoctoral fellowship to
substrate can compete for the substrate and at K. Surendra, Adv. Org. Syn. 1, 301 (2005). M.D.P. The authors thank D. Leung, G. Lalic, M. Seitz,
suitably high concentrations will completely 9. J. K. M. Sanders, Chem. Eur. J. 4, 1378 (1998). and C. Hastings for helpful discussions and
10. C. Marquez, W. M. Nau, Angew. Chem. Int. Ed. 40, 3155 H. van Halbeek and R. Nunlist for assistance with
displace the inhibitor, leading to competitive NMR experiments.
(2001).
inhibition. In order to test for competitive in- 11. X. Zhang, G. Gramlich, X. Wang, W. M. Nau, J. Am.
hibition for the hydrolysis of orthoformates Chem. Soc. 124, 254 (2002). Supporting Online Material
with 1, we measured the rates of hydrolysis of 12. J. Mohanty, A. C. Bhasikuttan, W. M. Nau, H. Pal, J. Phys.
Materials and Methods
triethyl orthoformate in the presence of a Chem. B 110, 5132 (2006).
Figs. S1 to S5
13. H. Bakirci, A. L. Koner, T. Schwarzlose, W. M. Nau,
varying amount of the strongly binding in- Chem. Eur. J. 12, 4799 (2006). References
hibitor NPr4+ [–DG° = 2.7(2) kcal mol–1]. The 14. F. Ortega-Caballero, C. Rousseau, B. Christensen, 12 December 2006; accepted 5 March 2007
lower binding constant of NPr4+ with respect T. E. Petersen, M. Bols, J. Am. Chem. Soc. 127, 3238 (2005). 10.1126/science.1138748

REPORTS
flow, which originates from the Northern Hemi-
The Deep Ocean During the sphere, results in ice-shelf bottom melting near
grounding lines (17). This melting process sub-
Last Interglacial Period stantially contributes to decreasing the stability of
the ice shelves that drain the grounded part of the
WAIS (18).
J. C. Duplessy,1* D. M. Roche,2 M. Kageyama3
To investigate whether the LIG ocean could
have helped to destabilize the West Antarctic ice
Oxygen isotope analysis of benthic foraminifera in deep sea cores from the Atlantic and Southern
shelves and ice sheet, we examined the oxygen
Oceans shows that during the last interglacial period, North Atlantic Deep Water (NADW) was
isotopic composition (d18O) of benthic forami-
0.4° ± 0.2°C warmer than today, whereas Antarctic Bottom Water temperatures were unchanged.
nifera. This quantity is a function of both tem-
Model simulations show that this distribution of deep water temperatures can be explained as a
perature and seawater d18O. On the one hand, the
response of the ocean to forcing by high-latitude insolation. The warming of NADW was transferred to
isotopic composition directly reflects isotopic
the Circumpolar Deep Water, providing additional heat around Antarctica, which may have been
variations in the ambient water (dw). On the
responsible for partial melting of the West Antarctic Ice Sheet.
other, the fractionation between the carbonate
shell and the water increases by about 0.25 per
he climate of the last interglacial (LIG) modern conditions, the meridional overturn- mil (‰) for each degree that the water is cooled.
T period, from 129,000 to 118,000 years

ago (1, 2), was slightly warmer than
today’s and is often viewed as an analog of
ing circulation carries warm surface waters
northward in the Atlantic, replacing the export
of the relatively cold North Atlantic Deep
Because the global ocean circulation during the
LIG period was very similar to the present cir-
culation (19), d18O differences between benthic
the climate expected during the next few cen- Water (NADW). However, NADW is warmer foraminifera from the LIG period and from the
turies. Recent assessments of the LIG climate than the cold bottom water formed around Ant- Holocene in a core reflect both the temperature
have provided strong evidence that sea level arctica. NADW therefore carries heat to the and d18O differences experienced by the same
was 4 to 6 m above the present level, due to Southern Ocean. It mixes with recirculated deep water mass as the one that is present today at
partial melting of both Greenland and the deep water from the Indian and Pacific Oceans, the core location.
West Antarctic Ice Sheet (WAIS) (3, 4). At peak forming a relatively warm deep water mass, The LIG d18O records are characterized by
interglacial conditions, summer temperatures the Circumpolar Deep Water (CDW), charac- quasi-constant values forming a plateau, in agree-
were 2° to 5°C warmer than today in the North terized by a temperature maximum of ~+2°C ment with coral reef data showing that the LIG
Atlantic (5) over Greenland (6) and the Arctic around 600 m depth and overlying the cold high–sea-level period lasted at least 7000 years
(7). The Norwegian-Greenland Sea experi- Antarctic Bottom Water. CDW floods the floor (1, 2, 20). In all cores, the LIG plateau exhibits
enced large variability, but during the warmest of the Amundsen and Bellinghausen Sea con- d18O values that are significantly lighter than
period, the Arctic oceanic front was located tinental shelves and reaches the undersides of those of the Holocene (Fig. 1). To accurately
west of its present location (8). Consequently, ice shelves flowing from the WAIS. This heat determine the d18O difference between these two
the Arctic climate was warm enough to explain
the shrinking of the Greenland Ice Sheet during
the LIG (9). Fig. 1. Comparison of
In the Southern Hemisphere, an ~2°C warm- three d18O benthic fora-
minifer records for the
ing occurred over the Antarctic Plateau during
past 135,000 years, ob-
the LIG (10), but it could not have resulted in any
tained from deep-sea
melting because local air temperature was still cores raised from the
extremely cold (~–50°C). In the Southern Ocean, major water masses
summer sea surface temperatures were about 2°C found in the Atlantic
higher than during the Holocene (11, 12). Over and Southern Oceans.
New Zealand and Tasmania, the LIG warming The upper panel shows
was between 0° and 2°C (13, 14). Such increases a Norwegian Sea benthic
in surface water or air temperature seem too small stack record (36), the
to have resulted in substantial melting of the middle panel is core SU
WAIS (15). 90-39 (52°32′N, 21°56′W,
However, the WAIS is sensitive to deep ocean depth 3955 m) from
temperatures (16). Indeed, the volume of this ice the North Atlantic, and
sheet is related to the efficiency of ice shelves in the lower panel is core
blocking the ice flow from the central part of the MD 00-2374 (46°04′S,
ice sheet outward, and ice shelves are themselves 96°48′E, depth 3250 m)
sensitive to deep ocean temperatures. Under from the Southern Oce-
an. All records encom-
1
Laboratoire des Sciences du Climat et de l’Environnement/ pass the LIG (135,000 to
Institut Pierre Simon Laplace (LSCE/IPSL), Laboratoire 110,000 years ago) and
Commissarial à l’Energie Atomique/CNRS/Université de the present interglacia-
Versailles Saint Quentin (CEA/CNRS/UVSQ), Parc du CNRS, tion (since 8000 years
91198 Gif sur Yvette, France. 2Department of Paleoclima-
tology and Geomorphology, Vrije Universiteit Amsterdam,
ago), together with the
De Boelelaan 1085, NL-1081 HV Amsterdam, Netherlands. last glacial period. The
3
Laboratoire des Sciences du Climat et de l’Environnement, gray line in each panel
LSCE/IPSL, Laboratoire CEA/CNRS/UVSQ, CE Saclay, l'Orme shows the mean Holo-
des Merisiers, 91191 Gif-sur-Yvette Cedex, France. cene d18O value. kyr,
*To whom correspondence should be addressed. E-mail: thousand years.
[email protected]

REPORTS
interglacial periods, we compared the records Light d18O values of LIG benthic foramini- global sea-level rise. This rate is likely to be an
of benthic foraminifera from the same species fera are partly explained by partial melting of both upper estimate for interglacial conditions, be-
in 42 cores (table S1). The mean d18O value of Greenland and the WAIS, which injected iso- cause ice melting at the end of the glaciation
LIG benthic foraminifera in these cores is 0.11 ± topically light meltwater into the ocean. To esti- originated from snow deposited at low temper-
0.01‰ lighter than during the Holocene, but mate the seawater d18O variation associated with ature and highly depleted in 18O. In contrast, ice
with significant regional variations: The d18O LIG sea level, which was 4 to 6 m above the melting during an interglacial climate would
difference between the LIG period and the Hol- present level, we note that between the last gla- originate from snow deposited at temperatures
ocene varies from 0.15 ± 0.02‰ in the Norwe- cial maximum and today, the sea level rose by higher than those of glacial climate and less de-
gian Sea to 0.11 ± 0.01‰ in the Atlantic Ocean 130 ± 10 m (21) and seawater d18O decreased by pleted in 18O. We conclude that the 4- to 6-m LIG
and to only 0.04 ± 0.03‰ in the Southern Ocean 1.05 ± 0.20‰ (22, 23). This implies that seawater sea-level rise would have resulted in a seawater
(table S2). d18O decreases by 0.008 ± 0.002‰ per meter of d18O decrease of 0.03 to 0.06‰. This change
was uniform in all water masses, because the
turnover of the ocean (about 1500 years) is much
Fig. 2. Calcite d18O shorter than the LIG duration. Therefore it should
anomaly (LIG – mod-
be recorded in all benthic foraminiferal d18O
ern) simulated by
values. We then computed a local d18O anomaly
CLIMBER-2 (in per
mil) in the Atlantic as the difference between the measured forami-
Ocean (computed be- niferal d18O value and that owing to the input of
tween the averages meltwater. This local d18O anomaly is only due
over the last 100 to changes in deep water d18O and temperature.
years of the simula- For the deep Southern Ocean (1900 to 3500 m),
tions). Calcite d18O is the anomaly is not statistically different from
computed as d18Oc = zero. This result implies that during the LIG,
21.9 − 0.27 þ d18Ow −ffi
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi bottom water formed on the Antarctic continental
3 10:61 þ 1 0T shelf at the freezing point, as it does today. In the
where T is the oceanic Norwegian Sea and the Atlantic Ocean, the anom-
temperature (37). The aly is significantly different from zero at the
global d18O change 1s level (–0.15 and –0.11‰, respectively).
in seawater composi- However, it is small and can be considered to
tion of –0.045‰ due be a minor departure from modern conditions.
to sea-level change at Such small changes in the physical properties of
the LIG is taken into water masses tend to develop at almost constant
account. density, as observed in hydrographic data
Fig. 3. Annual mean temperature anomalies (LIG – modern) simulated by LOVECLIM (in °C) along the western boundary of the Atlantic Ocean. The
field is averaged over the last 100 years of the simulation. The inset map shows the path chosen for the section.

REPORTS
collected during the past 50 years (24, 25). At changes compensate for each other (table S2). candidate for an associated sea-level rise of the needed
present, the temperature and salinity of NADW Both models therefore confirm the hypothesis magnitude (34, 35).
5. E. Cortijo et al., Paleoceanography 14, 23 (1999).
are close to 3°C and 34.96 practical salinity at the basis of our interpretation of the benthic 6. North Greenland Ice Core Project Members, Nature 431,
units (psu). To maintain a constant density, sa- d18O data. Furthermore, a nearly constant den- 147 (2004).
linity must increase by 0.12 psu for each degree sity explains why the deep oceanic circulation 7. A. V. Lozhkin, P. M. Anderson, Quat. Res. 43, 147 (1995).
that the water is warmed. Thus, we can com- is little affected by the insolation changes at 8. T. Fronval, E. Jansen, H. Haflidason, H. P. Sejrup,
Quat. Sci. Rev. 17, 963 (1998).
pute that the d18O anomalies measured in LIG 126,000 years before the present. Nordic Sea 9. B. L. Otto-Bliesner, S. J. Marshall, J. T. Overpeck,
benthic foraminifera result from a 0.37° ± 0.20°C surface waters, fed by warmer and saltier North G. H. Miller, A. Hu, Science 311, 1751 (2006).
warming of NADW, compensated for by a 0.04 Atlantic surface waters, form a deep water 10. F. Vimeux, K. M. Cuffey, J. Jouzel, Earth Planet. Sci. Lett.
increase in salinity. Similarly, a 0.57° ± 0.2°C mass, also slightly warmer and saltier than today 203, 829 (2002).
11. L. Labeyrie et al., Paleoceanography 11, 57
warming compensated for by a salinity in- [change in temperature (DT ) = 0.6°C and change (1996).
crease of 0.07 psu occurred in the Norwegian in salinity (DS) = 0.04 psu for the CLIMBER-2 12. K. Pahnke, R. Zahn, H. Elderfield, M. Schulz, Science
Sea (table S2). model; DT = 0.5°C and DS = 0.06 psu for the 301, 948 (2003).
Among the factors responsible for a LIG LOVECLIM model, see table S2]. This water 13. E. A. Colhoun, J. S. Pola, C. E. Barton, H. Heijnis,
Quat. Int. 57/58, 5 (1999).
warmer than today, insolation changes are the invades the North Atlantic as a large NADW
14. M. J. Vandergoes et al., Nature 436, 242 (2005).
best candidates. Indeed, Earth’s orbital parame- mass (Fig. 3), which then flows to the Southern 15. M. Oppenheimer, Nature 393, 325 (1998).
ters during the LIG favored warm Northern Ocean. 16. E. Rignot, S. S. Jacobs, Science 296, 2020 (2002).
Hemisphere summers as compared to the present. As a result, the Antarctic Circumpolar Waters 17. S. S. Jacobs, H. H. Hellmer, A. Jenkins, Geophys. Res. Lett.
LIG atmospheric greenhouse gas concentrations are slightly warmer than today. Our LIG simu- 23, 657 (1996).
18. A. Shepherd, D. Wingham, E. Rignot, Geophys. Res. Lett.
were not significantly different from preindustrial lations show a ~0.1° to 0.5°C warming in the 31, L23402 (2004).
values. To simulate the LIG atmosphere/ocean upper 500 m of the Southern Ocean close to the 19. J. C. Duplessy et al., Quat. Res. 21, 225 (1984).
state, we therefore forced our climate models Antarctic coast (Fig. 3 and fig. S2). This value is 20. C. Israelson, B. Wohlfarth, Quat. Res. 51, 306
with the insolation values for 126,000 years be- similar to the 0.5°C warming simulated by the (1999).
21. K. Lambeck, J. Chappell, Science 292, 679 (2001).
fore the present (supporting online text). We used National Center for Atmospheric Research mod- 22. J. C. Duplessy, L. Labeyrie, C. Waelbroeck, Quat. Sci. Rev.
two Earth System models of intermediate com- el for the upper 200 m of the Southern Ocean 21, 315 (2002).
plexity: CLIMBER-2 (26) and LOVECLIM (3). Although apparently modest, this warming 23. D. P. Schrag et al., Quat. Sci. Rev. 21, 331 (2002).
(27). In CLIMBER-2, the ocean is represented should not be underestimated. Recent results ob- 24. R. Dickson et al., Science 416, 832 (2002).
25. R. Curry, C. Mauritzen, Science 308, 1772 (2005).
by three latitude/depth basins, and the computed tained with satellite radar interferometry reveal 26. D. Roche, D. Paillard, A. Ganopolski, G. Hoffmann,
calcite d18O can be directly compared with mea- that bottom melting near an ice-shelf grounding Earth Planet. Sci. Lett. 218, 317 (2004).
surements. LOVECLIM includes a full-ocean line is strongly influenced by the temperature of 27. E. Driesschaert, thesis, Université Catholique de
general circulation model but no oceanic oxygen the surrounding seawater, and that the melting Louvain-la-Neuve, Belgium (2005).
28. N. Groll, M. Widmann, J. Jones, F. Kaspar, S. J. Lorenz,
isotope calculation. rate at the base of ice shelves increases by 1 m per J. Clim. 18, 4032 (2005).
The CLIMBER-2 model simulates negative year for each 0.1°C rise in ocean temperature 29. F. Kaspar, N. Kuhl, U. Cubasch, T. Litt, Geophys. Res. Lett.
calcite d18O anomalies (LIG –control) in all ocean (16). Thus, in addition to the higher sea level re- 32, L11703 (2005).
basins (Fig. 2). The magnitude of these anomalies sulting from the partial melting of the Greenland 30. C. Kubatzki, M. Montoya, S. Rahmstorf, A. Ganopolski,
M. Claussen, Clim. Dyn. 16, 799 (2000).
decreases from the Nordic Seas (–0.16‰), ice sheet, the 0.1° to 0.5°C warming that we have 31. S. S. Jacobs, C. F. Giulivi, P. A. Mele, Science 297, 386
through the Atlantic (–0.12‰), into the Southern estimated for LIG NADW and CDW may have (2002).
Ocean (–0.06‰). There is good agreement with affected vulnerable WAIS grounding lines and 32. J. H. Mercer, Nature 271, 321 (1978).
data (table S2), in particular for the Atlantic further weakened the ice shelves by causing 33. R. P. Scherer et al., Science 281, 82 (1998).
34. A. W. Droxler, R. Z. Poore, L. H. Burckle, Eds.,
Ocean, where the reconstructions are the most thinning from below. AGU Monogr. 137, 240 (2003).
robust. Furthermore, both models simulate sea Our data show that changes in climate in the 35. D. Q. Bowen, Eos 87 (fall meeting suppl.), abstr.
surface temperature changes consistent with re- high-latitude North Atlantic could have triggered PP51B-1139 (2006).
constructions and numerical studies for the LIG some ice sheet melting in Antarctica, but they 36. L. D. Labeyrie, J. C. Duplessy, P. L. Blanc, Nature 327,
477 (1987).
(9, 28–30) (supporting online text). Sea surface provide no information on the speed at which the 37. N. J. Shackleton, in Les Methodes Quantitatives d’Etude
temperatures increase at nearly all latitudes, with a WAIS shrank during the LIG. Although it is not des Variations du Climat au Cours du Pleistocène (CNRS,
maximum increase at high northern latitudes and our goal to predict the future of the WAIS, we Gif sur Yvette, France, 1974), pp. 203–209.
a secondary maximum in the Southern Ocean. note that recent ocean temperatures directly 38. D.M.R. is supported by the Netherlands Organization for
Scientific Research (NWO). J.-C.D. and M.K. are funded by
Maxima occur at high latitudes because of re- seaward of Antarctica's continental shelf have CNRS, CEA, Institut Polaire, the European Union Pole-Ocean-
duced sea-ice cover. The increased summer bo- already increased by ~0.2°C (31), a warming Pole (EVK-2000-00089) project, the Impairs project of the
real insolation is responsible for a significant comparable to that of the LIG period. Conse- Institut des Sciences de l’Univers and the “Intégration des
melting of the northern sea ice, which translates quently, the future evolution of the WAIS might contraintes Paléoclimatiques: réduire les Incertitudes sur
l’évolution du Climat des périodes Chaudes (PICC)” project of
into a year-round warming of the ocean due to its be a key component of sea-level change result- the Agence Nationale de la Recherche. The authors thank the
large thermal inertia. As a consequence of these ing from anthropogenic warming, as Mercer Potsdam Institute for Climate Impact Research for providing
higher sea surface temperatures, ocean evapora- warned more than 25 years ago (32). the CLIMBER-2 model and Hugues Goosse at the Université
tion increases. This increase is not compensated Catholique de Louvain-la-Neuve for technical assistance with
the LOVECLIM model, A. Friend and J. Overpeck for their
for by an increase in precipitation. Consequently, References and Notes reviews of an early version of the manuscript, and two
salinity increases, which agrees with the paleoceano- 1. J. H. Chen, H. A. Curran, R. White, G. J. Wasserburg, anonymous reviewers for their suggestions.
graphic reconstructions (5). Hence, the model re- Geol. Soc. Am. Bull. 103, 82 (1991).
2. C. H. Stirling, T. M. Esat, K. Lambeck, M. T. McCulloch,
sults are in broad agreement with the surface Earth Planet. Sci. Lett. 160, 745 (1998).
hydrographical reconstructions and the benthic 3. J. T. Overpeck et al., Science 311, 1747 (2006). www.sciencemag.org/cgi/content/full/316/5821/89/DC1
SOM Text
d18O data, the latter being simply interpreted as 4. A WAIS contribution to the high sea level of the LIG is
Figs. S1 and S2
the response of the ocean to the LIG insolation supported by diatoms and 10Be data collected from
sediments below the ice-stream region of the Ross Tables S1 and S2
values. Embayment, indicating that the central WAIS was
References
Simulated density is quasi-constant because probably smaller during the Pleistocene (33). The LIG 8 December 2006; accepted 5 March 2007
the impacts of the temperature and salinity and not an earlier interglaciation is the most likely 10.1126/science.1138582

REPORTS
report here on data collected during the south-
Subsurface Radar Sounding of the ern hemisphere nightside campaign of Mars Ex-
press between November 2005 and April 2006.
South Polar Layered Deposits of Mars Data were collected during more than 300 orbits,
with MARSIS primarily operating in a two-
frequency mode using bands centered on 3.0, 4.0,
Jeffrey J. Plaut,1 Giovanni Picardi,2 Ali Safaeinili,1 Anton B. Ivanov,1 Sarah M. Milkovich,1 or 5.0 MHz.
Andrea Cicchetti,2 Wlodek Kofman,3 Jérémie Mouginot,3 William M. Farrell,4 Roger J. Phillips,5 The NPLD were observed previously by
Stephen M. Clifford,6 Alessandro Frigeri,7 Roberto Orosei,8 Costanzo Federico,7 MARSIS during two orbits in June 2005 (11).
Iwan P. Williams,9 Donald A. Gurnett,10 Erling Nielsen,11 Tor Hagfors,11 Essam Heggy,6 The MARSIS signals appeared to penetrate to the
Ellen R. Stofan,12 Dirk Plettemeier,13 Thomas R. Watters,14 Carlton J. Leuschen,15 Peter Edenhofer16 base of the deposit, which was estimated to be
1.8 km deep in the thickest area observed, near
The ice-rich south polar layered deposits of Mars were probed with the Mars Advanced Radar for the periphery of the deposits. The very low atten-
Subsurface and Ionospheric Sounding on the Mars Express orbiter. The radar signals penetrate deep uation of the MARSIS signals in the NPLD
into the deposits (more than 3.7 kilometers). For most of the area, a reflection is detected at a time materials suggests that they contain only a few
delay that is consistent with an interface between the deposits and the substrate. The reflected power percent dust mixed with pure water ice. The basal
from this interface indicates minimal attenuation of the signal, suggesting a composition of nearly pure interface was seen to remain essentially horizon-
water ice. Maps were generated of the topography of the basal interface and the thickness of the tal beneath the NPLD, showing that flexural
layered deposits. A set of buried depressions is seen within 300 kilometers of the pole. The thickness downwarping due to the load does not exceed the
map shows an asymmetric distribution of the deposits and regions of anomalous thickness. The total estimated detection limit of several hundred me-
volume is estimated to be 1.6 × 106 cubic kilometers, which is equivalent to a global water layer ters and implying a thick lithosphere in that re-
approximately 11 meters thick. gion of the planet (11).
In a typical MARSIS observation over the
he polar regions of Mars are covered Martian PLD were first identified in orbital SPLD (Fig. 1), the echo from the surface splits
T with extensive finely layered deposits that

contain a record of climate variations of an
unknown time span (1). Although the precise
images obtained by the Mariner and Viking
spacecraft (3–6). They were noted to consist of
dozens of layers of contrasting albedo, with
into two continuous traces as the spacecraft passes
over the margin of the deposits. The surface trace
follows a profile expected from MOLA topog-
composition of the deposits is unknown, it is be- thicknesses down to the resolution of the avail- raphy. The bright lower trace occurs at a time
lieved that they are predominantly water ice and able images (about 10 m). Higher-resolution im- delay consistent with a continuation of the sur-
that they represent the largest known reservoir of ages acquired by the Mars Orbital Camera on rounding surface topography beneath the SPLD,
H2O on the planet (2). We applied a technique Mars Global Surveyor (MGS) indicated that the assuming a nominal value of the refractive index
commonly used to study the interior of ice sheets scale of layering extends down to the resolution of water ice. The lower interface is interpreted as
and glaciers on Earth—radar echo sounding—to of that camera; that is, a few meters (7). Topo- the boundary between the base of the ice-rich
study the south polar layered deposits (SPLD) of graphic data obtained by the Mars Orbiter Laser SPLD materials and the predominantly lithic
Mars. We report here on observations of the Altimeter (MOLA) on MGS showed that the substrate. The interface is detected beneath most
SPLDs by the Mars Advanced Radar for Sub- north PLD (NPLD) and the SPLD are similar in of the SPLD, although in places it becomes
surface and Ionospheric Sounding (MARSIS) gross morphology and thickness (2). Both the discontinuous, indistinct, or absent. It is generally
instrument on the Mars Express orbiter. The data NPLD and SPLD units are roughly domical in lower in backscatter intensity than the surface
were used to characterize the electrical properties shape and about 1000 km across, with maximum above it, but in places it appears equivalent to or
of the deposits in order to understand their com- relief relative to the surrounding terrain of about brighter than the surface echo. Propagation of the
position, map the topography of the bed of the 3.5 km. MOLA data were used to estimate the signal in the SPLD medium can be described
deposits, and measure the total volume of the volume of the PLD, which is equivalent to a with a simple two-layer homogeneous model,
SPLD. global layer 16 to 22 m thick (2). Although the using reflection and absorption coefficients that
relationship of the layering to climate variations are appropriate for materials expected on Mars.
1
Jet Propulsion Laboratory, California Institute of Technol- is not well understood, it is believed that the As in the case of the NPLD (11), the strong return
ogy, Pasadena, CA 91109, USA. 2Infocom Department, “La
Sapienza” University of Rome, 00184 Rome, Italy.
rhythmic nature of the deposits is related to oscil- from the basal interface indicates very low
3
Laboratoire de Planetologie de Grenoble, 38041 Grenoble lations in Mars’ orbital parameters (8). The al- attenuation values within the SPLD. If the
Cedex, France. 4NASA/Goddard Space Flight Center, Green- bedo variations among layers are thought to be material is assumed to be “dirty” water ice over-
belt, MD 20771, USA. 5Department of Earth and Planetary caused by varying mixtures of ice and dust. The lying a basaltic substrate, effective loss tangent
Sciences, Washington University, St. Louis, MO 63130, USA.
6 mixing ratio of ice and dust cannot be precisely values between 0.001 and 0.005 are obtained for
Lunar and Planetary Institute, Houston, TX 77058, USA.
7
Dipartimento di Scienze della Terra, Università degli Studi measured from optical data, but it has been the SPLD material (12). This corresponds to
di Perugia, 06123 Perugia, Italy. 8Istituto di Fisica dello shown that only a small fraction (<10%) of dust water ice with a dust contamination of 0 to 10%
Spazio Interplanetario, Istituto Nazionale di Astrofisica, is needed to lower the albedo of pure ice to the (13). The general behavior of the surface and
00133 Rome, Italy. 9Astronomy Unit, School of Mathema- observed levels (9). subsurface echoes over most of the SPLD is
tical Sciences, Queen Mary University of London, Mile End
Road, London E1 4NS, UK. 10Department of Physics and MARSIS is a multifrequency synthetic- consistent with a composition of water ice that is
Astronomy, University of Iowa, Iowa City, IA 52242, USA. aperture orbital sounding radar (10). In its sub- relatively free of impurities, overlying a typical
11
Max Planck Institute for Solar System Resarch, 37191 surface modes, MARSIS operates in frequency Martian regolith and crust.
Katlenburg-Lindau, Germany. 12Proxemy Research, Laytons- bands between 1.3 and 5.5 MHz, with a 1-MHz An extended area of unexpectedly bright
ville, MD 20882, USA. 13Fakultaet fuer Elektrotechnik und
Informationstechnik, Technische Universitaet Dresden, D- instantaneous bandwidth that provides free-space basal reflections occurs in an area between the
01062 Dresden, Germany. 14Center for Earth and Planetary range resolution of approximately 150 m. Lateral thickest part of the SPLD (~3.7 km) and the
Studies, National Air and Space Museum, Smithsonian spatial resolution is 10 to 30 km in the cross-track nearby SPLD margin, from 310° to 0° east
Institution, Washington, DC 20560, USA. 15Center for direction; and the along-track footprint, narrowed longitude (Fig. 1). The returns are often brighter
Remote Sensing of Ice Sheets, University of Kansas,
Lawrence, KS 66045, USA. 16Fakultaet fuer Elektrotechnik
by onboard synthetic-aperture processing, is 5 to than the surface return, which is not expected for
und Informationstechnik Ruhr-Universitaet Bochum, D- 10 km. Processing includes a correction for phase propagation through a lossy medium. Although a
44780 Bochum, Germany. distortion and delay in the ionosphere (11). We strong contrast in dielectric constant at the base

REPORTS
may be responsible, we deem it highly unlike- the ice, further arguing against basal melting. MARSIS radargrams of the SPLD (Fig. 1). The
ly that liquid water [from basal melting (14)] Nevertheless, we cannot completely rule out un- banding consists of bright continuous reflectors,
causes the bright return, because it occurs usual geothermal conditions or an exotic com- sometimes hundreds of kilometers long, alter-
below thin (as well as thick) sections of the position of the substrate in these anomalously nating with lower-backscatter bands. The band-
SPLD that are among the coldest places on the bright areas. ing is certainly related to the layered structure of
surface of Mars. The low attenuation is con- A pattern of banding commonly occurs be- the SPLD, possibly due to contrasts in dust con-
sistent with very low temperatures throughout tween the surface and basal interface traces in tent or density, but the precise mechanism that
Fig. 1. (A) MARSIS data from orbit 2753,

showing typical features of the SPLD. (B) MOLA
topography along the ground track. The lower
echo trace (arrows) is interpreted as the SPLD
basal interface with the substrate. The basal
reflector becomes indistinct at the right of center.
The central area shows multiple continuous
bands internal to the SPLD, where the estimated
SPLD thickness is 1.6 km. (C) MARSIS data from
orbit 2682, showing a bright basal reflector
(arrow). (D) MOLA topography along the ground
track. The reflector extends from the margin of
the SPLD (left of center) to below a 3.5-km-thick
section of the SPLD. The basal reflector abruptly
disappears for unknown reasons. (E) MOLA
surface elevations (black line) and MARSIS
measured basal elevations (blue symbols),
assuming a refractive index of ice. The basal
reflector is at a fairly constant elevation between
1000 and 1500 m. The apparent curvature of the
reflector in (C) is an artifact of the time rep-
resentation of the data. The vertical dimension in
(A) and (C) is round-trip travel time. See Fig. 2 for the location of ground tracks and the MOLA elevation scale.
Fig. 2. (left). Topography of the south polar region of Mars from MGS 87°S (dark circle in upper center). Fig. 3 (right). Same as Fig. 2, with
MOLA data, with locations of MARSIS measurements of the SPLD thickness topography at the SPLD basal interface shown, based on MARSIS
shown as open circles. The SPLD unit as mapped by (15) is outlined in measurements of SPLD thickness. A indicates a depression below a distal
black. Red lines indicate ground tracks of the orbits in Fig. 1. Apparent SPLD lobe. B indicates relative highs within the remnant Prometheus basin
gaps in coverage are due to the lack of a discernible basal interface, and (the basin rim is indicated with arrows). C indicates depressions in the
not to gaps in observations. No MARSIS data are available poleward of near-polar region.

REPORTS
creates the bands is unknown. The position and were combined with MOLA elevations along elastic lithosphere in the south polar region is
brightness of the bands sometimes vary with the margin of the SPLD unit as mapped by very thick (>150 km), as was inferred for the
the frequency of the MARSIS observation, sug- (15), where the thickness of the unit is con- north polar region (11).
gesting that the bands may be due to interfer- sidered to be zero. The results of a “natural- A thickness map of the SPLD was generated
ence effects that depend on the relative scales neighbor” interpolation (16, 17) of these data by subtracting the elevations of the interpolated
of the radar wavelength and the internal layer- are shown in Fig. 3. The map of the sub-SPLD basal topography from the high-resolution
ing of the SPLD. topography is generally consistent with that MOLA surface topography (Fig. 4). The distri-
Detection of the basal interface below most of expected from simple interpolation of MOLA bution of SPLD thickness reflects the asymmetry
the SPLD allows us to generate a map of the data from the margins of the unit (18). The sur- of the south polar geology, with the thickest
topography of this interface and to provide new face is typically low in relief, with broad areas portions offset from the pole near 0°E longitude
estimates of the thickness and volume of the of higher and lower topography. A pronounced and the much more areally extensive but thinner
SPLD. The methodology used is as follows: The low area is seen near the SPLD margin around portion centered near 180°E. The newly dis-
time delay was measured between the peak of 72° to 74°S, 130° to 145°E, and elevated re- covered near-polar depressions show clearly as
the surface reflection and the peak of the last gions within the remnant Prometheus impact anomalously thick areas, as do several of the
bright continuous reflector, which is assumed to basin are seen to continue below the SPLD at distal lobes. The maximum measured thickness is
be the basal interface. Over 1800 points from 60 78° to 82°S, 100° to 130°E, and from 70° to 3.7 ± 0.4 km, under the highest elevations of the
high-quality MARSIS orbits were used. The 90°E poleward of about 83°S. An unexpected SPLD near 0°E. Our estimate of the integrated
orbits were chosen to provide coverage that was feature of the basal topography is a series of de- volume of the entire SPLD is 1.6 ± 0.2 × 106 km3
sufficiently dense to generate medium-resolution pressions at the highest latitudes (84° to 87°S). (19). This translates to an equivalent global water
maps of basal topography and SPLD thickness. These occur discontinuously from longitudes layer thickness of 11 ± 1.4 m (assuming an SPLD
Data from two frequencies were evaluated for 95° to 295°E. The depressions range in width composition of nearly pure ice) and is within
most points. All points were verified to be actual from 50 to 200 km and reach a depth as much the range estimated by previous workers using
subsurface reflectors and not surface “clutter,” as 1 km below the surrounding sub-SPLD to- MOLA data alone (2, 18, 20). Knowledge of
using simulations of surface echoes based on pography. The basal reflection within the de- the basal topography now allows us to esti-
MOLA topography (11). The vertical resolution pressions is typically dimmer than under the mate the volume with a much smaller range of
of the data used was about 100 m in ice, with an rest of the SPLD. This fact and the position of uncertainty.
estimated uncertainty on a given measurement the depressions in the near-polar areas suggest MARSIS data do not allow us to distin-
less than 200 m. To convert time delay to depth, that there may be processes unique to this sub- guish a component of CO2 ice in the SPLD
we used the refractive index of pure ice with a SPLD area. The depressions may be the result material, but there is no corroborative evidence
real dielectric constant of 3. We could reasonably of differential compaction of megaregolith in for such a component. Spectral and albedo ob-
expect this estimate to be off by ±0.5, which response to the SPLD load. Alternatively, they servations of the surface of the SPLD indicate
translates into an additional error in our depth may be a group of buried impact craters or other an optically thick lag of dust or rocky material,
estimate of somewhat less than 10%. Figure 2 preexisting topography such as the ~1-km-deep but this layer is “optically” thin at MARSIS
shows a map of the locations of the measured pits (“Cavi”) in the nearby plains. On a regional wavelengths. Similarly, MARSIS detects no dif-
points. scale, the basal interface is relatively flat. The ference in surface or subsurface echoes from
To obtain a map of the basal interface, the lack of evidence of regional downwarping in areas covered by the residual (“perennial”) CO2-
MARSIS measured elevations of the interface response to the SPLD load suggests that the rich ice unit, which is consistent with recent
analyses indicating that it is a deposit no more
than a few tens of meters thick (21, 22).
Fig. 4. Map of the SPLD
thickness, based on MARSIS
measurements and MOLA References and Notes
surface topography. An 1. P. C. Thomas, K. Herkenhoff, A. Howard, B. Murray, in
anomalous thick section Mars, H. H. Kieffer et al., Eds. (Univ. of Arizona Press,
Tucson, AZ, 1992), pp. 767–795.
appears at lower right 2. D. E. Smith et al., J. Geophys. Res. 106, 23689
(see A in Fig. 3). The (2001).
thickest areas occur be- 3. B. C. Murray et al., Icarus 17, 328 (1972).
neath the highest eleva- 4. J. A. Cutts, J. Geophys. Res. 78, 4231 (1973).
tions of the SPLD (red 5. K. R. Blasius, J. A. Cutts, A. D. Howard, Icarus 50, 140
areas near the top) and in (1982).
6. A. D. Howard, J. A. Cutts, K. R. Blasius, Icarus 50, 161
association with the near- (1982).
polar depressions (see C 7. M. C. Malin, K. S. Edgett, J. Geophys. Res. 106, 23429
in Fig. 3). (2001).
8. O. B. Toon, J. B. Pollack, W. Ward, J. A. Burns, K. Bilski,
Icarus 44, 552 (1980).
9. H. H. Kieffer, J. Geophys. Res. 95, 1481 (1990).
10. G. Picardi et al., in Mars Express: A European Mission to
the Red Planet [SP-1240, European Space Agency (ESA)
Publications Division, European Space Research and
Technology Centre, Noordwijk, Netherlands, 2004],
pp. 51–69.
11. G. Picardi et al., Science 310, 1925 (2005).
12. Loss tangent in this context is defined as (e''/e') where
e''and e' are the imaginary and real parts, respectively,
of the complex dielectric permittivity.
13. E. Heggy et al., in 4th International Conference on Mars
Polar Science and Exploration (Lunar and Planetary
Institute, Houston, TX, 2006), abstr. 8105.
14. S. M. Clifford, J. Geophys. Res. 92, 9135 (1987).

REPORTS
15. K. Tanaka, E. Kolb, U.S. Geol. Surv. Open-File Rep. 19. The error on this estimate includes a 10% uncertainty in Agency) for the operations of MARSIS and Mars Express.
2005-1271 (2005). refractive index and an additional factor due to gaps in We thank Y. Gim for clutter simulation. Some of the
16. R. Sibson, in Interpolating Multivariate Data (Wiley, New coverage and detectability of the basal interface. research described in this publication was carried out
York, 1981), pp. 21–36. 20. P. M. Schenk, J. M. Moore, J. Geophys. Res. 105, 24529 at the Jet Propulsion Laboratory, California Institute
17. We used the natural-neighbor interpolation as implemented (2000). of Technology.
in the ArcMap GIS software from ESRI (Redlands, CA). The 21. S. Byrne, A. P. Ingersoll, Science 299, 1051 (2003).
interpolation applies a weighted moving average and is a 22. J.-P. Bibring et al., Nature 428, 627 (2004). 8 January 2007; accepted 27 February 2007
preferred method for irregularly distributed data sets (16). 23. The authors acknowledge the support of the space Published online 15 March 2007;
18. C. Davies, B. A. Murray, J. Byrne, Eos 85, Fall Meeting agencies of Italy (Agenzia Spaziale Italiana), the 10.1126/science.1139672
Supplement, abstr. P13A-0971 (2004). United States (NASA), and Europe (European Space Include this information when citing this paper.
Synchronized Oscillation in Coupled interest. A two-oscillator system demonstrates

inherently rich linear and nonlinear dynamics,
which contrast with its deceptive simplicity (1, 11).
Nanomechanical Oscillators After the historical observation of synchroniza-
tion of two pendulum clocks by Huygens,
Appleton (12) and van der Pol (13) showed that
Seung-Bo Shim,* Matthias Imboden, Pritiraj Mohanty†
the frequency of a triode generator can be
entrained, or synchronized, to an external drive;
We report measurements of synchronization in two nanomechanical beam oscillators coupled by a
their work was motivated by the potential ap-
mechanical element. We charted multiple regions of frequency entrainment or synchronization by
plication in radio communication. The first sys-
their corresponding Arnold’s tongue diagrams as the oscillator was driven at subharmonic and
tematic studies of synchronization in biological
rational commensurate frequencies. Demonstration of multiple synchronized regions could be
systems (and, in particular, human physiology)
fundamentally important to neurocomputing with mechanical oscillator networks and
started with Peskin’s attempt to model self-
nanomechanical signal processing for microwave communication.
synchronization of cardiac pacemaker cells to
understand the generation of a heartbeat (4). In
he concept of synchronized oscillation in with Huygens’ discovery of the phenomenon in biological neurocomputing, neural networks
T coupled systems is pervasive in both na-

ture (1) and human physiology (2). Ex-
amples of synchronization include rhythmic
two coupled pendulum clocks (6) and leading to
modern-day experiments on coherent radiation in
coupled spin-torque nano-oscillators (7, 8) and
show rhythmic behavior, exemplified in many
Department of Physics, Boston University, 590 Common-

wealth Avenue, Boston, MA 02215, USA.
blinking of fireflies (3), the activity of pacemaker parametric resonance in mechanical oscillators
cells in the sinoatrial node of a human heart (4), (9, 10). *Present address: Center for Strongly Correlated Materials
Research and School of Physics and Astronomy, Seoul
and the spin-orbit resonance of the planet Mer- Frequency entrainment, a class of synchroni- National University, Seoul, South Korea.
cury (5). In physical systems, synchronization zation, of coupled micro- and nanomechanical †To whom correspondence should be addressed. E-mail:
has been studied for over three centuries, starting oscillators is of fundamental and technical [email protected]
A Mode 1 Mode 2
60 60
50 B C 50
40 40
a b 30
E
E 30
D D
20 C C 20
B B
10 A A 10
c d 0 0
15.23 15.24 15.25 15.26 16.06 16.07 16.08 16.09
f (MHz) f (MHz)
Force (nN) Force (nN)
0.003 0.01 0.1 0.5 1 2 5 0.003 0.01 0.1 0.5 1 2 5
20
Fig. 1. Device micrograph, magnetomotive characterization, and mode 1000 D E
E E
10
shape. (A) Scanning electron micrograph of the coupled nanomechanical 100 D D
5
Pinduced (pW)
oscillator. Two main beams, each 10 mm in length, are doubly clamped to

10 C
contact pads a and b and to c and d, respectively. The main beams are C 1
1 0.5
mechanically coupled near the center by a 5-mm-long beam. The beams are
500 nm wide and thick. The electrical leads (colored yellow) are selectively B B
0.1
keff = 712N/m keff = 797N/m 0.1
deposited such that contacts a and b are electrically isolated from contacts c 0.01 A A
and d. We observe two separate modes in the coupled nanomechanical -100 -90 -80 -70 -60 -50 -40 -30 -100 -90 -80 -70 -60 -50 -40 -30
resonator, labeled below as mode 1 and mode 2 with resonance frequencies Pdr (dBm) Pdr (dBm)
of 15.241 and 16.071 MHz, respectively. (B) Linear and nonlinear response
of the device structure in mode 1. The response has Lorentzian shape in the Plots of induced power (Pinduced) as a response of Pdrive at 5 T. Pinduced is a
linear regime. Asymmetric responses and hysteresis occur when Pdrive is measure of the resonant displacement, and Pdrive corresponds to the driving
greater than −60 dBm (trace D). The inset shows the mode shape from force. From the log-log plot of driving force versus displacement, we
finite element simulation. Vemf is the voltage measured by the network calculate the effective linear-response spring constants keff = 712 and 797
analyzer. (C) Linear and nonlinear response of the device structure in mode N/m for modes 1 and 2, respectively. Dx, the central displacement of the
2. Nonlinearity commences at similar powers as in the mode 1. (D and E) beam. See SOM for technical details.

REPORTS
brain subsystems, in which the pattern recog- Another representation of synchronization is field. The structure is driven magnetomotively
nition properties are similar to those of oscilla- frequency entrainment, where two or many (17) with alternating current along the beam
tor networks. Therefore, with the use of networks different frequencies of the oscillators, upon length and perpendicular to magnetic field B.
of nanomechanical oscillators, it might be pos- coupling, entrain or lock to a single frequency, Using a network analyzer, we identify two res-
sible to build a neurocomputer with associative determined by either the dynamics of the onances at 15.241 MHz (mode 1) and 16.071
memory in which the network can store and oscillator network or an external drive. In our MHz (mode 2). Corresponding mode shapes are
retrieve complex oscillatory patterns as syn- experiment, we study entrainment of a given analyzed by means of finite element simulation.
chronized states (14). oscillator mode f0 by observing its response as Each mode has a well-defined linear and non-
Synchronized states are often represented by another part of the structure is driven at a linear response (Fig. 1, B and C), as well as
Arnold’s tongues, which are regions of frequency fractional frequency, fd = (m/n) f0. the expected B2 dependence (18). Low driving
locking in the parameter space. In mathematical Our coupled oscillator consists of two elec- power Pdrive measurements in the linear regime
models, these regions appear (11, 15) whenever trically independent doubly clamped beams result in effective spring constants keff of 712
the drive frequency fd is a rational fraction of the (10 mm long by 500 nm wide by 500 nm thick), and 797 N/m for modes 1 and 2, respectively.
resonance frequency f0, such that fd = (m/n)f0, which are coupled by a 5-mm-long beam of From a simple point particle and spring-coupled
where m and n are respective integer winding the same width and thickness that is attached oscillator model, the keff value of the coupling
numbers of two oscillators. As the coupling in- to the centers of the main beams (Fig. 1A). Se- beam is estimated to be 39 N/m [see the sup-
creases, the frequency-locking regimes widen, lectively evaporated gold electrodes on each porting online material (SOM) for more technical
which give the appearance of a tongue shape. In beam serve as electrical connections for mea- details].
another graphical representation, the Devil’s surements. Because the beams are electrically To observe the frequency entrainment, we
staircase depicts the winding number of the isolated, the coupling between the beams is drove one of the beams with a frequency gen-
synchronized regions as a function of fd. This purely mechanical. The device is fabricated from erator, and the response of the second beam was
monotonic increase contains plateaus where the single-crystal silicon by electron-beam lithog- measured with a spectrum analyzer at the first
response frequency is locked to a given winding raphy and a combination of dry and wet etch resonant mode [characterized by the resonant
number. In general, the simplest fractions have processes. For all experimental data, the sample frequency of 15.241 MHz and full width at half
the largest frequency-locked synchronized is cooled to 280 mK with a 3He cryostat and maximum (FWHM) of 1.5 kHz]. Figure 2 shows
regions (16). placed at the center of a 5 T in-plane magnetic the response at f0 when driving the beam at sub-
-50 -20 Fig. 2. Synchronization at subharmonic driving.

A Pout(dBm) B Pout(dBm)
Frequency-power sweep with subharmonic fd ( f0/n,
7,875 -77.00 313 -105.0
-22 n = 1, 2,…,7). A signal generator drives one beam,
-60 2,876 -85.70 192 -109.3
and the response of the second beam is measured
Pdrive (dBm)
Pdrive (dBm)
1,050 -94.40 118 -113.6

-24 with a spectrum analyzer. The contours represent
-70 394 -103.1 70 -117.9 the response in dBm. The synchronized regions
144 -111.8 -26 43 -122.2 become visible in the contour plots when the
-80 53 -120.5 26 -126.5 response exceeds the noise level of −136 dBm. We
19 -129.3 -28 14 -130.8 drive one of the beams at a frequency f0/n and
5 -138.0 10 -135.0 record the response of the second beam at the
-90 -30 fundamental frequency f0. The coupling is purely
15.232 15.240 15.248 7.616 7.620 7.624
fdrive (MHz) fdrive (MHz) mechanical as a result of the center beam (Fig. 1A).
(A) Frequency-power sweep at resonant frequency
-10 Pout(dBm) n/m = 1. Pout, power output. (B) Synchronization
C 99.2 -115.0 with fd = f0/2. (C) Frequency-power sweep for
-14 74.4 -117.5 subharmonic excitations ( f0/n, n = 3, 4, 5, 6, 7).
55.8 -120.0 For these measurements, a minimum of 200-dB
Pdrive (dBm)
-18 41.8 -122.5 isolation is ensured so that the higher harmonic

31.6 -125.0 outputs from the RF source no longer contribute. (D)
-22 23.5 -127.5 Response extracted from contour plot for fd = f0/4.
17.6 -130.0 Each plot has Lorentzian shape and shows a linear
-26 13.2 -132.5 increase with Pdrive. (E) Devil’s staircase plot depicting
9.9 -135.0 the width of the Lorentzian of each synchronized
-30 region. For clarity, the plateaus are greatly magnified.
1/7 1/6 1/5 1/4 1/3 We measure FWHM (d f ) of the Lorentzian peaks at
fdrive/fo Pdrive = −12 dBm [dashed line in (C)]. The y axis (n/m)
0.40 represents the winding number between driving
-118 D Pdrive (dBm) E frequency and fundamental frequency.
0.35
fdrive/fo=1/4 -18 Pdrive = -12 dBm
Response (dBm)
-122 -20 0.30

716 Hz
n/m
-22
0.25
-126 -24 463 Hz
-26 0.20
306 Hz
-130
-28 362 Hz
0.15
-134
0.10
3.806 3.810 3.814 0.10 0.15 0.20 0.25 0.30 0.35 0.40
f drive (MHz) f drive/fo

REPORTS
harmonic frequencies. The observed Arnold’s here rather loosely, as a synonym for resonant represents the approximate winding number. The
tongues appear at all subharmonic drive fre- actuation, in which the response is measured at plateaus, representing the region of synchroniza-
quencies fd = (1/n)f0 with n ranging from mode 1 and not at a single frequency; the use of tion, vary in size. They tend to be larger for drive
1 (resonance) to 7, which means that, when the the term “locked” is justified because the frequencies closer to f0. However, for fd = f0/7, the
actuation frequency is an integer fraction of f0 of response frequency is correlated to a respective synchronized regime is larger than the regime for
the first mode, this mode of the structure starts fd. Resonant actuation is not observed when the fd = f0/6. This is not unexpected because, in a
to oscillate. drive frequency is not in the proximity of fd = f0/n Devil’s staircase, the plateau sizes do not grow
Figure 2A shows the Arnold’s tongue m/n = or other well-defined drive frequencies as is seen monotonously.
1/1 for which fd sweeps over f0. This is essentially below. The Arnold’s tongues only appear with The maximum responses in Arnold’s tongues
the same plot as the linear part of Fig. 1B. In a high Pdrive where the structure is known to have shift to lower drive frequencies for higher driving
typical measurement, the drive-frequency reso- nonlinear response when driven at the resonant powers. Within a frequency-locked regime, the
lution is 40 Hz, and Pdrive is increased in 2-dBm frequency (Fig. 1). locked frequency, observed close to resonance,
steps, which provides 10,521 averaged measure- Figure 2E displays the Devil’s staircase plot, shifts upward with increasing fd over a 10- to
ments taken over 12 hours. For the remaining which maps the widths of the Arnold’s tongues, 20-kHz range. In idealized systems, Arnold’s
Arnold’s tongues (Fig. 2, B and C), low-pass defined as the FWHM for a given Pdrive. tongues are expected for every rational number
filters with high isolation are used to ensure that Essentially, this is a slice for a given power level m/n, where small m and n values are preferred.
there is no higher harmonic output from the out of the Arnold’s tongue contour plots. The For this structure, drive frequencies such as fd =
frequency generator, because it would drive the actual width of frequency locking is much greater ( 2=3 )f0, ( 3=4 )f0, ( 3=2 )f0, and similar fractions could
structure on resonance. Because we do not than the FWHM of the fundamental resonant not be detected within the experimental limita-
measure the phase of the response, synchroniza- mode; however, the electronic noise floor tions. It is possible that the missing Arnold’s
tion is detected purely through frequency prevents detection far from the center of the tongues are present but are either too weak in
locking. The term “frequency locking” is used Lorentzian. The x axis indicates fd, and the y axis amplitude (below the noise floor of −136 dBm)
Fig. 3. Synchronization at non-subharmonic driving frequency. Synchro- and (L) are staircase plots indicating the widths of 16 synchronization
nization regions other than f0/n were discovered for a large range of regions. As in Fig. 2E, the plateaus have been magnified for clarity. There
frequencies by means of the same experimental procedure as in Fig. 2. In are two classes of synchronization regions that are characterized by their
most cases [(A to C), (E) and (F), and (I) and (J)], these regions are narrow behavior when sweeping Pdrive. In some cases, the synchronization regions
with a small response. However, two synchronization regions [(G) and (K)] appear at lower frequencies as Pdrive increases {[(A) and (B)], (E), (G), (I), and
were discovered to have a very prominent response, which is comparable to (K)} (i.e., the tongues bend to the left). However, there is also a substantial
the f0/3 result. (G) shows subharmonic synchronization at fd = 8.155 MHz, number of regions with the opposite behavior: the fd at which frequency
and (K) indicates superharmonic synchronization at fd = 18.360 MHz. No locking occurs increases with increasing Pdrive {[(B) and (C)], (F), and (J)} (i.e.,
obvious integer fractions can be associated with these frequencies. (D), (H), the tongues bend to the right).

REPORTS
or too narrow in frequency span to be detected lators as discussed in the SOM. The Devil’s through one beam and pump on the other beam.
with our setup. Alternatively, they simply may staircase is also plotted for these off-harmonic This enables us to explore purely mechanical
not exist in this non-idealized setup. Arnold’s tongues. The sizes of the frequency- amplification effects. In our experiment, we mod-
In addition to the Arnold’s tongues shown in locked regimes appear to fluctuate randomly, and ulate the spring constant of the structure by ap-
Fig. 2, synchronized regions are also found at no pattern could be established. A large number plying a high-amplitude pump signal at twice the
frequencies with no obvious commensurate of frequency-locked regimes would be essential resonance frequency. We apply a pump signal
relation to f0. These are depicted in Fig. 3. A to the realization of neural computers based on ( fpump = 2f0) on one beam (c to d, Fig. 1A) while
drive-frequency range of 370 kHz starting at 8.01 oscillator networks. driving at f0 through the other beam (a to b,
MHz, well above the fd = f0/2 frequency, reveals Another notable aspect of nonresonant exci- Fig. 1A). Two referenced signal generators are
34 Arnold’s tongues, with the possibility of more, tation is parametric amplification, which enables used for the drive and pump signals. The pre-
where the limitations are given by the frequency the enhancement of small signals in individual amplified induced voltage is detected with an RF
resolution and preamplifier noise. A smaller oscillator modes in a network. In our structure, lock-in amplifier. A Lorentzian fit to the data is
drive span of 180 kHz starting at 18.24 MHz on-chip amplification of small mechanical used to extract f0 and the amplitude A.
(well above f0) reveals a similar Arnold’s tongue signals can be performed by parametric down- We observe that f0 shifts down by 1.7 kHz
count of 24. Not only is resonant actuation conversion in which the small signal generated in with an −8-dBm pump signal (Fig. 4A), possibly
observed, but some of these frequency-locking one beam can be amplified by coupling it to a as a result of the applied strain from the pump.
regimes (Fig. 3, G and K) are also larger than the second beam that provides the necessary pump- This corresponds to a 0.4% change in keff. Here,
synchronized regimes illustrated in Fig. 2. For ing for signal amplification. In mechanical the gain is defined as the ratio of the response
Apump-on
both sub- and superharmonic drive frequencies, structures, parametric down-conversion or am- with and without pumping: G ¼ Apump . Figure
-off
some Arnold’s tongues bend down (maximum plification has been studied in optical setups with 4A illustrates an example of two Lorentzian
response is observed at decreasing fd) (Fig. 3, A, cantilever (19), torsional (20), and disk resonators responses with the pump on and off (Apump-on =
E, G, I, and K) and some Arnold’s tongues bend (21). Here, we report parametric amplification of 95.6 nV and Apump-off = 33.1 nV), yielding a gain
up (maximum response is observed at increasing mechanical signals in a nanometer-scale me- of 2.9. In this coupled oscillator structure, the
fd) (Fig. 3, C, F, and J) for growing Pdrive. It is not chanical resonator with a distinct two-oscillator maximum phase-sensitive gain (19, 22) is found
clear whether there is a pattern that can be or two-beam structure. Parametric amplification to be 3.3 at a relative phase of 0°. At a relative
established in the data. The response frequency is defined by a gain in the mechanical response phase of 90°, parametric attenuation is observed
(i.e., the frequency at which mode 1 is observed) on resonance (frequency f0) when modulating a as expected (Fig. 4B). In the experimental setup,
actually decreases as fd increases for the Arnold’s parameter of the oscillator such as the spring the phase between the drive and pump signals
tongues that bend up. This effect is the opposite constant, for instance, by adding parametric can be locked because they represent f and its
of that for the Arnold’s tongues that bend down modulation (pumping) at twice the resonance harmonic 2f. The phase locking between the two
as Pdrive increases. It is noteworthy that the frequency (pump frequency 2f0). Because the signals is monitored with the use of an oscillo-
bending of Arnold’s tongues is also observed in two beams on each side of the structure are scope. Figure 4C shows the measured phase and
mathematical simulations of phase-locked oscil- electrically isolated, we can drive the structure amplitude relationship between the two signals.
The mechanically coupled two-oscillator
120 structure demonstrates a large number of fre-
Pump ON 3.0 quency synchronization regions, as illustrated by
90
A B Arnold’s tongues. Furthermore, we demonstrate
Ppump = -8 dBm
2.0 phase-sensitive mechanical parametric amplifi-
Gain
60 cation of small signals. Future work would

involve an array of mechanically coupled nano-
1.0 mechanical oscillators with the ability to self-
30
oscillate. Even though synchronization resulting
Amplitude (nV)
0 0.0 from reactive coupling and nonlinear frequency

-40 -35 -30 -25 -20 -15 -10 -5 pulling has been studied in a model of a large
120 Ppump (dBm) array (23), a comprehensive study of two coupled
Pump OFF nanomechanical oscillators will elucidate further
50
90 aspects of the data in our experiments.
C
Amplitude (a.u.)
-7dBm 45 Demonstration of a large number of synchro-

60 nization regions provides exciting opportunities
-10dBm 40 for practical realization of pattern recognition
30 with the use of oscillator networks. Storage and
35
-20dBm retrieval of complex patterns through the cor-
0 30 responding synchronized states will enable the
feasibility of constructing nanomechanical neu-
-30 25 rocomputers. Small size, high speed, and low
16.066 16.070 16.074 16.078 0
power consumption in these structures further
f (MHz) add to the fundamental benefit of an on-chip
Fig. 4. Parametric amplification. (A) Response of the device structure with (top) and without (bottom) nanomechanical oscillator network with distrib-
pumping. Ppump, pumping power. (B) Dependence of gain for varying Ppump. At a relative phase of 0° uted on-chip signal processing at microwave
between driving and pumping signals, we measure parametric amplification, and, at a relative phase of frequencies (24). The advantage of such a device
90°, we observe parametric attenuation. The maximum gain observed is 3.3 with possible indication of would be its scalable architecture, based on
oscillation death. fw – f2w is the phase difference between the pump and drive signals. (C) Response as standard lithography and semiconductor process-
relative phase varies between the drive and the pump signal (monitored with an RF oscilloscope) for ing techniques. Beyond neural network models,
three different pumping powers. We observe a notable change in response as the two signals sweep nanomechanical oscillator networks capable of
from in phase to out of phase. a.u., arbitrary units. individual-oscillator addressing and global cou-

REPORTS
pling could provide paradigms by enabling ex- 5. S. F. Dermott, Nature 429, 814 (2004). 18. A. N. Cleland, M. L. Roukes, Appl. Phys. Lett. 69, 2653
perimental realization of physical models that 6. C. Huygens, Horologium Oscillatorium [The Pendulum (1996).
Clock] (Apud F. Muguet, Paris, 1673) [R. J. Blackwell, 19. D. Rugar, P. Grutter, Phys. Rev. Lett. 67, 699 (1991).
show phase transition, glassy states, and other Transl. (Iowa State Univ. Press, Ames, IA, 1986)]. 20. D. W. Carr, S. Evoy, L. Sekaric, H. G. Craighead, J. M. Parpia,
collective behavior. If two coupled oscillators 7. F. B. Mancoff, N. D. Rizzo, B. N. Engel, S. Tehrani, Nature Appl. Phys. Lett. 77, 1545 (2000).
demonstrate such rich collective behaviors, a 437, 393 (2005). 21. M. Zalalutdinov et al., Appl. Phys. Lett. 79, 695 (2001).
network of nanomechanical oscillators could 8. S. Kaka et al., Nature 437, 389 (2005). 22. A. N. Cleland, New J. Phys. 7, 235 (2005).
9. M. Zalalutdinov et al., Appl. Phys. Lett. 78, 3142 (2001). 23. M. C. Cross, A. Zumedieck, A. Lifshitz, J. L. Rogers,
possibly realize the complexity and intelli- 10. K. L. Turner et al., Nature 396, 149 (1998). Phys. Rev. Lett. 93, 224101 (2004).
gence, if not of the human brain subsystems, 11. V. I. Arnold, Mathematical Methods of Classical 24. R. L. Badzey, P. Mohanty, Nature 437, 995 (2005).
then at least those of pacemaker cells of a hu- Mechanics, K. Vogtmann, A. Weinstein, Transl. (Springer- 25. This work was supported by NSF under grant number
man heart or the rhythmic blinking of a con- Verlag, New York, ed. 2, 1989). CCF-0432089 and partially under grant number
12. E. V. Appleton, Proc. Cambridge Philos. Soc. 21, 231 DMR-0449670. We thank N. Kopel, Y. D. Park, J. Wei,
gregation of fireflies. (1922). and M. Grifoni for their valuable comments.
13. B. van der Pol, Philos. Mag. 3, 65 (1927).
References and Notes 14. E. M. Izhikevich, F. C. Hoppensteadt, SIAM J. Appl. Math.
1. A. T. Winfree, The Geometry of Biological Time (Springer- 63, 1935 (2003).
Verlag, New York, 1980). 15. C. Hayashi, Nonlinear Oscillations in Physical Systems
(Princeton Univ. Press, Princeton, 1985). Materials and Methods
2. S. Strogatz, Sync: The Emerging Science of Spontaneous
SOM Text
Order (Hyperion, New York, 2003). 16. A. Pikovsky, M. Rosenblum, J. Kurths, Synchronization:
Figs. S1 to S5
3. J. Buck, E. Buck, Science 159, 1319 (1968). A Universal Concept in Nonlinear Sciences (Cambridge
Univ. Press, Cambridge, 2001). References
4. C. S. Peskin, Mathematical Aspects of Heart Physiology
(Courant Institute of Mathematical Sciences, New York 17. A. Gaidarzhy, G. Zolfagharkhani, R. L. Badzey, P. Mohanty, 8 November 2006; accepted 14 February 2007
University, New York, 1975). Phys. Rev. Lett. 94, 030402 (2005). 10.1126/science.1137307
Giant Fluctuations of Coulomb Electron-electron scattering, and the result-

ing momentum transfer between the layers,
usually creates a so-called positive Coulomb
Drag in a Bilayer System drag, where the current in the active layer
produces a charge flow in the passive layer in
the same direction. There are also some cases
A. S. Price,1 A. K. Savchenko,1* B. N. Narozhny,2 G. Allison,1 D. A. Ritchie3 where unusual, “negative” Coulomb drag is
observed: e.g., between 2D layers in the pres-
The Coulomb drag in a system of two parallel layers is the result of electron-electron interaction ence of a strong, quantizing magnetic field
between the layers. We have observed reproducible fluctuations of the drag, both as a function (6); and between two dilute, 1D wires where
of magnetic field and electron concentration, which are a manifestation of quantum interference of electrons are arranged into a Wigner crystal
electrons in the layers. At low temperatures the fluctuations exceed the average drag, giving rise (11). All previous studies of the Coulomb
to random changes of the sign of the drag. The fluctuations are found to be much larger than drag, however, refer to the macroscopic (aver-
previously expected, and we propose a model that explains their enhancement by considering age) drag resistance. Recently there have been
fluctuations of local electron properties. theoretical predictions of the possibility to
observe random fluctuations of the Coulomb
n conventional measurements of the re- Because Coulomb drag originates from e-e drag (12, 13), where the sign of the frictional
I sistance of a two-dimensional (2D) layer,

an electrical current is driven through the
layer and the voltage drop along the layer is
interactions, it has become a sensitive tool for
their study in many problems of contempo-
rary condensed-matter physics. For example,
force will change randomly from positive to
negative when either the carrier concentra-
tion, n, or applied (very small) magnetic field,
measured. In contrast, Coulomb drag studies Coulomb drag has been used in the search for B, are varied.
are performed on two closely spaced but Bose-condensation of interlayer excitons (9), Drag fluctuations originate from the wave
electrically isolated layers, where a current I1 the metal-insulator transition in 2D layers nature of electrons and the presence of dis-
is driven through one of the layers (active (10), and Wigner crystal formation in quan- order (impurities) in the layers. Electrons travel
layer) and the voltage drop V2 is measured tum wires (11). around each layer and interfere with each other,
along the other (passive) layer (Fig. 1). The
origin of this voltage is electron-electron (e-e) Fig. 1. Schematic showing
interaction between the layers, which creates the origin of the drag signal I1
a “frictional” force that drags electrons in the V2 induced by the current
second layer. The ratio of this voltage to the I1. The fluctuations of the
driving current RD = −V2/I1 (the drag resist- drag arise from the inter-
ance) is a measure of e-e interaction between ference of electron waves in
the layers. The measurement of Coulomb drag each layer, before the two
in systems of parallel layers was first pro- electrons take part in the
posed in (1, 2) and later realized in a number interlayer interaction. l
of experiments (3–7) [for a review, see (8)].
1
School of Physics, University of Exeter, Stocker Road,
Exeter EX4 4QL, UK. 2The Abdus Salam International Δr
Centre for Theoretical Physics, Strada Costiera 11, Trieste
I-34100, Italy. 3Cavendish Laboratory, University of Cam-
bridge, Madingley Road, Cambridge CB3 0HE, UK.
[email protected]
V2

REPORTS
after collisions with impurities, over the char- the drag, which is demonstrated in the inset to drag fluctuations (at low T, when the fluc-
acteristic area ~ Lϕ2, where Lϕ is the coher- Fig. 3 where the temperature dependence of the tuations exceed the average) in the diffusive
ence length (Fig. 1). This interference is very drag is shown at two different values of n2. The regime is
important for the conductive properties of drag is seen first to decrease as the temperature
electron waves. For example, the interference is decreased, but then become either increasing-
pattern is changed when the phase of electron ly positive or increasingly negative, dependent e4 ET ðLÞtϕ ln kd
waves is varied by a small magnetic field, upon n2. The reproducible fluctuations of the 〈Ds2D 〉 ≈ A ð1Þ
ℏ2 g 4 ℏðkdÞ3
producing universal conductance fluctuations drag resistivity have also been observed as a
(UCF) seen in small samples with size L ~ Lϕ. function of magnetic field (Fig. 3B). For a fixed
There is, however, an important difference temperature, the magnitude of the drag fluctua- where sD ≈ rD/(r1r2), and r1 and r2 are the
between UCF and the fluctuations of the drag tions as a function of n2 is roughly the same as active- and passive-layer resistivities, respective-
resistance. The former are only a small cor- that as a function of B. ly; ET(L) is the Thouless energy, ET(L) = ħD/L2,
rection to the average value of the conductance: The theory of (12) calculates the variance of where D is the diffusion coefficient; tϕ is the
In our experiment, the single-layer resistance drag fluctuations in the so-called diffusive decoherence time; k is the inverse screening
fluctuates by ~200 milliohm around an average regime, l < d. In this case the drag is determined length; A = 4.9 × 10−3; and g = h/(e2r) is the
resistance of ~500 ohm. In contrast, the drag by global properties of the layers, averaged dimensionless conductivity of the layers. When
fluctuations, although small in absolute mag- over a region Dr >> l. The expected variance of the parameters of our system are used, this
nitude (~20 milliohm), are able to change ran-
domly but reproducibly the sign of the Coulomb
drag between positive and negative. Surprisingly, Fig. 2. Drag resistivity as a
we have found that these fluctuations of the function of passive-layer carrier
Coulomb drag, observed at temperatures below concentration for different tem-
1 K, are four orders of magnitude larger than peratures: T = 5, 4, 3, 2, 1, 0.4,
predicted in (12). and 0.24 K, from top to bottom.
Our explanation of the giant drag fluctuations Inset (A): rD as a function of T 2.
takes into account that, unlike the UCF, the drag Inset (B): rD as a function of n2,
fluctuations are not only an interference effect with n1 = 1.1 × 1011 cm−2;
dashed line is a n2−1.5 fit.
but also fundamentally an interaction effect. In
conventional drag structures, the electron mean
free path l is much larger than the separation d
between the layers, and therefore large momen-
tum transfers ħq between electrons in the layers
become essential. According to the quantum
mechanical uncertainty principle, Dr Dq ~ 1,
electrons interact over small distances Dr << l
when exchanging large values of momentum
(Fig. 1). As a result the local properties of the
layers, such as the local density of electron states
(LDOS), become important in the interlayer e-e
interaction. These local properties at the scale
Dr << l exhibit strong fluctuations (14) that di-
rectly manifest themselves in the fluctuations of
the Coulomb drag.
The samples used in this work are AlGaAs-
GaAs double-layer structures, in which the
carrier concentration of each layer can be
independently controlled by gate voltage. The
two GaAs quantum wells of the structure, 200 Å
in thickness, are separated by an Al0.33Ga0.67As
layer of thickness 300 Å. Each layer has a Hall-
bar geometry, 60 mm in width and with a distance
between the voltage probes of 60 mm (15).
Figure 2 shows the appearance of the fluc-
tuations in the drag resistivity, rD, at low temper-
atures. At higher temperatures, the drag resistance
changes monotonically with both T and n; the
insets to Fig. 2 show that rD increases with
increasing temperature as T 2 and decreases with
increasing passive-layer carrier concentration as
n2b, where b ≈ −1.5. These results are consistent Fig. 3. (A) Drag resistance measured at low temperatures as a function of passive-layer concentration;
with existing experimental work on the average T = 1, 0.4, and 0.24 K, from top to bottom. (Inset) rD as a function of T for two values of n2 denoted by
Coulomb drag (4, 16). the dotted lines in Fig. 3A; solid line is the expected T2 dependence of the average drag. (B) rD as a
Figure 3A shows a magnified view of the function of B; T = 0.4, 0.35, and 0.24 K, from top to bottom. (Graphs for higher T are vertically offset
reproducible fluctuations as a function of n2. for clarity.) Single-layer concentration for each layer is 5.8 × 1010 cm−2. (Inset) The UCF of the single
These fluctuations result in an alternating sign of layer, with an average background resistance of 500 ohm subtracted.

REPORTS
expression gives a variance of ~6 × 10−11 mS2, q-values: q < 1/l and 1/l < q < 1/d. We have seen interfering electrons, however, is ET(Lϕ) (17),
which is approximately eight orders of magni- that small q values cannot explain the large which is much smaller than EF, and therefore in
tude smaller than the variance of the observed fluctuations of the drag (12), and so argue that a mesoscopic system e-h asymmetry produces a
drag fluctuations. The fluctuations in rD have it is large momentum transfers with q > 1/l that larger effect.
been measured in two different samples, and their give rise to the observed effect. In this case the Under the condition of large momentum
variance is seen to be similar in magnitude and T two electrons interact at a distance Dr that is transfer between the layers, fluctuations in G are
dependence, confirming the discrepancy with the smaller than the average impurity separation enhanced by a factor of g compared with (12),
theoretical prediction (12). and, therefore, it is the local electron properties similarly to the fluctuations of the LDOS, which
The expected fluctuations of the drag con- of the layers that determine e-e interaction. In can be estimated as dn2 ~ (n2/g)ln[max(Lϕ, LT)/l]
ductivity share the same origin as the UCF in (14), it is shown that the fluctuations of the (14). Also, the interaction in the ballistic regime
the conventional conductivity: coherent elec- local properties are larger compared to those of can be assumed to be constant, Dij ≈ −1/nkd,
tron transport over Lϕ in the layers before e-e the global properties that are responsible for the because q is limited by q ≤ 1/d. Finally, to
interaction between the layers (Fig. 1). There- drag in the diffusive case. average fluctuations of the drag over the sample
fore, we have compared the drag fluctuations A theoretical expression for the drag conduc- with size L, we should divide it into coherent
with the fluctuations seen in the single-layer tivity is obtained by means of a Kubo formula patches of size Lϕ that fluctuate independently
resistivity of the same structure (Fig. 3B, analysis (18–21) (see detailed description in the and thus decrease the total variance: 〈DsD2〉 =
inset), which have shown the usual behavior supporting text). For a qualitative estimate, three 〈DsD2(Lϕ)〉(Lϕ/L)2. If kBT > ET(Lϕ), fluctuations
(17). We estimate the expected variance of the factors have to be taken into account: (i) the are further averaged on the scale of ~ kBT, and
single-layer conductance fluctuations using interlayer matrix elements of the Coulomb therefore the variance is suppressed by an
the relation 〈Dsffixx2〉 = (e2/h)2(LT/L)2, where
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi interaction Dij; (ii) the phase space (the number additional factor of ET(Lϕ)/kBT. Combining the
LT = ℏD=kB T is the thermal length (17). of electron states available for scattering); and above arguments we find
This expression produces a value of 0.8 mS2, (iii) the electron-hole (e-h) asymmetry in both
which is in good agreement with the measured layers. Point (iii) takes into account that in a
value of 0.6 mS2. The typical “period” of the quantum system, the current is carried by both e4 ðkB TÞ2 l 4 L2ϕ
drag fluctuations (the correlation field, DBc) is electron-like (above the Fermi surface) and hole- 〈Ds2D 〉 ¼ N ; ð2Þ
g 2 ℏ2 ðkdÞ4 ET2 ðLϕ Þ d 4 L2
similar to that of the UCF (15), indicating that like (below the Fermi surface) excitations. If they
both depend upon the same Lϕ and have the were completely symmetric with respect to each
same quantum origin. other, then the current-carrying state of the active where N is a numerical coefficient.
To address the question of the discrepancy layer would have zero total momentum, and thus Compared to the diffusive situation (Eq. 1),
between the magnitude of drag fluctuations in no drag effect would be possible. The physical the fluctuations described by our model are
theory (12) and our observations, we stress that quantity that measures the degree of e-h greatly enhanced. Equations 2 and 1 differ
the theoretical prediction for the variance, Eq. 1, asymmetry is the nonlinear susceptibility G of because in the ballistic regime, electrons are not
was obtained under the assumption of diffusive the 2D layer. Theoretically, the drag conductivity scattered by impurities between events of e-e
motion of interacting electrons, with small is represented in terms of the nonlinear suscep- scattering. Large momentum transfers corre-
interlayer momentum transfers, q << 1/l. Because tibilities of each layer and dynamically screened spond to short distances, and thus in the ballistic
the layers are separated by a distance d, the e-e interlayer Coulomb interaction Dij(w) as sD º regime, drag measurements explore the local (as
interactions are screened at distances Dr > d. ∫dwD12(w)G2(w)D21(w)G1(w) (indices 1 and 2 opposed to averaged over the whole sample)
Therefore, in all regimes the maximum momen- correspond to the two layers) (18, 12). The e-h nonlinear susceptibility. This leads to the appear-
tum transfers are limited by q < 1/d. In the asymmetry appears in G as a derivative of the ance of three extra factors in Eq. 2: (i) the factor
diffusive regime, l < d, this relation also means density of states n and the diffusion coefficient D: l 4/d 4 [which is also present in the average drag in
that q < 1/l, that is, interlayer e-e interactions G º ∂(nD)/∂m, and it is this quantity that allows the ballistic regime (18)]; (ii) the phase space
occur at distances Dr > l and involve scattering drag fluctuations to exceed the average. Because factor T/ET (which appears because the interac-
by many impurities in the individual layers. Dn ~ g and the typical energy of electrons is the tion parameters Dij become energy independent);
In the opposite situation, l >> d, the trans- Fermi energy, EF, we have ∂(nD)/∂m ~ g/EF for and (iii) the extra factor g2 due to fluctuations of
ferred momenta will include both small and large the average drag. The typical energy scale for the the local nonlinear susceptibility. Local fluctua-
tions are enhanced because the random quantity
G becomes averaged over a small part of the
Fig. 4. The variance of the drag con- ensemble, allowing one to detect rare impurity
ductivity fluctuations (squares) plotted
configurations.
against temperature. The solid and
Our model not only explains the large
dashed lines are calculated from Eq. 2
with the diffusive and ballistic asymp- magnitude of the fluctuations but also predicts a
totes of tϕ, respectively. (Inset) tϕ ex- nontrivial temperature dependence of their mag-
tracted from the correlation magnetic nitude. The latter comes from the change in the
field of the single-layer fluctuations, temperature dependence of Lϕ (22): At low
plotted against temperature. temperatures, kBTt/ħ << 1, the usual result is
Lϕ º T −1/2, whereas for kBTt/ħ > 1, the temper-
ature dependence changes to Lϕ º T −1 (23).
Consequently, the temperature dependence of the
variance of the drag fluctuations is expected to
change from T −1 at low T to T −4 at high T. This
temperature dependence is very different from
the T dependence of drag fluctuations in the
diffusive regime, 〈DsD2〉 º T −1. To test the
prediction of Eq. 2, we have analyzed the T
dependence of 〈DsD2〉 (Fig. 4). The variance is

REPORTS
calculated in the limits of both the diffusive tϕ drag. One of the important developments in the 15. The details of the structures, measurements, and the
(solid line, tϕ−1 º T ) and ballistic tϕ (dashed field of Coulomb drag fluctuations can be their model are available as supporting material on Science
Online.
line, tϕ−1 º T 2), with N ≅ 10−4. In fitting the study in quantizing magnetic fields, including 16. M. Kellogg, J. P. Eisenstein, L. N. Pfeiffer, K. W. West,
drag variance, we have found tϕ to agree with the regimes of integer and fractional quantum- Solid State Commun. 123, 515 (2002).
theory to within a factor of 2 (15), which is typi- Hall effects. 17. B. L. Al’tshuler, P. A. Lee, R. A. Webb, Eds., Mesoscopic
cal of the agreement found in other experiments Phenomena in Solids (North-Holland, New York, 1991).
References and Notes 18. A. Kamenev, Y. Oreg, Phys. Rev. B 52, 7516 (1995).
on determining tϕ (24). (The single-layer values 1. M. B. Pogrebinskii, Sov. Phys. Semicond. 11, 372 19. L. Zheng, A. H. MacDonald, Phys. Rev. B 48, 8203 (1993).
of tϕ found from our analysis of the UCF agree 1977). 20. A.-P. Jauho, H. Smith, Phys. Rev. B 47, 4420 (1993).
with theory to within a factor of 1.5.) Thus, the 2. P. J. Price, Physica 117B, 750 (1983). 21. K. Flensberg, B. Y.-K. Hu, A.-P. Jauho, J. M. Kinaret,
temperature dependence of the observed drag 3. P. M. Solomon, P. J. Price, D. J. Frank, D. C. La Tulipe, Phys. Rev. B 52, 14761 (1995).
Phys. Rev. Lett. 63, 2508 (1989). 22. B. N. Narozhny, G. Zala, I. L. Aleiner, Phys. Rev. B 65,
fluctuations strongly supports the validity of our 4. T. J. Gramila, J. P. Eisenstein, A. H. MacDonald, 180202 (2002).
explanation. L. N. Pfeiffer, K. W. West, Phys. Rev. Lett. 66, 1216 23. B. L. Altshuler, A. G. Aronov, Electron-Electron
We have observed reproducible fluctuations (1991). Interactions in Disordered Systems, A. L. Efros, M. Pollak,
of the Coulomb drag and demonstrated that they 5. U. Sivan, P. M. Solomon, H. Shtrikman, Phys. Rev. Lett. Eds. (North-Holland, Amsterdam, 1985).
68, 1196 (1992). 24. C. W. J. Beenakker, H. van Houten, in Solid State Physics,
are an informative tool for studying wave
6. M. P. Lilly, J. P. Eisenstein, L. N. Pfeiffer, K. W. West, H. Ehrenreich, D. Turnbull, Eds. (Academic Press, San
properties of electrons in disordered materials, Phys. Rev. Lett. 80, 1714 (1998). Diego, 1991).
and the local properties in particular. Contrary to 7. J. G. S. Lok et al., Phys. Rev. B 63, 041305 (2001). 25. We thank I. L. Aleiner, M. Entin, I. L. Lerner, A. Kamenev,
UCF, which originate from quantum interfer- 8. A. G. Rojo, J. Phys. Condens. Matter 11, R31 (1999). and A. Stern for numerous helpful discussions.
ence, fluctuations of drag result from an inter- 9. D. Snoke, Science 298, 1368 (2002).
10. R. Pillarisetty et al., Phys. Rev. B 71, 115307
play of the interference and e-e interactions. (2005). Supporting Online Material
More theoretical and experimental work is re- 11. M. Yamamoto, M. Stopa, Y. Tokura, Y. Hirayama, S. Tarucha, www.sciencemag.org/cgi/content/full/316/5821/99/DC1
quired to study their manifestation in different Science 313, 204 (2006).
SOM Text
situations. For instance, similarly to the pre- 12. B. N. Narozhny, I. L. Aleiner, Phys. Rev. Lett. 84, 5383
(2000). Figs. S1 to S3
vious extensive studies of the evolution of UCF 13. N. A. Mortensen, K. Flensberg, A.-P. Jauho, Phys. Rev. B References
with increasing magnetic field, such experi- 65, 085317 (2002). 22 December 2006; accepted 6 March 2007
ments can be performed on the fluctuations of 14. I. V. Lerner, Phys. Lett. A 133, 253 (1988). 10.1126/science.1139227
Direct-Current Nanogenerator enhanced the conductivity of the electrode,

but also created a Schottky contact at the
interface with ZnO. The NWs were grown on
Driven by Ultrasonic Waves either GaN substrates (Fig. 1B) or sapphire
substrates that were covered by a thin layer of
ZnO film (14, 15), which served as a common
Xudong Wang, Jinhui Song, Jin Liu, Zhong Lin Wang*
electrode for directly connecting the NWs with
an external circuit. The density of the NWs was
We have developed a nanowire nanogenerator that is driven by an ultrasonic wave to produce
~10/mm2, and the height and diameter were
continuous direct-current output. The nanogenerator was fabricated with vertically aligned zinc
~1.0 mm and ~40 nm, respectively. The top
oxide nanowire arrays that were placed beneath a zigzag metal electrode with a small gap. The
electrode was composed of parallel zigzag
wave drives the electrode up and down to bend and/or vibrate the nanowires. A piezoelectric-
trenches fabricated on a (001) orientated Si
semiconducting coupling process converts mechanical energy into electricity. The zigzag electrode
wafer (16) and coated with a thin layer of Pt
acts as an array of parallel integrated metal tips that simultaneously and continuously create,
(200 nm in thickness) (Fig. 1C). The electrode
collect, and output electricity from all of the nanowires. The approach presents an adaptable,
was placed above the NW arrays and manipu-
mobile, and cost-effective technology for harvesting energy from the environment, and it offers a
lated by a probe station under an optical micro-
potential solution for powering nanodevices and nanosystems.
scope to achieve precise positioning; the
spacing was controlled by soft-polymer stripes
he operation of nanodevices fabricated mechanical energy into electric energy by between the electrode and the NWs at the four
T with one-dimensional nanostructures

[such as nanowires, nanotubes, and nano-
belts (1–8)] usually requires very low power
piezoelectric zinc oxide (ZnO) nanowire (NW)
arrays (13). By deflecting the aligned NWs with
a conductive atomic force microscopy (AFM)
sides. The resistance of the nanogenerator was
monitored during the assembly process to en-
sure a reasonable contact between the NWs and
(9–11), which is provided by an external source, tip in contact mode, the mechanical energy the electrode by tuning the thickness of the
such as a battery that may have to be replaced or created by the deflection force was converted polymer film. Then the assembled device was
recharged regularly. The reliance on an external into electricity with the use of the piezoelectric sealed at the edges to prevent the penetration of
power source may present a limitation for these properties of the NWs. To improve the power liquid. A cross-sectional image of the packaged
systems. Various approaches have been devel- generation capabilities of the system, it is NW arrays is shown in Fig. 1D; it displays a
oped for energy scavenging with applications in necessary to replace the AFM tip with a simpler “lip/teeth” relationship between the NWs and
wireless electronics, such as thermoelectric, source of mechanical energy that can actuate all the electrode. Some NWs are in direct contact
piezoelectric thin-film, and vibrational canti- the NWs simultaneously and continuously. We with the top electrode, but some are located
levers (12). We have recently demonstrated an solved these problems by using ultrasonic between the teeth of the electrode. The inclined
innovative approach for converting nanoscale waves to drive the motion of the NWs, leading NWs in the scanning electron microscopy (SEM)
to the production of a continuous current. image were primarily caused by the cross sec-
School of Materials Science and Engineering, Georgia The experimental setup is schematically tioning of the packaged device. The packaged
Institute of Technology, Atlanta, GA 30332-0245, USA. shown in Fig. 1A. An array of aligned ZnO device was supported by a metal plate that was
*To whom correspondence should be addressed. E-mail: NWs was covered by a zigzag Si electrode direct in contact with water contained in the
[email protected] coated with Pt. The Pt coating not only cavity of an ultrasonic generator. The operation

REPORTS
frequency of the ultrasonic wave was ~41 kHz. deformation occurs. This discharging process, of the voltage is consistent with the mechanism
The output current and voltage were measured if substantial, may also contribute to the presented in Fig. 2C. The signal-to-noise ratio of
by an external circuit at room temperature. measured current. In each of the four cases the current is substantially better than that of the
The experimental design relies on a unique described in Fig. 2, A to C, all of the currents output voltage for the following reasons: Be-
coupling between piezoelectric and semicon- are added up in the same phase. cause the resistance of the current meter (ideally,
ducting properties of the aligned ZnO NWs An equivalent electric circuit is shown in zero) plus Rc (20 to 30 ohms) was only ~1/1000
(13, 17, 18). The asymmetric piezoelectric po- Fig. 2D to illustrate the measurements and out- of Rw when the current was measured, the cur-
tential across the width of a ZnO NW and the puts of the nanogenerator. The NWs producing rent generated by the nanogenerator can be safely
Schottky contact between the metal electrode current in the nanogenerator are equivalent to a assumed to be bypassing Rw; the current path is
and the NW are the two key processes for voltage source Vs plus an inner resistance Ri that indicated by a solid blue curve in Fig. 2D, so
creating, separating, preserving, accumulating, also contains the contact resistance between the the measured current is IA ≈ Vs /(Rc + Ri).
and outputting the charges [see figure 3 in (13)]. active NWs and the electrode. On the other However, because Rw was very much smaller
A top electrode is designed to achieve the cou- hand, there are a lot of NWs that are in contact than the inner resistance of a voltage meter
pling process and to replace the role played by with the electrode but cannot be bent or move (ideally, infinity) when the voltage was mea-
the AFM tip, and its zigzag trenches act as an freely; thus, they do not actively participate in sured, a loop was formed between the power-
array of aligned AFM tips (fig. S2 and Fig. 2A). the current generation, but they do provide a generating portion of the system and Rw, as
When subject to the excitation of an ultrasonic path for conducting current. These NWs are shown by a solid pink curve in Fig. 2D. In this
wave, the zigzag electrode can move down and simply represented by a resistance Rw that is case, the current is IV and the measured voltage
push the NW, which leads to lateral deflection parallel to the portion that generates power. A V is that across the power-generating portion,
of NW I. This, in turn, creates a strain field resistance Rc is introduced to represent the V ≈ –VsRw/(Ri + Rw). During the ultrasonic
across the width of NW I, with the NW’s outer contact resistance between the electrode and vibration, for the unstable contacts between the
surface being in tensile strain and its inner the external measurement circuit. The capaci- NWs and the electrodes, IA is affected by the
surface in compressive strain. The inversion of tance in the system is ignored in the circuit in instability of Vs and Ri (but mainly by Vs
strain across the NW results in an inversion of order to simplify the discussion about dc because Rc is a constant), and V is affected by
piezoelectric field Ez along the NW (fig. S1), measurement. the instability of Vs, Ri, and Rw. As a result, V
which produces a piezoelectric-potential inver- The current and voltage outputs of the has about two times the noise level of IA, con-
sion from V – (negative) to V + (positive) across nanogenerator are shown in Fig. 2, E and F, re- sistent with the observations displayed in Fig. 2,
the NW (Fig. 2B). When the electrode makes spectively, with the ultrasonic wave being turned E and F. On the other hand, because the
contact with the stretched surface of the NW, on and off regularly. A jump of ~0.15 nA was voltages created by all of the NWs are in par-
which has a positive piezoelectric potential, the observed when the ultrasonic wave was turned allel, the output voltage is effectively the volt-
Pt metal–ZnO semiconductor interface is a on, and the current immediately fell back to age created by one NW; thus, it appears
reversely biased Schottky barrier, resulting in the baseline once the ultrasonic wave was turned relatively unstable and with a larger noise level
little current flowing across the interface. This is off. Correspondingly, the voltage signal ex- than that of IA (Fig. 2F). Furthermore, the output
the process of creating, separating, preserving, hibited a similar on and off trend but with a voltage is naturally smaller than that created by
and accumulating charges (13). With further negative output of ~–0.7 mV. The negative sign deflecting a NW by AFM, because it is limited
pushing by the electrode, the bent NW I will
reach the other side of the adjacent tooth of the Fig. 1. Nanogenerators
zigzag electrode (Fig. 2C). In such a case, the driven by an ultrasonic
electrode is also in contact with the compressed wave. (A) Schematic dia-
side of the NW, where the metal/semiconductor gram showing the design
interface is a forward-biased Schottky barrier, and structure of the nano-
resulting in a sudden increase in the output generator. Aligned ZnO NWs
electric current flowing from the top electrode grown on a solid/polymer
into the NW. This is the discharge process. substrate are covered by a
Figure 2, A to C, shows four possible con- zigzag electrode. The sub-
figurations of contact between a NW and the strate and the electrode are
zigzag electrode. Analogous to the situation de- directly connected to an
scribed for NW I, the same processes apply to external load. (B) Aligned
the charge output from NW II. NW III is chosen ZnO NWs grown on a GaN
to elaborate on the vibration/resonance induced substrate. The gold catalyst
by an ultrasonic wave. When the compressive particles used for the growth
side of NW III is in contact with the electrode, had been mostly vaporized;
the same discharge process as that for NW I thus, the final NWs were
purely ZnO with flat top
occurs, resulting in the flow of current from the
ends. (C) Zigzag trenched
electrode into the NW (Fig. 2C). NW IV, which
electrode fabricated by the
is short in height, is forced (without bending) standard etching technique
into compressive strain by the electrode. In such after being coated with 200
a case, the piezoelectric voltage created at the nm of Pt. The surface fea-
top of the NW is negative [see figure 4 in (13)]. tures are due to nonuniform
Thus, across the electrode–ZnO interface, a etching. (D) Cross-sectional
positively biased Schottky barrier is formed; SEM image of the nano-
hence, the electrons can flow freely across the generator, which is com-
interface. As a result, electrons flow from the posed of aligned NWs and
grounded substrate electrode into the NW and the zigzag electrode. (Inset)
then into the top zigzag electrode as the A typical NW that is forced by the electrode to bend.

REPORTS
by the smaller degree of deflection amplitude of strated a cost-effective prototype technology for from the output power of the nanogenerator.
the NW, as induced by an ultrasonic wave in fabricating the nanogenerator. (ii) We have in- From our previous study [see supporting online
comparison to that induced by the AFM tip tegrated an array of tips into a zigzag electrode material for (13)], the NW deformed by AFM
(13). Finally, the output current is a sum of the for the simultaneous creation, collection, and produced an electric energy of DEAFM ~ 0.01 fJ
currents produced by many NWs. Therefore, the output of electricity generated by many NWs, for each cycle of discharge, which lasted for
current signal is more stable and continuous, establishing the principle for raising the output 0.1 ms; thus, the power generated by one NW
and it has been used to characterize the power. (iii) We have achieved a continuous and was DWAFM ~ 0.1 pW. In the current experimen-
performance of the dc nanogenerator in this fairly stable dc output with this system. The tal design, the vibrational amplitude of the NW
study. The resistance of the entire nanogenerator principle demonstrated here has set a platform for was much smaller than that of the NW directly
was also measured with and without turning harvesting energy from the environment to power deflected by an AFM tip; thus, the output
on the ultrasonic wave (Fig. 2G). The resist- in vivo biosensors, wireless and remote sensors, voltage was ~1 mV, which is about 5 to 10
ance remained very stable at R = 3.560 ± 0.005 and nanorobots, and it has also established the times smaller than that received when AFM was
kilohms. This measurement indicates that the basis for building zero-power force/pressure used as the deformation tool (13). In this case,
jump in current could not be due to the variation sensors. the output power of a NW, as driven by ultra-
in resistance, as caused by the vibration of the The number of NWs that was effective for sonic wave, would be DWwave = 1 to 4 fW. The
NWs (19), suggesting that the current signal producing output electricity can be estimated output-power volume density per NW is ~1 to
presented in Fig. 2E was created by the
nanogenerator.
The output electricity of the nanogenerator is
continuous and reasonably stable. A continuous
output current is generated when the ultrasonic
wave is turned on, and the current disappears
when the wave is turned off (Fig. 3A). The
output current is in the nanoampere range. The
current signal shows no direct coupling with
the frequency of the ultrasonic wave, because the
wave frequency is ~80 times smaller than the
resonance frequency of the NWs (~3 MHz) (20).
The size of the nanogenerator is ~2 mm2 in
effective substrate surface area. The number of
NWs that were actively contributing to the
observed output current is estimated to be 250
to 1000 in the current experimental design. The
nanogenerator worked continuously for an
extended period of time of beyond 1 hour
(Fig. 3B).
The experimental design [as presented in
(Fig. 1)] has been tested in comparison to the
experiments conducted using different materials
or configurations. Using the design shown in
Fig. 1A, simply by replacing a ZnO NW array
with an array of carbon nanotubes (CNTs), no
jump in current was observed when the ultra-
sonic wave was turned on (Fig. 4A). This is
because the CNTs are not piezoelectric. In a
system with a ZnO NW array but in which the
top electrode was replaced with a flat, thin Pt
film that totally covered the tips of the NWs
(Fig. 4B), no jump in output current was
observed. This is because the design does not
follow the mechanism of the nanogenerator (fig. Fig. 2. The mechanism of the nanogenerator driven by an ultrasonic wave. (A) Schematic
S1) (13). A clear jump was observed only when illustration of the zigzag electrode and the four types of NW configurations. (B) Piezoelectric
the top electrode was in a zigzag shape and potential created across NWs I and II under the push or deflection of the electrode, as driven by the
when ZnO NWs were present (Fig. 4C). These ultrasonic wave, but without the flow of current because of the reversely biased Schottky barrier at
the electrode/NW interface. NW III is in vibration under the stimulation of the ultrasonic wave. NW
experiments may rule out possible contributions
IV is in compressive strain without bending. (C) Once the NWs touch the surface of the adjacent
from electronic noise and/or measurement error
teeth, the Schottky barrier at the electrode/NW interface becomes forward-biased, and piezoelectric
or artifacts in producing the output current, and discharge occurs, resulting in the observation of current flow in the external circuit. (D) Equivalent
they consistently support the process proposed circuit of the nanogenerator and the setup for measuring the output current I, output voltage V,
in Fig. 2, A to C, for piezoelectric NWs. and resistance R. (E to G) I, V, and R measured with the connections shown in (D), respectively,
In comparison to our previous work (13), when the ultrasonic wave was turned on and off purposely. The baseline of the current signal was
our current work has achieved three major ob- produced by the electronic measurement system and the interference from the ultrasonic-wave
jectives: (i) We have replaced the expensive and source. Because the voltages created by all of the NWs are in parallel, the output voltage is
sophisticated AFM tip with ultrasonic waves/ effectively the voltage created by one NW; thus, it appears to be relatively unstable and with a
vibrations to induce the elastic deformation and larger noise level than that of IA. A pixel averaging was applied for (F). The inset in (G) is an
vibration of the NWs, and we have demon- enlargement of the resistance before and after the wave was turned on.

REPORTS
4 W/cm3, which is more than two orders of bution on the substrate surface; thus, the arrays matching the design of the electrode
magnitude higher than that produced by a vi- output current was rather small in the present (fig. S2), and an improved packaging technol-
brational microgenerator (21). The output power design. In addition, some of the NWs directly ogy to keep a precise control on the spacing
of the nanogenerator fabricated with a sub- pushed the electrode at the top edges/apexes and alignment between the electrode and the
strate of area = 2 mm2 is Wwave = IAV ≈ 1 pW of the zigzag trenches, preventing the other NW arrays. If the area taken by each metal tip
(Fig. 2, C and D). Therefore, the number of NWs from reaching and contacting the elec- is 0.5 by 0.5 mm (fig. S2), the grown density
NWs that was active for producing electricity trode to produce electricity. These technical of the NWs is ~109/cm2. If one NW produces
in Fig. 2E was N = Wwave/DWwave ≈ 250 to difficulties could be overcome by an opti- 10 fW of power by optimizing its size and
1000 NWs. As limited by the multiple contacts mized design to improve nanogenerator effi- shape, the output power per unit of area could
between the NWs and the electrode in the ciency. For example, nanogenerator efficiency be 10 mW/cm2. The power used to operate a
present design (Fig. 1D), the large majority of could be improved with the use of patterned- device fabricated with one NW or nanotube is
the NWs did not produce electricity because tip arrays as the electrode, the designed and ~10 nW (9–11); thus, the nanogenerator built
of their nonuniformity in height and distri- patterned growth of high-quality uniform NW with the NWs grown on an area of 1 cm2
could operate up to 1000 of such nanodevices,
Fig. 3. Continuous dc output of the based on our current study. We anticipate that
nanogenerator, as characterized by the performance will be improved by two to
the current signal. (A) Reproducible three orders of magnitude with improved
and highly repeatable current output design and optimization of the nanogenerator
of the nanogenerator when the ultra- (22–23).
sonic wave was turned on and off. (B)
Continuous current output of the
nanogenerator for an extended peri- 1. F. Patolsky et al., Science 313, 1100 (2006).
od of time. The data are displayed 2. Y. Li, F. Qian, J. Xiang, C. M. Lieber, Mater. Today 9, 18
after they were corrected for the (2006).
background introduced by electronic 3. F. Patolsky, C. M. Lieber, Mater. Today 8, 20 (2005).
drift. The baseline of the current sig- 4. P. Pauzauskie, P. Yang, Mater. Today 9, 36 (2006).
nal was produced by the electronic 5. A. Javey, J. Guo, Q. Wang, M. Lundstrom, H. J. Dai,
Nature 424, 654 (2003).
measurement system and the in- 6. J. W. R. Hsu et al., Nano Lett. 5, 83 (2005).
terference from the ultrasonic-wave 7. Z. R. R. Tian et al., Nat. Mater. 2, 821 (2003).
source. The size of the nanogenerator 8. Z. W. Pan, Z. R. Dai, Z. L. Wang, Science 291, 1947
is ~2 mm2 in effective substrate sur- (2001).
face area. 9. Y. Huang et al., Science 294, 1313 (2001).
10. A. Bachtold, P. Hadley, T. Nakanishi, C. Dekker, Science
294, 1317 (2001).
11. J. Chen et al., Science 310, 1171 (2005).
12. J. A. Paradiso, T. Starner, IEEE Pervasive Comput. 4, 18
(2005).
13. Z. L. Wang, J. Song, Science 312, 242 (2006).
14. X. D. Wang et al., J. Am. Chem. Soc. 127, 7920 (2005).
15. X. D. Wang, C. J. Summers, Z. L. Wang, Nano Lett. 4, 423
(2004).
16. J. Frühauf, S. Krönert, Microsyst. Technol. 11, 1287
(2005).
17. J. H. Song, J. Zhou, J. Liu, Z. L. Wang, Nano Lett. 6, 1656
(2006).
18. X. D. Wang et al., Nano Lett. 6, 2768 (2006).
19. Among the 200,000 NWs on the substrate surface, less
than 0.5% were actively creating current. A large
percentage of the NWs were serving as current paths
between the top and bottom electrodes (Fig. 1D). The
resistance introduced by the active NWs was less than
2% of the total resistance, when considering the NWs
that were not in contact with the electrodes; thus, it could
not produce the large jump in current as observed
experimentally.
20. X. D. Bai, P. X. Gao, Z. L. Wang, E. G. Wang, Appl. Phys.
Lett. 82, 4806 (2003).
21. P. D. Mitcheson et al., J. Microelectromech. Syst. 13, 429
(2004).
22. P. X. Gao, J. H. Song, J. Liu, Z. L. Wang, Adv. Mater. 19,
67 (2007).
23. Z. L. Wang, MRS Bull. 32, 109 (2007).
24. We thank the Emory Georgia Tech Center of Cancer
Nanotechnology Excellence funded by NIH, the Defense
Advanced Research Projects Agency, and NSF (grant DMR
Fig. 4. Output of the nanogenerator with different materials and design configurations. The 9733160) for support. We also thank C. M. Lieber and
designs and true-device SEM images are shown on the right-hand side, and the corresponding P. Gao for many stimulating discussions.
current curve is on the left-hand side. (A) Nanogenerator based on arrays of CNTs with a zigzag top Supporting Online Material
electrode, showing no jump in current when the ultrasonic wave was turned on. (B) Nanogenerator www.sciencemag.org/cgi/content/full/316/5821/102/DC1
based on arrays of ZnO NWs but with a flat top electrode, showing no jump in current while SOM Text
stimulated by an ultrasonic wave. (C) Nanogenerator based on arrays of ZnO NWs with a zigzag top Figs. S1 and S2
electrode, showing a large jump in current when the ultrasonic wave was turned on. The baseline in 28 December 2006; accepted 1 March 2007
the current signal was produced by the measurement electronics. 10.1126/science.1139366

REPORTS
equaled or exceeded that of full-length Vip1
A Conserved Family of (fig. S3A). With full-length or truncated Vip1
proteins, we observed that IP6 kinase activity
Enzymes That Phosphorylate was pH dependent, with optimum activity oc-
curring at pH 6.2 (fig. S3B). These data dem-
Inositol Hexakisphosphate onstrate that the ATP-grasp domain of Vip1 is
sufficient to encode the IP6 kinase activity.
To determine whether Vip1 functioned to
Sashidhar Mulugu,1* Wenli Bai,1 Peter C. Fridy,1 Robert J. Bastidas,1 James C. Otto,1 produce IP7 in cells, we analyzed IP production
D. Eric Dollins,1 Timothy A. Haystead,2 Anthony A. Ribeiro,3 John D. York1† in yeast mutants. The predominant IPs observed
in kcs1 ddp1 null cells (kD dD) are IP6 and PP-IP5
Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular (Fig. 1C) (16). Deletion of Vip1 in this back-
roles remain an active area of study. We report a previously uncharacterized class of inositol ground (vip1 kcs1 ddp1 triple null, kD dD vD)
pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of resulted in the ablation of PP-IP5 synthesis and
actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode increased the amount of IP6 (Fig. 1C). Production
inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in of PP-IP5 could be restored by introduction of a
kinase activity led to defects in cell growth, morphology, and interactions with ARP complex plasmid encoding the VIP1 promoter and cod-
members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity ing sequence (Fig. 1C). We also identified that
through metabolism of IP6. Vip1 was required to synthesize PP-IP4b in cells
(fig. S3C). These observations are consistent
ctivation of phosphoinositide-specific enriched by a factor of more than 3000. Partially with Vip1 functioning as a cellular inositol pyro-
A phospholipase C (PLC) generates an

ensemble of intracellular inositol phos-
phate (IP) second messengers involved in diverse
purified peak fractions were separated by SDS–
polyacrylamide gel electrophoresis, and individ-
ual protein bands were excised and subjected
phosphate synthase that phosphorylates both IP6
and I(1,2,3,4,5)P5 and indicate that Vip1 encodes
activities previously designated as Ids1 (16) and
cellular processes (1–3). Among these is inositol to microsequence analysis. Numerous proteins Ips1 (15).
1,4,5-trisphosphate, which functions as a regu- were identified, so we obtained and purified To identify catalytic residues in the kinase
lator of calcium release (4) and as a precursor to individual candidate proteins from the tandem- domain of Vip1, we used structure-based thread-
more highly phosphorylated IP molecules, in- affinity purification (TAP)–tagged yeast collec- ing software [Protein Homology/analogY Rec-
cluding inositol tetrakis- (IP4), pentakis- (IP5), tion and tested each for kinase activity (17). Of ognition Engine (PHYRE) server, www.sbg.bio.
hexakis- (IP6), and inositol pyrophosphates the 40 proteins examined, only the purified TAP- ic.ac.uk/phyre]. Within the ATP-grasp domain,
(PP-IPs) (5). The PP-IPs have more phosphates tagged Vip1 protein showed IP6 kinase activity we identified aspartate 487 as a putative active-
than IP6 (6, 7) and were chemically identified as (Fig. 1A). We inserted the entire coding sequence site catalytic residue (fig. S1B) and, indeed,
diphosphoryl inositol species (7, 8). Cloning of of Vip1 into a glutathione S-transferase (GST) substitution with alanine (Vip1D487A) rendered
the inositol pyrophosphate synthase designated expression plasmid and produced the protein in the mutant kinase defective in vivo (Fig. 1C) and
IP6 kinase (IP6K in metazoans and Kcs1 in bacteria (which lack endogenous IP6 kinase in vitro. Similar analysis of the acid-phosphatase
budding yeast) (9, 10) provided a molecular basis activity). Purified recombinant GST-Vip1 also domain identified histidine 548 and 993 as in-
for the synthesis of PP-IP5 (also known as IP7 or had IP6 kinase activity (Fig. 1A). These data in- variant residues (fig. S2B). Substitution of H548
InsP7) through the coupled activities of PLC and dicate that Vip1 functions as an intrinsic inositol in Vip1 with alanine (Vip1H548A) did not appear
inositol phosphate kinases (IPKs) (11–13). Loss pyrophosphate synthase or IP6 kinase. to alter IP6 kinase activity in vitro (fig. S3A).
of IP6K results in pleiotropic cellular defects The protein information annotation for Vip1 Reintroduction of Vip1H548A into kcs1 ddp1 vip1
including aberrant DNA recombination, vacuolar (Saccharomyces Genome Database), and its mutant yeast cells restored PP-IP5 and IP6 levels
morphology, gene expression, chemotaxis, os- Schizosaccharomyces pombe ortholog Asp1 to those observed in kcs1 ddp1 mutant cells (Fig.
motic stress, protein phosphorylation, and telo- (18), identified two regions with relevance to 1C). These data identify a key catalytic residue
mere length (5, 14). Cells also have a second IP6 inositol signaling enzymes (Fig. 1B): a “rimK” or for kinase activity and are consistent with Vip1
kinase detected in kcs1 deficient cells whose adenosine triphosphate (ATP)–grasp superfamily functioning as a regulator of IP6 and PP-IP5 lev-
molecular identity has yet to be reported (15, 16). domain SSF56059 (residues 200 to 525) and a els in cells.
We used a biochemical approach to purify histidine acid-phosphatase or phytase PF00328 To determine the molecular structure of the
and clone the remaining IP6 kinase activity. To domain (residues 530 to 1025). The ATP-grasp Vip1 kinase reaction product, samples were
reduce interference by contaminating IP6 kinase fold is present in certain inositol signaling kinases subjected to nuclear magnetic resonance (NMR)
and PP-IP pyrophosphatase activities, initial (19), and the acid-phosphatase fold is present in analysis. Comparison of proton-decoupled phos-
protein extracts were prepared from kcs1D microbial and fungal phytase enzymes that phorus NMR spectra of high-performance liquid
ddp1D–deficient cells and then fractionated on a hydrolyze IP6 to release inorganic phosphate chromatography (HPLC)–purified IP6 standard
series of ion-exchange chromatography columns (Pi) and IPs (20). Sequence alignments of Vip1 or (Fig. 2A) or PP-IP5 Vip1 product (Fig. 2B)
(tables S1 and S2). IP6 kinase activity was Asp1 genes revealed that orthologs having both revealed the appearance of five singlet peaks of
amino- and carboxyl-terminal domains were monophosphates and two upfield phosphorus-
1
present in all eukaryotic genomes from yeast to phosphorus–coupled doublets corresponding to
Howard Hughes Medical Institute, Department of Pharma-
man (figs. S1 and S2). Thus, the Vip1 and Asp1 the b- and a-phosphates of a pyrophosphate
cology and Cancer Biology, Duke University Medical Center,
DUMC 3813, Durham, NC 27710, USA. 2Department of Phar- proteins define a distinct class of IP6 kinase with moiety. The spectrum observed for the Vip1
macology and Cancer Biology, Duke University Medical a conserved dual-domain structure. PP-IP5 product is nearly identical to the reported
Center, Durham, NC 27710, USA. 3Department of Biochem- Our analyses indicated that the kinase do- spectrum for 4-PP-I(1,2,3,5,6)P5 or its enantiomer
istry, NMR Center, Duke University Medical Center, Durham, main of Vip1 might reside within the amino- 6-PP-I(1,2,3,4,5)P5 purified from Dictyostelium
NC 27710, USA.
terminal portion of the protein. Therefore we (21). Two-dimensional 1H– 31P–NMR correlation
*Present address: Bharathidasan University, Tiruchirappalli,
620 024, India.
expressed a deletion mutant GST-Vip11-535 analysis was reported, allowing for unequivocal
†To whom correspondence should be addressed. E-mail: (residues 1 to 535) and performed kinase reac- assignment of the pyrophosphate (21). We per-
[email protected] tions. GST-Vip11-535 had IP6 kinase activity that formed NMR analysis on the PP-IP5 product of

REPORTS
Fig. 1. Characterization of inositol hexakisphosphate IP6 kinase
enzyme, Vip1. (A) IP6 kinase activity of purified yeast TAP-Vip1 and
the production of IP7 (left). Dose-dependent (ng of protein added)
activity of recombinant GST-Vip1 (middle) and sensitivity of IP7 and
IP8 products of TAP-Kcs1 and GST-Vip1 to the diphospho-inositol
phosphatase GST-Ddp1 (right). (B) Schematic of the dual-domain
structure of Vip1 and Asp1. Evolutionarily conserved residues from
the ATP-grasp (IP6K) and putative acid phosphatase domains from
either budding or fission yeast proteins are shown. (C) Function of
Vip1 to regulate PP-IP5 and IP6 levels in cells. Loss of vip1 in kcs1
ddp1–null yeast ablated PP-IP5 production and increased IP6 by a
factor of 2, as monitored by HPLC analysis of 3H-inositol–labeled
mutant yeast strains (10 mM AP increased linearly to 1.7 M AP over
12 min, followed by an isocratic flow at 1.7 M AP for 48 min) and in-
line radioactivity detection. Synthesis of PP-IP5 was restored in cells
expressing either GFP-tagged full-length Vip1 (kDdDvD + pVip1) or
kinase-only (kDdDvD + pvip1H548A), but not kinase-deficient protein
(kD dD vD + pvip1D487A).
Fig. 2. Identification of Vip1 PP-IP5 product by 31P NMR analysis and kinase active forms of Vip1 showed robust activity and were able to convert
inositol heptakisphosphate kinase activity. Proton-decoupled phosphorus 5-PP-I(1,2,3,4,6)P5 to 4,5-PP2-IP4 (IP8) at neutral pH. Recombinant human
NMR spectrum of 2 mg of purified (A) IP6 standard, (B) Vip1 product PP-IP5, GST IP6K fusion protein was also examined and was found to
or (C) human IP6K1 PP-IP5 product. All analyses were performed on phosphorylate 4-PP-IP5 to 4,5-PP2-IP4 (right). For simplicity, we have
HPLC-purified samples at pH 5.8. Structures of relevant IP6 and IP7 assigned the product of the Vip1 reactions to be the D-4 species. Given our
species are shown: 4-PP-I(1,2,3,5,6)P5 and 6-PP-I(1,2,3,4,5)P5 are non- analysis, we cannot exclude that these may also be the enantiomer species
superimposable mirror images (enantiomers). Resonances have been harboring a D-6 pyrophosphate. (E) Schematic diagram of IP6 metabolism
assigned based on similarity to previous reports (21, 23). P, phosphate; pathway in Saccharomyces cerevisiae and production of IP7 and IP8
PP, pyrophosphate. (D) Characterization of Vip1 and human IP6K IP7 through the actions of Vip1 and Kcs1. Kcs1 functions as a D-5 kinase that
kinase activity. Purified recombinant GST-Vip1 fusion proteins (ng of may phosphorylate IP6 and 4-PP-IP5 substrates. Vip1 functions as an IP6
protein added are shown) were incubated with radiolabeled 32PP-IPs, ATP kinase to generate 4- or 6-PP-IP5 and phosphorylates the IP7 product of
at pH 6.2, and reactants were visualized after separation by Kcs1 to form IP8. Ddp1 is an inositol pyrophosphate phosphatase that
polyethyleneimine cellulose–thin-layer chromatography (left). All three dephosphorylates IP7 or IP8 to IP6.

REPORTS
human IP6K, which is reported to have a similar 4,5-PP2-I(1,2,3,6)P4 or 5,6-PP2-I(1,2,3,4)P4, or trol (p3X), full-length protein (pASP1), or proteins
activity to that of Kcs1 (14, 16, 22). The spectrum both (Fig. 2D). Similarly, when we incubated with mutations in the kinase (pasp1D333A) or
of IP7 produced from an HPLC-purified IP6K radiolabeled 4-PP-IP5 or 6-PP-IP5, ATP and re- putative acid-phosphatase (pasp1H397A) domains.
reaction was distinct from Vip1 product (Fig. 2C). combinant human GST-IP6K, we observed con- The temperature-sensitive growth and morpho-
Tentative assignment was again made based on version to PP2-IP4 product (Fig. 2D). These data logical defects exhibited by asp1 null cells were
this spectrum’s appearing to be similar to that indicate that Vip1 and IP6K (Kcs1) are the rescued by low-level expression (medium with
reported for 5-PP-I(1,2,3,4,6)P5 (23). Overall, our enzymes required for the synthesis of IP8 or thiamine) of either pASP1 or pasp1H397A, con-
NMR analysis confirms that the IP7 produced by PP2-IP4 from IP6 (Fig. 2E). sistent with a role for IP6 kinase in mediating
Vip1 is structurally distinct from that produced Clues into possible functional roles for the IP6 these phenotypes (Fig. 3, A and B). When these
by the IP6K class of proteins, and that Vip1 kinase activity of Vip1 came from studies of the strains were induced to high-level expression
appears to harbor D-4 or D-6 kinase activity, or S. pombe ortholog, Asp1, which may participate (medium lacking thiamine), the kinase-only
both (fig. S3A). in actin cytoskeleton and cellular morphology protein, pasp1H397A, prevented cell growth at
Given the specificities of the Vip1 and through the regulation of actin-related protein both normal and high temperatures (Fig. 3, A and
IP6K (Kcs1) kinases, we sought to examine their (Arp) complexes (18). Arp2/3 multiprotein com- B). Cells overexpressing kinase-only protein
roles as IP7 kinases capable of generating IP8 plexes are involved in cellular signaling–dependent underwent rapid lysis when analyzed by light
(PP2-IP4), which has been reported in various control of nucleation of branched-actin-filament microscopy (Fig. 3B). These data indicate that
eukaryotic cells (7, 8, 21, 24, 25). In IP6 kinase networks required for regulating cell motility and loss of IP6 kinase activity accounts for asp1
reactions with both GST-Vip1 and purified endocytosis (26, 27). Activation of Arp2/3 re- temperature sensitivity and morphological de-
TAP-Kcs1, we observed the formation of IP7 quires additional nucleation-promoting factors fects and that too much IP6 kinase is cytotoxic.
and IP8, which were hydrolyzed to IP6 in the such as Wiskott-Aldrich syndrome protein (WASP) To probe whether ARP complex function was
presence of the inositol pyrophosphatase Ddp1 (28, 29). required for the observed effects, we examined
(Fig. 1A). Incubation of 5-PP-IP5 along with We therefore examined whether these pheno- the expression of wild-type or mutant ASP1
ATP and recombinant GST-Vip1, GST-Vip11-535, types depended on IP6 kinase activity. Wild-type proteins in a cold-sensitive mutant Arp3 strain
or GST-Vip1H548A protein, resulted in the produc- and asp1 null strains were transformed with (arp3 cl). The toxicity induced by overexpression
tion of PP2-IP4, consistent with Vip1 function- thiamine-regulated (nmt1 promoter) GFP-tagged of IP6 kinase-only activity (pasp1H397A) was
ing as a 5-PP-IP5 kinase capable of generating expression plasmids encoding empty vector con- dependent on a functional Arp3 protein, as
Fig. 3. A role for IP6

kinase activity in regu-
lating cell morphology,
growth, and ARP com-
plex function. (A) Asp1
IP6 kinase activity regu-
lates cell growth of fission
yeast. Loss of IP6 kinase
inhibited growth at in-
creased temperatures (left
two panels). Overproduc-
tion of Asp1 IP6 kinase-
only (pasp1H397A) protein
(without thiamine) was
toxic to cell growth at nor-
mal and elevated temper-
atures (middle and right
two panels). Appropriate
strains were serially di-
luted, spotted onto solid
medium, and grown at
the indicated tempera-
ture. (B) IP6 kinase activity
regulates cell morpholo-
gy, vacuole biogenesis,
and cell lysis. Cells lack-
ing Asp1 kinase activity
(asp1 D + 3X or asp1 D +
aspD333A) had rounded,
vacuolated, and irregular
cell morphology as compared with kinase-competent cells (asp1 D + ASP1, Expression of Asp1 IP6 kinase-dead protein (pasp1D333A) results in Arp3-
asp1 D + asp1H397A). Overproduction of IP6 kinase-only protein (without dependent defects in cell-wall deposition, as shown by microscopic analysis of
thiamine) resulted in the inhibition of cell growth and promoted cellular lysis cells stained by calcofluor white. Staining of cells overexpressing full-length
(ASP1 + pasp1H397A). (Insets) Zoomed images. (C) Toxicity induced by IP6 Asp1 show normal cell wall synthesis regardless of Arp3 function. (E)
kinase-only overexpression and suppression by loss of arp3. Cells overproducing Requirement of Vip1 IP6 kinase activity for genetic interactions with ARP
asp1H397A fail to grow at 30°C (Arp3+); however, they were able to grow when complex member, Las17. A temperature-induced growth defect was observed
shifted to a cold temperature (arp3–). In contrast, when arp3 function is lost upon loss of both Vip1 and Las17 (vDlD, top), the WASP component of the ARP
(19°C), cells overexpressing Asp1 IP6 kinase-dead protein (pasp1D333A) were complex. This synthetic growth defect was rescued in the vip1 las17 double-
unable to grow, and this defect is rescued by restoring Arp3 function (30°C). (D) mutant strain by restoring Vip1 IP6 kinase activity (bottom).

REPORTS
judged by growth at 19°C (arp3–) versus death dual-domain structure of the Vip1 class of en- 20. E. J. Mullaney, A. H. Ullah, Biochem. Biophys.
at 30°C (Arp3+) (Fig. 3C). Furthermore, when zymes is evolutionarily conserved, raising the Res. Commun. 312, 179 (2003).
21. T. Laussmann, R. Eujen, C. M. Weisshuhn, U. Thiel,
Arp3 function was lost, the overexpression of possibility that there may be interplay between G. Vogel, Biochem. J. 315, 715 (1996).
kinase-deficient Asp1 was now lethal, as deter- these two regions of the proteins to control 22. A. Saiardi, C. Sciambi, J. M. McCaffery, B. Wendland,
mined by a failure of pasp1D333A-transformed intracellular signaling pathways. S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 99, 14206
cells to grow under induced conditions at 19°C (2002).
23. T. Laussmann et al., FEBS Lett. 426, 145 (1998).
(Fig. 3C). Analysis of these cells using the cell- References and Notes 24. M. C. Glennon, S. B. Shears, Biochem. J. 293, 583 (1993).
wall stain calcofluor white (CFW) revealed that 1. R. F. Irvine, M. J. Schell, Nat. Rev. Mol. Cell Biol. 2, 327 25. S. B. Shears, N. Ali, A. Craxton, M. E. Bembenek,
overproduction of kinase-deficient Asp1 resulted (2001). J. Biol. Chem. 270, 10489 (1995).
in aberrant appearance of cell-wall material at the 2. P. W. Majerus, Annu. Rev. Biochem. 61, 225 (1992). 26. E. D. Goley, M. D. Welch, Nat. Rev. Mol. Cell Biol. 7, 713
3. S. B. Shears, Biochim. Biophys. Acta 1436, 49 (2006).
growing ends rather than at the septum midline (1998). 27. T. D. Pollard, G. G. Borisy, Cell 112, 453 (2003).
(Fig. 3D). We also examined interactions be- 4. M. J. Berridge, R. F. Irvine, Nature 341, 197 (1989). 28. H. N. Higgs, T. D. Pollard, J. Cell Biol. 150, 1311 (2000).
tween Vip1 and Las17 (the yeast WASP protein) 5. J. D. York, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 29. R. Rohatgi et al., Cell 97, 221 (1999).
in budding yeast cell growth. Deletion of vip1 1761, 552 (2006). 30. Y.-S. Lee, S. Mulugu, J. D. York, E. K. O'Shea, Science
6. G. N. Europe-Finner, B. Gammon, P. C. Newell, Biochem. 316, 109 (2007).
alone did not cause temperature sensitivity; Biophys. Res. Commun. 181, 191 (1991). 31. We thank members of the York laboratory for helpful
however, when combined with a loss in las17, 7. F. S. Menniti, R. N. Miller, J. W. Putney Jr., S. B. Shears, discussions; J. Rudolph for assistance with kinetic
the double mutant was growth-compromised at J. Biol. Chem. 268, 3850 (1993). analysis; K. Gould (Vanderbilt University) for published
high temperatures (Fig. 3E). The temperature- 8. L. Stephens et al., J. Biol. Chem. 268, 4009 (1993). strains; M. Tsieh (Cell Signals, OH) for IP standards for
9. A. Saiardi, H. Erdjument-Bromage, A. M. Snowman, NMR; and E. O’Shea and Y.-S. Lee (Harvard University)
sensitive growth was rescued by restoring Vip1 P. Tempst, S. H. Snyder, Curr. Biol. 9, 1323 (1999). for sharing unpublished data, helpful discussions, and
IP6 kinase activity (Fig. 3E). Overall, our func- 10. A. Saiardi, R. Bhandari, A. C. Resnick, A. M. Snowman, comments on the manuscript. This work was supported by
tional analyses indicate that IP6 kinase activity S. H. Snyder, Science 306, 2101 (2004). funds from the Howard Hughes Medical Institute ( J.D.Y. ),
of Asp1 and Vip1 is required for maintaining 11. A. R. Odom, A. Stahlberg, S. R. Wente, J. D. York, Science and from NIH grants R01-HL-55672 ( J.D.Y.), R33-DK-
287, 2026 (2000). 070272 ( J.D.Y. ), 2-P30-CA14236-3 (T.A.H.), and NCI
cellular integrity, temperature-dependent growth,
12. A. Saiardi, J. J. Caffrey, S. H. Snyder, S. B. Shears, P30-CA-14236 (A.A.R.). Instrumentation in Duke NMR
rod-shape morphology, and genetic interactions FEBS Lett. 468, 28 (2000). Center was funded by NSF, NIH, the North Carolina
with ARP complex components. 13. J. D. York, A. R. Odom, R. Murphy, E. B. Ives, S. R. Wente, Biotechnology Center, and Duke University. The authors
We identified Vip1 and Asp1 as members Science 285, 96 (1999). have no conflicting financial interests.
of a class of IP6 kinase that is responsible for 14. M. Bennett, S. M. Onnebo, C. Azevedo, A. Saiardi,
Cell. Mol. Life Sci. 63, 552 (2006). Supporting Online Material
producing signaling molecules 4-PP-IP5 or 6-PP- 15. A. M. Seeds, R. J. Bastidas, J. D. York, J. Biol. Chem. 280, www.sciencemag.org/cgi/content/full/316/5821/106/DC1
IP5, or both, that regulate cell function and yeast 27654 (2005). Materials and Methods
phosphate-responsive signaling (30). We show 16. S. J. York, B. N. Armbruster, P. Greenwell, T. D. Petes, Figs. S1 to S3
that the Vip1 and Kcs1 (IP6K) kinases generate J. D. York, J. Biol. Chem. 280, 4264 (2005). Tables S1 and S2
17. S. Ghaemmaghami et al., Nature 425, 737 (2003). References
distinct inositol pyrophosphate products and that 18. A. Feoktistova, D. McCollum, R. Ohi, K. L. Gould, Genetics
both can function as IP7 kinases, together 152, 895 (1999). 20 December 2006; accepted 5 March 2007
capable of converting IP6 to IP8 (PP2-IP4). The 19. N. V. Grishin, J. Mol. Biol. 291, 239 (1999). 10.1126/science.1139099
Regulation of a Cyclin-CDK-CDK of Pho80-Pho85 activity in response to Pi-

limitation (6).
We used a biochemical strategy to identi-
Inhibitor Complex by fy cellular components that influence Pho80-
Pho85-Pho81 activity. The Pho80-Pho85-Pho81
Inositol Pyrophosphates complex immunopurified from cells grown in
Pi-rich conditions actively phosphorylated Pho4
in vitro, whereas the complex isolated from
Young-Sam Lee,1 Sashidhar Mulugu,2 John D. York,2 Erin K. O’Shea1* Pi-starved cells was less active (6) (Fig. 1A). To
identify regulators of Pho80-Pho85-Pho81, we
In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin–cyclin- added extracts from cells grown in Pi-rich and
dependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes Pi-starved conditions to active (from Pi-rich)
involved in nutrient homeostasis. We isolated myo-D-inositol heptakisphosphate (IP7) as a cellular and inactive (from Pi-starved) immunopurified
component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP7 is necessary for Pho80-Pho85-Pho81 complexes and performed
Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP7 kinase assays with recombinant Pho4 serving as
increased upon phosphate starvation, and yeast mutants defective in IP7 production failed to the substrate. Extracts from Pi-starved cells effi-
inhibit Pho80-Pho85 in response to phosphate starvation. These observations reveal regulation of a ciently inactivated Pho80-Pho85-Pho81, whereas
cyclin-CDK complex by a metabolite and suggest that a complex metabolic network mediates the Pi-rich cell extract had little effect (Fig. 1B).
signaling of phosphate availability. When the extract of Pi-starved cells was frac-
tionated into fractions of large (>3 kD) and
n response to nutrient limitation, cells in- grown in medium that is rich in Pi, Pho80-Pho85
I crease transcription of starvation response

genes. Many proteins that function in nu-
trient response systems have been identified,
is active and phosphorylates the transcription
factor Pho4 (4), resulting in its export from the
nucleus (5). Upon Pi starvation, Pho80-Pho85
1
Howard Hughes Medical Institute, Faculty of Arts and
Sciences Center for Systems Biology, Department of
Molecular and Cellular Biology, Harvard University, 7
Divinity Avenue, Cambridge, MA 02138, USA. 2Howard
but the molecular details of their regulation are kinase activity is decreased, leading to nuclear
Hughes Medical Institute, Department of Pharmacology
not well understood (1). The Saccharomyces accumulation of Pho4 and transcription of PHO and Cancer Biology, Duke University Medical Center,
cerevisiae Pho80-Pho85 cyclin-CDK complex is genes. In vivo, the Pho81 CDK inhibitor (CKI) Durham, NC 27710, USA.
a key regulator in the phosphate (Pi)–responsive is bound to Pho80-Pho85 regardless of Pi *To whom correspondence should be addressed. E-mail:
(PHO) signaling pathway (2, 3). When cells are conditions and is required for the inhibition [email protected]

REPORTS
small (<3 kD) molecular size, most inhibitory method (8) and tested its effect on the Pho80- mately 3% or less of the IP6 concentration (Fig. 3)
activity was found in the fractions of small Pho85 complex. When the Pho80-Pho85-Pho81 (12). However, upon Pi limitation, IP7 levels
molecular size. This activity had little effect on complex that had been immunopurified from Pi- increased to more than 20% of the levels of IP6
Pho80-Pho85 purified from pho81D cells (Fig. rich cells was treated with synthetic IP7, kinase (Fig. 3). This increase in abundance of IP7 is not
1C), indicating that, as is true in vivo (6), the activity was inhibited with an apparent median due to Pho4-dependent transcriptional activation
inhibitory activity requires Pho81 for its function. inhibitory concentration (IC50) of ~50 mM (Fig. (fig. S3) (7), and the time course and depen-
To determine the identity of the inhibitory 2A). IP7 had little or no effect on the kinase dence of the IP7 increase on extracellular Pi are
activity, we enriched it through several fraction- purified from pho81D cells, suggesting that IP7- comparable to those observed for PHO pathway
ation steps and characterized its properties (fig. mediated inactivation of Pho80-Pho85 requires regulation (fig. S3). Assuming that the IP6 con-
S1) (7). The low–molecular weight inhibitor Pho81. Inactivation of Pho80-Pho85-Pho81 by centration is ~100 mM in cells (9) and that the
contained acid-labile Pi (8), and characteriza- IP7 appears to be specific, given that inositol IP6 concentration does not change in response to
tion by nuclear magnetic resonance and mass and several inositol derivatives did not affect Pi limitation, the concentration of IP7 may reach
spectrometry suggested that it was inositol kinase activity (Fig. 2B). ~30 mM during Pi limitation (fig. S3), a value
heptakisphosphate (9), also known as diphospho- We next tested whether IP7-mediated in- similar to the apparent IC50 measured in vitro
myo-D-inositol pentakisphosphate, PP-InsP5, activation of Pho80-Pho85-Pho81 is direct or (Fig. 2).
PP-IP5, InsP7, or IP7 (Fig. 1D). whether it requires other factors from yeast. Nei- Many gene products involved in yeast ino-
Inositol polyphosphates are signaling mol- ther synthetic IP7 nor the activity from Pi-starved sitol polyphosphate metabolism are known (Fig.
ecules found in all eukaryotic cells. They are cell extract could inactivate recombinant Pho80- 4A) (9). Kcs1 is a homolog of IP6 kinases in
produced through the action of a conserved set Pho85 purified from Escherichia coli (Fig. 2C). other organisms (19) and is a major IP6 kinase
of inositol polyphosphate kinases from inositol Similarly, addition of recombinant Pho81 mini- in yeast (12). However, IP7 is still observable in
1,4,5-trisphosphate (IP3) (9). IP3, which regu- mum domain (Pho81-MD) (18), a fragment that kcs1D cells (16, 20), suggesting that another IP6
lates Ca2+ release from the endoplasmic reticu- functionally substitutes for full-length Pho81 in kinase exists. A second yeast IP6 kinase, Vip1,
lum (10), can be sequentially phosphorylated to vivo, did not cause inhibition of recombinant generates an isomer of IP7 distinct from that
generate IP4, IP5, inositol hexakisphosphate Pho80-Pho85 in vitro, even in two- to threefold
(IP6), and inositol pyrophosphates such as IP7. molar excess (Fig. 2C). Because Pho81-MD
Loss-of-function studies have implicated IP7 in binds to Pho80-Pho85 (apparent dissociation con-
recombination (11), vacuole function (12), gene stant Kd ~ 170 nM, fig. S2), we concluded that
expression, endo- and exocytosis (13, 14), os- neither Pho81-MD nor IP7 is sufficient for the
motic stress (12), and telomere length control inactivation of Pho80-Pho85. However, when
(15, 16), but it is unclear whether IP7 directly both IP7 and Pho81-MD were added, Pho80-
regulates these processes. One well-characterized Pho85–Pho81-MD was inactivated (Fig. 2C).
role for IP7 is the regulation of chemotaxis in Therefore, IP7 directly inactivates Pho80-Pho85-
Dictyostelium, in which intracellular concentra- Pho81 and this inactivation requires Pho81.
tions of IP7 increase upon chemoattractant addi- To test whether the cellular concentration
tion and IP7 competes with phosphatidylinositol of IP7 increases when cells are starved of Pi,
3,4,5-trisphosphate (PIP3) to bind to proteins we monitored inositol polyphosphate levels in
containing a pleckstrin homology domain (17). cells labeled with [3H]-inositol and grown in
To test whether the inhibitory activity was Pi-rich or Pi-limiting conditions (12). In Pi-rich
IP7, we synthesized IP7 by a nonenzymatic conditions, the IP7 concentration was approxi-
Fig. 2. Inhibition of Pho80-Pho85-Pho81 by syn-

thetic IP7. (A) Synthetic IP7 was serially diluted and
added to immunopurified Pho80-Pho85 isolated
Fig. 1. Purification of a cellular component that inactivates Pho80-Pho85 in a Pho81-dependent from wild-type (WT) or pho81D cells that had been
manner. (A) Phosphorylation of recombinant Pho4 by Pho80-Pho85-Pho81 immunopurified from grown in Pi-rich medium (Fig. 1C). Kinase activity
Pi-rich (High) and Pi-starved (Low) cells expressing Pho80–hemagglutinin tag (3HA). (B) Effects of cell was then measured. (B) Serial dilutions of inositol,
fractions on Pho80-Pho85 activity. Extracts from cells (3 × 109 cells per ml extract) grown in high-Pi myo-D-inositol 1,4,5-triphosphate (IP3), IP6, or IP7
medium (High Pi extract) or starved for phosphate (Low Pi extract) were added either directly or after were added to Pho80-Pho85-Pho81 immunopuri-
filtration (molecular weight cut-off 3 kD; Low and High indicate low– and high–molecular weight fied from wild-type cells grown in Pi-rich medium,
fractions, respectively) to Pho80-Pho85-Pho81 immunopurified from Pi-rich cells, and kinase assays and kinase activity was measured. (C) Synthetic
were done as in (A). U, untreated Pho80-Pho85-Pho81 from the same gel. (C) The low-Pi low–molecular IP7 was added to recombinant Pho80-Pho85 or
weight fraction in (B) was serially diluted and added to Pho80-Pho85 immunopurified from wild-type Pho80-Pho85–Pho81-MD complex (50 nM Pho80-
(WT) and pho81D cells that had been grown in Pi-rich medium. Kinase assays were then performed Pho85 and 250 nM Pho81-MD) and incubated
using recombinant Pho4 as a substrate. (D) Structures of relevant IP7 isomers. 4-PP-IP5 and 6-PP-IP5 20 min at room temperature. Kinase activity was
are mirror images of each other. P, phosphate; PP, pyrophosphate. then measured.

REPORTS
produced by the mammalian IP6 kinase and manner, whereas extract from vip1D cells did regulation of Pho80-Pho85-Pho81. We moni-
likely distinct from that produced by Kcs1 (21). not, suggesting that Vip1 mediates synthesis of tored the activity of Pho80-Pho85 by measuring
To determine which IP6 kinase is involved in the IP7 relevant to the PHO system. the subcellular localization of its substrate, Pho4
PHO pathway, we assayed the ability of cellular To further assess the physiological role of (Fig. 4C). In the ipk1D and vip1D strains, Pho4
extracts from Pi-starved wild-type, kcs1D, and Vip1-generated IP7 in Pi signaling in vivo, we was localized to the cytoplasm even in condi-
vip1D cells to inactivate Pho80-Pho85-Pho81 in determined whether yeast strains with mutations tions of Pi starvation, suggesting that the kinase
vitro (Fig. 4B). The extract from kcs1D cells in- in enzymes involved in inositol polyphosphate cannot be inactivated. In the kcs1D strain, Pho4
activated Pho80-Pho85 in a Pho81-dependent metabolism (Fig. 4A) (9) had defects in the became localized to the nucleus upon Pi star-
vation, consistent with the model that Kcs1 does
Fig. 3. Increase in IP7 upon Pi star- not generate the IP7 that inhibits Pho80-Pho85-
vation. Strong anion exchange chro- Pho81. A fraction of the kcs1D cells grown in
matograms of extracts derived from high-Pi medium exhibited nuclear localization
wild-type (WT) cells labeled with of Pho4; the cause of this phenotype is not un-
[3H]-inositol grown in medium con- derstood (22, 23). Pho4 was constitutively nu-
taining high Pi (10 mM) or low Pi clear in ddp1D cells (16), which have high
(5 mM). Cells were starved for 2 hours levels of IP7, but was constitutively cytoplasmic
at 30°C before lysis and analysis. in kcs1Dvip1Dddp1D and arg82D cells, which
CPM, counts per minute. cannot produce IP7 and IP5, respectively (fig.
S4A). Pho4 was constitutively nuclear in a
vip1Dpho80D strain and was constitutively cy-
toplasmic in ddp1Dpho81D cells, suggesting that
IP7 acts upstream of Pho80-Pho85-Pho81 (fig.
S4B). Additionally, IP7 enzymatically synthesized
by recombinant Vip1 inhibited Pho80-Pho85–
Pho81-MD in a manner dependent on Pho81-
MD, but IP7 enzymatically synthesized by the
human homolog of Kcs1, hIP6K1, did not (Fig.
4D; apparent IC50 ~ 50 mM; fig. S4C) (21).
We conclude that IP7 produced by Vip1 acts
as a signaling molecule that controls the yeast
PHO signaling pathway: IP7 concentrations in-
crease in response to Pi limitation, and IP7 is re-
quired for inhibition of Pho80-Pho85-Pho81 in
vivo and in vitro. This work illuminates the mo-
lecular mechanisms underlying previously reported
genetic interactions between inositol polyphos-
phate metabolism and the phosphate-responsive
signaling pathway (22, 24). Our work reveals
regulation of a cyclin-CDK-CDK inhibitor com-
plex by a small molecule, IP7. It is unclear whether
IP7 acts by allosterically regulating Pho80-Pho85-
Pho81 or by covalently modifying one or more
of these components, as it has been demonstrated
to do for other yeast and mammalian proteins (25).
IP7 may be a more general signal for nutrient
limitation given that Pi starvation of budding
yeast and nutrient limitation of Dictyostelium
(17) leads to elevation of IP7 levels. In addition,
considering that a mammalian IP6 kinase is known
to stimulate Pi uptake (26), a phenotype similar to
Pi-starved yeast cells, it is possible that connec-
tions between phosphate metabolism and inositol
polyphosphate are conserved in other organisms.
1. W. A. Wilson, P. J. Roach, Cell 111, 155 (2002).
Fig. 4. Effect of disrupted inositol polyphosphate metabolism on Pho80-Pho85 activity, monitored by 2. M. E. Lenburg, E. K. O’Shea, Trends Biochem. Sci. 21,
383 (1996).
Pho4 localization. (A) Schematic diagram showing inositol polyphosphate metabolism in S. cerevisiae. 3. A. S. Carroll, E. K. O’Shea, Trends Biochem. Sci. 27, 87
(B) Wild-type (WT), kcs1D, or vip1D cell extracts from Pi-starved cells were added to immunopurified (2002).
Pho80-Pho85 from Pi-rich wild-type (top) or pho81D (bottom) cells, and kinase activity was measured. 4. A. Kaffman, I. Herskowitz, R. Tjian, E. K. O’Shea, Science
U, untreated. (C) Fluorescence microscopic (100× magnification) analysis of wild-type, ipk1D, kcs1D, 263, 1153 (1994).
vip1D, or ddp1D cells expressing Pho4–green fluorescent protein, grown in medium containing high 5. E. M. O'Neill, A. Kaffman, E. R. Jolly, E. K. O’Shea,
Science 271, 209 (1996).
(top) or low (bottom) concentrations of Pi. (D) Isomer specificity of the IP7-mediated Pho80-Pho85
6. K. R. Schneider, R. L. Smith, E. K. O’Shea, Science 266,
inactivation. Pho80-Pho85 (8 nM) in the presence (top) and the absence (bottom) of His6–Pho81-MD 122 (1994).
(250 nM) was incubated with IP7 isomers synthesized with recombinant Vip1 (top) and human IP6 7. Materials and methods are available as supporting
kinase 1 (bottom), and kinase activity was measured. material on Science Online.

REPORTS
8. G. W. Mayr, T. Radenberg, U. Thiel, G. Vogel, 18. S. Huang, D. A. Jeffery, M. D. Anthony, E. K. O’Shea, comments on the manuscript; and D. Kahne and his
L. R. Stephens, Carbohydr. Res. 234, 247 (1992). Mol. Cell. Biol. 21, 6695 (2001). laboratory members for access to equipment. This work was
9. R. F. Irvine, M. J. Schell, Nat. Rev. Mol. Cell Biol. 2, 327 19. A. Saiardi, H. Erdjument-Bromage, A. M. Snowman, supported by NIH R01 GM051377 (E.K.O.), DK070272
(2001). P. Tempst, S. H. Snyder, Curr. Biol. 9, 1323 (1999). (J.D.Y.), and HL055672 (J.D.Y.); the David and Lucile
10. H. Streb, R. F. Irvine, M. J. Berridge, I. Schulz, Nature 20. A. M. Seeds, R. J. Bastidas, J. D. York, J. Biol. Chem. 280, Packard Foundation (E.K.O.); and the Howard Hughes
306, 67 (1983). 27654 (2005). Medical Institute (E.K.O. and J.D.Y.). The authors have no
11. H. R. Luo et al., Biochemistry 41, 2509 (2002). 21. S. Mulugu et al., Science 316, 106 (2007). conflicting financial interest.
12. E. Dubois et al., J. Biol. Chem. 277, 23755 (2002). 22. C. Auesukaree, H. Tochio, M. Shirakawa, Y. Kaneko,
13. W. Ye, N. Ali, M. E. Bembenek, S. B. Shears, E. M. Lafer, S. Harashima, J. Biol. Chem. 280, 25127 (2005). Supporting Online Material
J. Biol. Chem. 270, 1564 (1995). 23. S. Huang, E. K. O’Shea, Genetics 169, 1859 (2005). www.sciencemag.org/cgi/content/full/316/5821/109/DC1
14. B. Fleischer et al., J. Biol. Chem. 269, 17826 24. J. S. Flick, J. Thorner, Genetics 148, 33 (1998). Materials and Methods
(1994). 25. A. Saiardi, R. Bhandari, A. C. Resnick, A. M. Snowman, SOM Text
15. A. Saiardi, A. C. Resnick, A. M. Snowman, B. Wendland, S. H. Snyder, Science 306, 2101 (2004). Figs. S1 to S5
S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 102, 1911 26. M. J. Schell et al., FEBS Lett. 461, 169 (1999). Tables S1 and S2
(2005). 27. We thank Y. Liu for preparation of strains; P. Fridy for References
16. S. J. York, B. N. Armbruster, P. Greenwell, T. D. Petes, reagents; B. Stern, H. Kim, C. Leimkuhler, and D. Schwarz
J. D. York, J. Biol. Chem. 280, 4264 (2005). for comments on the manuscript; members of the J.D.Y. 19 December 2006; accepted 22 February 2007
17. H. R. Luo et al., Cell 114, 559 (2003). laboratory for unpublished data, helpful discussions, and 10.1126/science.1139080
A Single IGF1 Allele Is a Major and four small PWDs and in nine dogs from
small and giant breeds (<9 and >30 kg average
breed mass, respectively). We then measured the
Determinant of Small Size in Dogs association between 116 SNPs and skeletal size
in a sample of 463 PWDs and identified a single
peak within 300 kb of the insulin-like growth
Nathan B. Sutter,1 Carlos D. Bustamante,2 Kevin Chase,3 Melissa M. Gray,4 Keyan Zhao,5 factor 1 gene (IGF1) (Fig. 1A), confirming the
Lan Zhu,2 Badri Padhukasahasram,2 Eric Karlins,1 Sean Davis,1 Paul G. Jones,6 FH2295 QTL. IGF1 is an excellent candidate
Pascale Quignon,1 Gary S. Johnson,7 Heidi G. Parker,1 Neale Fretwell,6 Dana S. Mosher,1 gene known to influence body size in both mice
Dennis F. Lawler,8 Ebenezer Satyaraj,8 Magnus Nordborg,5 K. Gordon Lark,3 and humans (15–17).
Robert K. Wayne,4 Elaine A. Ostrander1* Haplotype analysis of 20 SNPs spanning
IGF1 further supported a role for the locus in
The domestic dog exhibits greater diversity in body size than any other terrestrial vertebrate. We determining body size. We observed that 889 of
used a strategy that exploits the breed structure of dogs to investigate the genetic basis of size. the 926 (96%) PWD chromosomes carry one of
First, through a genome-wide scan, we identified a major quantitative trait locus (QTL) on just two haplotypes, termed B and I. Dogs
chromosome 15 influencing size variation within a single breed. Second, we examined genetic homozygous for haplotype B have a smaller
variation in the 15-megabase interval surrounding the QTL in small and giant breeds and found median skeletal size [Fig. 1B; P < 3.27 × 10−7,
marked evidence for a selective sweep spanning a single gene (IGF1), encoding insulin-like growth analysis of variance (ANOVA)] and mass (fig.
factor 1. A single IGF1 single-nucleotide polymorphism haplotype is common to all small breeds S1) than dogs homozygous for I and a lower
and nearly absent from giant breeds, suggesting that the same causal sequence variant is a major level of IGF1 protein in blood serum (Fig. 1C;
contributor to body size in all small dogs. P < 9.34 × 10−4, ANOVA). In PWDs, 15% of
the variance in skeletal size is explained by the
ize variation in the domestic dog is majority of dog breeds originated over the past IGF1 haplotype. Linkage disequilibrium around
S extreme and surpasses that of all other liv-

ing and extinct species in the dog family,
Canidae (1, 2). However, the genetic origin of
few hundred years (11). Understanding the
genetic basis for the rapid generation of ex-
treme size variability in the dog would provide
IGF1 in PWDs is too extensive to allow fine
mapping, presumably because of the breed’s
recent origin and small population size (18, 19).
this diversity is obscure. Explanations include critical tests of alternative genetic mechanisms However, if a mutation at IGF1 in general un-
increased recombination or mutation rates (3, 4), and insight into how evolutionary diversifica- derlies genetic differences in size among dog
a unique role of short repeat loci near genes (3), tion in size could occur rapidly during adaptive breeds, comparison of breeds of different sizes
expansion of specific short interspersed nuclear radiations (12). that have distinct genealogical histories may
elements (5), regulatory gene variation (6, 7), or a To investigate the genetic basis for size allow fine mapping of the mutation. Moreover,
readily altered developmental program (1, 6). variation in dogs and understand how change because size has been the target of strong se-
The domestic dog descended from the gray wolf in size might occur rapidly in dogs and other lection by dog breeders, we would expect to
at least 15,000 years ago (8–10), but the vast canids, we first initiated sequence-based marker find a signature of selection surrounding the
discovery across a 15–megabase (Mb) interval QTL in breeds of extreme small or giant size.
1
National Human Genome Research Institute, Building 50,
on chromosome 15 in the Portuguese water dog To test these predictions, we surveyed genet-
Room 5349, 50 South Drive MSC 8000, Bethesda, MD (PWD), a breed that is allowed large variation in ic variation for the same 116 SNPs in 526 dogs
20892–8000, USA. 2Department of Biological Statistics and skeletal size by the American Kennel Club (13). from 23 small (<9 kg) and 20 giant (>30 kg)
Computational Biology, Cornell University, Ithaca, NY 14850, Previously, based on 92 radiographic skeletal breeds. To obtain an empirical distribution of
USA. 3Department of Biology, University of Utah, Salt Lake
City, UT 84112, USA. 4Department of Ecology and Environmen-
measurements for size and shape, we found that our association mapping test statistics, we also
tal Biology, University of California, Los Angeles, CA 90095, two QTL (FH2017 at 37.9 Mb and FH2295 at surveyed variation in 83 SNPs with no known
USA. 5Department of Molecular and Computational Biology, 43.5 Mb) within this region were strongly association to body size on canine chromosomes
University of Southern California, Los Angeles, CA 90089, USA. associated with body size in 463 PWDs from a 1, 2, 3, 34, and 37. These data were analyzed
6
The WALTHAM Centre for Pet Nutrition, Waltham on the Wolds, well-characterized extended pedigree (13, 14). first to determine if intense artificial selection on
Leicestershire, LE14 4RT, UK. 7Department of Veterinary Patho-
biology, University of Missouri, Columbia, MO 65211, USA. We discovered 302 single-nucleotide polymor- body size has resulted in a “selective sweep”
8
Nestle Research Center (NRC-STL), St. Louis, MO 63164, USA. phisms (SNPs) and 34 insertion/deletion poly- (20), reducing variability and increasing allele
*To whom correspondence should be addressed. E-mail: morphisms by sequencing 338 polymerase frequency divergence near IGF1. We found a
[email protected] chain reaction (PCR) amplicons in four large marked reduction in marker heterozygosity and

REPORTS
Fig. 1. Relationships of increased genetic differentiation between small
skeletal size, SNP markers, and giant dogs centered on IGF1 (Fig. 2). Spe-
IGF1 haplotype, and serum cifically, near IGF1, average heterozygosity in
levels of the IGF1 protein in small dogs is only 25% of that in large dogs,
PWDs. (A) A mixed-model genetic differentiation (FST, where ST represents
test for association between subpopulation) peaks significantly at 0.6, and
size and genotype. The as- overall heterozygosity is sharply reduced (Fig.
sociation of three genotype 2B) (figs. S2 to S5). Together, these results
categories (A1A1, A1A2, and suggest that a narrow and precisely defined
A2A2) with skeletal size mea- genomic region holds the variant (or variants)
surements was calculated with
responsible for small size in a disparate set of
the use of all pairwise coef-
small dog breeds.
ficients of consanguinity for
376 dogs. Each point repre- We next tested for association between each
sents a single SNP position SNP and average breed size (Fig. 3A). The null
on canine chromosome 15 hypothesis of no association between body size
and negative log P value and marker frequency across breeds is rejected
for the association statistic. (Bonferroni-correct P value < 0.05) for 25
(B) PWD IGF1 haplotypes contiguous SNPs defining an 84-kb interval
and mean skeletal size. spanning the same region that shows evidence
Haplotypes were inferred of a selective sweep (chromosome 15 base pairs
for 20 markers spanning 44,199,850 to 44,284,186) (Figs. 2 and 3A).
the IGF1 gene (chromo- The Mann-Whitney U statistic provides a uni-
some 15: 44,212,792 to form distribution of P values for 83 genomic
44,278,140, CanFam1). Out control markers (fig. S6). Similarly, P values
of the 720 chromosomes from Fisher’s exact test of association across
with successful inference, individuals were smaller than 10−100 in the 84-kb
96% carry one of just two interval; although these P values are clearly
haplotypes, B and I, iden- biased by confounding population structure (fig.
tical to haplotypes inferred S6), as evidenced by the 83 genomic control
for small and giant dogs, markers [for which the minimum P value was
respectively (Fig. 3). Data are graphed as a histogram for each genotype: B/B (closed triangle, black line), B/I
10–20 (fig. S7)], the result is significant.
(open square, dashed line), and I/I (closed circle, gray line). (C) Serum levels of IGF1 protein (ng/ml) as a
Analysis of specific breed haplotypes shows
function of haplotype. Serum levels of IGF1 protein were assayed in 31 PWDs carrying haplotypes B and I. Box
plots show the median (center line in box), first and third quartile (box ends), and maximum and minimum that a 20-SNP haplotype spanning IGF1 is
values (whiskers) obtained for each category: homozygous B/B (n = 15), heterozygous B/I (n = 7), and shared by all 14 sampled small dog breeds
homozygous I/I (n = 9). (Fig. 3, B and C) and is identical to haplotype B
in small PWDs. This haplotype was observed in
only three of the nine giant breeds because most
giant dogs carry one or both of two distinct
haplotypes: F and I. SNP 5, located at base pair
position 44,228,468 (Fig. 3B), is the best can-
didate for being proximate to the causative
mutation for the following reasons: (i) It dis-
tinguishes haplotypes A, B, and C, associated
with small body size, from haplotypes D to L,
which are common in large breeds; (ii) an an-
cestral recombination graph suggests an ab-
sence of recombination between SNPs 4 and
5 (fig. S8); and (iii) marker analysis in the
golden jackal and gray wolf indicates that the
SNP 5 A allele of small breeds is the derived
condition (fig. S9) (table S1). To further as-
sess the association between body size and the
SNP 5 A allele, we genotyped six tagging
SNPs that distinguish all major IGF1 haplo-
types in a set of 3241 dogs from 143 breeds
Fig. 2. Signatures of recent selection on the IGF1 locus across 22 small and giant dog breeds. (A) (Fig. 4) (table S2). The frequency of the SNP
Heterozygosity ratio (HR) for small versus giant dogs. (B) Genetic differentiation (FST) for small versus giant 5 A allele is strongly negatively correlated with
dogs. For both (A) and (B), a sliding 10-SNP window across IGF1 was used. Dashed lines delimit the 95% breed average mass across this large sample
confidence intervals based on nonparametric bootstrap resampling. The IGF1 gene interval is indicated of breeds (Fig. 4, Spearman’s rank correlation
above the graphs as a red box drawn to scale. (C) Observed heterozygosity (HObs) of SNPs near IGF1 typed coefficient r = −0.773; P < 2.2 × 10–16; likeli-
in small breeds (<9 kg) and giant breeds (>30 kg). Small breeds have a reduction in observed hood ratio test = 2882.3, c2df=1 < 2 × 10−16,
heterozygosity compared with that of giant breeds. Red and blue points are average observed logistic regression of allele frequency on body
heterozygosity in small and giant breeds, respectively. Dashed lines are locally weighted scatterplot size). A strong negative correlation remains when
smoothing (LOWESS) best fit to the data. The IGF1 gene is shown as a black bar with exons indicated by the 22 breeds used to discover SNP 5 are removed
vertical lines. from the analysis (r = –0.729; P < 2.2 × 10−16,

REPORTS
Spearman’s rank correlation). Exceptions, such correlate with body size in toy, miniature, and in the PWDs (P < 1.4 × 10−6, ANOVA) and the
as the large Rottweiler or small whippet breeds, standard poodles (24). These studies did not small and giant breeds (P < 2.2 × 10−14, chi–
may carry compensatory mutations at other compare IGF1 genetic variation with differences square test; table S6). All of these variations
size QTL or recombinants that could aid fine in serum IGF1 protein concentrations; we ob- were in strong linkage disequilibrium and there-
mapping at IGF1. Our results show that a served that PWDs carrying the B haplotype of fore a causative variant could not be definitively
single IGF1 haplotype is common to a large the IGF1 gene have significantly lower serum identified by this approach. Given the difficulty
sample of small dogs and strongly imply that levels of IGF1 (Fig. 1C). of developing inbred dog lines segregating small
the same causal variant (or variants) is a ma- Finally, to identify possible causative var- size, future studies will focus on using knock-in
jor influence on the phenotype of diminished iants, we sequenced the exons of IGF1 in a mice to explore the effect of these variants on
body size. panel of nine small and giant dogs and found phenotypes.
The IGF1 gene is a strong genetic determi- only one variation in coding sequence, a syn- Our findings suggest that a single IGF1
nant of body size across mammals; mice onymous SNP in exon 3 [chromosome 15 base haplotype substantially contributes to size vari-
genetically deficient in IGF1 are just 60% nor- pair position 44,226,324, Canis familiaris ge- ation in the domestic dog. Because our sample
mal birth weight (15), and a human with a nome assembly 1 (CanFam1)]. Extensive rese- includes small breeds that are distantly related
homozygous partial deletion of the gene was quencing within introns and flanking genomic (25) and reproductively isolated, and because
born 3.9 SD below normal length (16, 17). sequence was also undertaken (table S3). Sev- the extent of haplotype sharing at IGF1 is rela-
IGF1 binds the type 1 IGF receptor, a tyrosine eral additional SNPs (table S4) and an antisense tively small, the sequence variant or variants prob-
kinase signal transducer. This interaction pro- oriented retrotransposon (table S5) unique to ably predate the common origin of the breeds
motes cell growth and organismal longevity (21) small breeds were identified. Alleles of a dinu- and likely evolved early in the history of dogs.
and induces cellular differentiation (22). Serum cleotide CAn microsatellite in the IGF1 promoter The early appearance of this allele may have
levels of IGF1 protein (23) have been found to were also significantly associated with body size facilitated the rapid genesis of size diversity in
the domestic dog. The first archaeological record
of dogs, beginning about 12,000 to 15,000 years
ago (9, 26), shows that size diversity was pre-
sent early in the history of domestication. For
example, dog remains from eastern Russia dated
to 14,000 to 15,000 years ago are similar in size
and conformation to great Danes, whereas slight-
ly younger dog remains from the Middle East
and Europe (10,000 to 12,000 years ago) are
similar in size to small terriers (9, 26, 27). The
early and widespread appearance of small size
suggests that an ancesral small dog IGF1 haplo-
type was readily spread over a large geographic
area by trade and human migration and was
maintained in local gene pools by selection.
Such early selection on dogs may have been
manifest as intentional artificial selection exer-
cised by early humans or as an adaptive trait for
coexistence with humans in the more crowded
confines of developing villages and cities (28).
Fig. 3. Evidence of association and IGF1 haplotypes for 14 small and 9 giant breeds. (A) Mann-Whitney
U (MWU) P values for tests of association between individual SNPs and body size (small versus giant) for
116 SNPs on chromosome 15 and 83 SNPs on five control chromosomes. The dashed line indicates
Bonferroni correction for multiple tests. Only breeds with data for at least 10 chromosomes were
included (14 small and 9 giant breeds). (B) Haplotypes for the 20 markers spanning the small breed
sweep interval near IGF1. The haplotypes were inferred independently in each breed. For each individual, Fig. 4. Association of body size and frequency of
fractional chromosome counts were summed for all haplotypes with at least 5% probability according to the SNP 5 A allele. Binomial regression of allele
the haplotype inference software program PHASE. Chromosome sums for each breed were rounded to frequency on square root of mean breed mass.
integer values; several breeds have odd numbers of chromosomes due to rounding error. Only inferred Dashed lines indicate the 95% confidence
haplotypes carried by at least three dog chromosomes total (i.e., >0.5% frequency overall) are shown. interval on the predicted equation line as esti-
Sequence reads collected from golden jackal (Canis aureus) were used to determine the ancestral allele mated from nonparametric bootstrap resampling.
for each SNP. The haplotypes are rows labeled A to L, and marker alleles are colored yellow for ancestral Between 5 and 109 (median = 22) dogs were
state (matching the nucleotide observed in the golden jackal) and blue for derived state. SNP positions genotyped for each of 143 breeds. The PWD is
within IGF1 are shown at the top with IGF1 introns (horizontal line) and exons (vertical bars) indicated. highlighted in red along with three giant breeds
(C) Breed name and the average size of adult males in kilograms are provided. Small breeds less than that have larger breed average masses than is
9 kg and giant breeds greater than 30 kg are grouped for totals shown at the far right. predicted by their SNP 5 allele frequency.

REPORTS
The ubiquitous occurrence of the IGF1 B 4. C. Webber, C. P. Ponting, Genome Res. 15, 1787 24. J. E. Eigenmann, D. F. Patterson, E. R. Froesch,
haplotype in a diverse panel of small breeds (2005). Acta Endocrinol. (Copenh.) 106, 448 (1984).
5. W. Wang, E. F. Kirkness, Genome Res. 15, 1798 (2005). 25. H. G. Parker et al., Science 304, 1160 (2004).
clearly does not support unorthodox explana- 6. R. K. Wayne, J. Zool. 210, 381 (1986). 26. H. Epstein, The Origin of the Domestic Animals of Africa
tions of phenotypic diversity in the dog such as 7. P. Saetre et al., Brain Res. Mol. Brain Res. 126, 198 (2004). (Africana Publishing, New York, 1971).
elevated mutation or recombination rates. Rath- 8. P. Savolainen, Y. P. Zhang, J. Luo, J. Lundeberg, 27. M. V. Sablin, G. A. Khlopachev, Curr. Anthropol. 43, 795
er, we show that a single IGF1 allele is a major T. Leitner, Science 298, 1610 (2002). (2002).
9. S. J. Olsen, Origins of the Domestic Dog (Univ. of Arizona 28. E. Tchernov, L. K. Horwitz, J. Anthropol. Archaeol. 10, 54
determinant of small size in dogs and that in- Press, Tucson, AZ, 1985). (1991).
tense artificial selection has left a signature in 10. C. Vila et al., Science 276, 1687 (1997). 29. We thank the hundreds of dog owners who contributed
the proximity of IGF1 that can readily be found 11. J. Sampson, M. M. Binns, in The Dog and Its Genome, samples; the AKC Canine Health Foundation;
by genomic scans of breeds sharing a common E. A. Ostrander, K. Lindblad-Toh, U. Giger, Eds. (Cold S. Hoogstraten-Miller and I. Ginty for assistance at dog
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, shows; P. Cruz for assistance with automated PCR primer
phenotype. The ability to identify a gene con- 2006), vol. 44, pp. 19–30. designs; S. Kim for analytical assistance, and R. Pelker
tributing to morphology without doing a genetic 12. B. Van Valkenburgh, X. Wang, J. Damuth, Science 306, for assistance with blood serum assays of IGF1. Funded
cross, but instead by using centuries of dog 101 (2004). by the National Human Genome Research Institute (E.A.O.,
breeding, highlights the contribution that the 13. K. Chase et al., Proc. Natl. Acad. Sci. U.S.A. 99, 9930 (2002). N.B.S., E.K., S.D., P.Q., H.G.P., and D.S.M.), the NSF
14. K. Chase, D. R. Carrier, F. R. Adler, E. A. Ostrander, (R.K.W.), NIH grant no. 5 T32 HG002536 (M.M.G.), NSF
study of canine genetics can make to an under-
K. G. Lark, Genome Res. 15, 1820 (2005). grant 0516310 (C.D.B. and L.Z.), NSF grant DBI 0606461
standing of mammalian morphogenesis. These 15. J. Baker, J. P. Liu, E. J. Robertson, A. Efstratiadis, Cell 75, (B.P.), NIH grant P50 HG002790 (K.Z. and M.N.), and the
results provide a precedent for future studies 73 (1993). National Institute of General Medical Sciences 063056, the
aimed at identifying the genetic basis for com- 16. K. A. Woods, C. Camacho-Hubner, D. Barter, A. J. Clark, Judith Chiara Charitable Trust, and the Nestle Purina
plex traits such as behavior and skeletal mor- M. O. Savage, Acta Paediatr. Suppl. 423, 39 (1997). Company (K.G.L.).
17. K. A. Woods, C. Camacho-Hubner, M. O. Savage,
phology in dogs and other species with small A. J. Clark, N. Engl. J. Med. 335, 1363 (1996). Supporting Online Material
populations that have experienced strong artifi- 18. N. B. Sutter et al., Genome Res. 14, 2388 (2004). www.sciencemag.org/cgi/content/full/316/5821/112/DC1
cial or natural selection. 19. K. Lindblad-Toh et al., Nature 438, 803 (2005). Materials and Methods
20. J. P. Pollinger et al., Genome Res. 15, 1809 (2005). Figs. S1 to S9
References and Notes 21. R. Kooijman, Cytokine Growth Factor Rev. 17, 305 Tables S1 to S6
1. R. K. Wayne, Evolution 40, 243 (1986). (2006). References
2. R. K. Wayne, J. Morphol. 187, 301 (1986). 22. P. Cohen, Horm. Res. 65, 3 (2006).
3. J. W. Fondon 3rd, H. R. Garner, Proc. Natl. Acad. Sci. U.S.A. 23. R. P. Favier, J. A. Mol, H. S. Kooistra, A. Rijnberk 1 November 2006; accepted 8 March 2007
101, 18058 (2004). J. Endocrinol. 170, 479 (2001). 10.1126/science.1137045
Binding of the Human Prp31 Nop in the U4 5′-SL (12) and is required for
subsequent recruitment of the human (h) Prp31
protein to the U4/U6 di-snRNP (13). hPrp31
Domain to a Composite RNA-Protein does not interact with either the 15.5K or the
RNA alone (13, 14), but it is not known whether
Platform in U4 snRNP 15.5K merely prestructures the RNA for subse-
quent binding of hPrp31 or whether 15.5K pro-
vides part of the hPrp31 binding site. hPrp31 is
Sunbin Liu,1* Ping Li,1,2* Olexandr Dybkov,1 Stephanie Nottrott,1 Klaus Hartmuth,1 essential for pre-mRNA splicing (15) and is a
Reinhard Lührmann,1† Teresa Carlomagno,2† Markus C. Wahl3† component of both major and minor spliceo-
somes. In the latter, the U4 snRNA is replaced by
the U4atac snRNA (Fig. 1A). Nevertheless, both
Although highly homologous, the spliceosomal hPrp31 and the nucleolar Nop56 and Nop58
snRNAs bind 15.5K, and both primary RNPs
(Nop56/58) proteins recognize different ribonucleoprotein (RNP) particles. hPrp31 interacts with
incorporate hPrp31 in a strictly hierarchical
complexes containing the 15.5K protein and U4 or U4atac small nuclear RNA (snRNA), whereas
manner (13, 16).
Nop56/58 associate with 15.5K–box C/D small nucleolar RNA complexes. We present structural and
The 15.5K protein also binds to a K turn in
biochemical analyses of hPrp31-15.5K-U4 snRNA complexes that show how the conserved Nop
box C/D small nucleolar (sno) RNAs (17, 18),
domain in hPrp31 maintains high RNP binding selectivity despite relaxed RNA sequence
but subsequently Nop56 and Nop58 (Nop56/58;
requirements. The Nop domain is a genuine RNP binding module, exhibiting RNA and protein
Nop5p in archaea) are recruited to the snoRNPs
binding surfaces. Yeast two-hybrid analyses suggest a link between retinitis pigmentosa and an
(Fig. 1A) (17, 19). Stem II of the snRNAs and
aberrant hPrp31-hPrp6 interaction that blocks U4/U6-U5 tri-snRNP formation.
snoRNAs (Fig. 1A) encompasses crucial identity
elements for secondary protein binding. In the
ost eukaryotic pre-mRNAs contain spliceosome activation, the U4-U6 interaction box C/D snoRNAs, stem II is longer by one base
M introns that are removed before trans-

lation by a multi-megadalton ribo-
nucleoprotein (RNP) enzyme, the spliceosome
is disrupted, U4 snRNA is released, and U6
snRNA forms short duplexes with U2 snRNA
and the pre-mRNA substrate (6). Understand-
1
Abteilung Zelluläre Biochemie, Max-Planck-Institut für
Biophysikalische Chemie, Am Faßberg 11, D-37077
(1–3). A spliceosome is assembled anew on ing this catalytic activation of the spliceosome Göttingen, Germany. 2AG Flüssig-NMR Spektroskopie,
each intron from small nuclear (sn) RNPs and requires detailed structural information on the Max-Planck-Institut für Biophysikalische Chemie, Am
non-snRNP splice factors (4, 5). The RNP net- snRNPs. Faßberg 11, D-37077 Göttingen, Germany. 3AG Makro-
work of the spliceosome is extensively restruc- As for other complex RNPs (10), the U4/U6 molekulare Röntgenkristallographie, Max-Planck-Institut
tured during its maturation (2, 6, 7), reflected by di-snRNP is built in a hierarchical manner. A U4 für Biophysikalische Chemie, Am Faßberg 11, D-37077
Göttingen, Germany.
changing RNA interactions. The U6 snRNA is 5′ stem loop (U4 5′-SL) between two base-paired
*These authors contributed equally to this work.
delivered to the pre-mRNA in a repressed state, stems of U4/U6 serves as a binding site for the †To whom correspondence should be addressed. E-mail:
in which catalytically important regions are highly conserved U4/U6-15.5K protein (11). [email protected] (R.L.); taco@nmr.
base-paired to the U4 snRNA (8, 9). During 15.5K binds to and stabilizes a kink turn (K turn) mpibpc.mpg.de (T.C.); [email protected] (M.C.W.)

REPORTS
pair, and no sequence deviation is tolerated resembled that of full-length hPrp31 [Support- globular module (residues 121 to 180; helices
(19–21). Somewhat paradoxically, both hPrp31 ing Online Material (SOM) text and fig. S2]. A a2 to a5). An oval-shaped Nop domain (res-
and Nop56/58 contain a conserved, ~120-residue reconstituted hPrp3178-333-15.5K-U4 5′-SL com- idues 215 to 333; helices a7 to a13) follows the
Nop domain (hPrp31215-333) (15, 22, 23) (fig. plex yielded a 2.6 Å resolution crystal struc- coiled-coil motif at the C terminus. hPrp3178-333
S1), which seems to mediate binding to the ture (table S1), in which residues 4 to 128 of contacts the primary RNP exclusively via its
different primary RNPs (24). 15.5K and 85 to 333 of hPrp31 (excluding Nop domain (Fig. 2A), suggesting that this
To delineate the structural basis for the residues 256 to 265 that form the tip of a flex- element is the most crucial RNP interacting
ordered and selective binding of hPrp31, we first ible loop) and all RNA residues (nucleotides 20 module in hPrp31.
probed whether hPrp31 engages in direct to 52 of U4 snRNA) could be traced (fig. S3). The Nop domain of hPrp3178-333 exhibits a
contacts with 15.5K in the context of the U4 The hPrp3178-333–15.5K–U4 5′-SL complex is flat surface formed by helices a9, a10, a12, and
snRNP by using nuclear magnetic resonance triangular, with one subunit at each vertex of a13 (Fig. 2, A and B). The lower part of this
(NMR) spectroscopy (25). [15N]15.5K protein the triangle and each subunit contacting the surface (helix a9 and the C-terminal half of helix
was bound to an RNA representing the entire U4 other two (Fig. 2A). The negatively charged a12) interacts with the a2 and a3 region of
5′-SL [residues 20 to 52 of U4 snRNA (Fig. RNA is sandwiched between positively charged 15.5K (contact regions a and b in Fig. 2B).
1A)], and NMR chemical shift changes were areas on hPrp3178-333 and 15.5K (fig. S4). The Details of the interactions between hPrp3178-333
monitored upon addition of hPrp31. Primarily region of hPrp3178-333 interacting with 15.5K and 15.5K (Fig. 3, A and B) agree well with full-
residues in helices a2 and a3 of 15.5K were exhibits alternating positively and negatively length hPrp31-15.5K contacts mapped by NMR
affected (Fig. 1B). Saturation transfer from the charged surface patches matched by a com- (Fig. 1B). The upper portion of the surface (helix
aliphatic protons of hPrp31 to the amide res- plementary set of patches on the 15.5K protein a10 and the N-terminal half of helix a12)
onances of 15.5K in a ternary complex con- (fig. S4). contacts the RNA on the side that is not
taining [15N,2D,1HN]15.5K confirmed direct hPrp3178-333 exhibits an all-helical fold associated with 15.5K (contact region 1 in Fig.
contacts between hPrp31 and helices a2 and a3 with three domains (Fig. 2A and fig. S1). Res- 2B) and in the major groove of stem II (region 2).
of 15.5K (Fig. 1B). idues 85 to 120 (helix a1) and 181 to 215 (helix A loop following helix a10 interacts with the
By using limited proteolysis, we defined a6) form two branches of an extended coiled capping pentaloop (region 3). The surfaces of
fragment hPrp3178-333, whose binding activity coil, which is interrupted at the tip by a small 15.5K and of the RNA buried upon binding of
Fig. 1. (A) Schematics of the 5′-SLs of

U4 snRNA (left), U4atac snRNA (mid-
dle), and the K-turn region of box C/D
snoRNAs (right). N indicates any nucleo-
tide; R, purine. Binding of 15.5K and
the secondary binding proteins is
indicated. Stem II of the K turn in the
box C/D snoRNAs is longer by one base
pair (20), and a single additional base
pair in stem II is known to interfere with
hPrp31 binding (21). Box C/D snoRNPs
act as sequence-specific 2'-O methyl-
transferases, which posttranscription-
ally modify several functional RNAs.
(B) (Left) 1H-15N heteronuclear single-
quantum coherence spectra of 15.5K
in the binary 15.5K–U4 5′-SL complex
(black) and in the ternary complex
containing full-length hPrp31 (red).
Assignments of selected resonances
are indicated. ppm, parts per million.
(Middle) Mapping of NMR chemical
shift changes elicited by the addition
of hPrp31 on the structure of the
15.5K-RNA complex [coordinates
from (12); PDB ID 1E7K]. Dashed
line is the disordered pentaloop of
the RNA; 15.5K, light gray; and RNA,
dark gray. All structure figures were
prepared with PyMOL (34). (Right)
Mapping of saturation transfer from
hPrp31 to RNA-bound 15.5K, indi-
cating residues of 15.5K that are
directly contacted by hPrp31. Appar-
ent contacts to the central b sheet of
15.5K arise from spin diffusion.
Degrees of chemical shift changes
and saturation transfer are color-coded: red, strong; orange, intermediate; and yellow, weak. A similar picture is obtained when mapping the contacts of
hPrp3178-333 on 15.5K in the framework of the 15.5K–U4 5′-SL complex (SOM text), confirming that the hPrp3178-333 fragment interacts with 15.5K in the
same way as the full-length protein.

REPORTS
the Nop domain are comparable (550 to 650 Å2 The conformation of the core 15.5K-RNA (27). In addition, the short helix a10 of the Nop
each) and are confluent (fig. S5). Thus, the Nop complex is unaffected by the addition of domain lines the major groove of stem II of
domain presents an RNP recognition motif, as hPrp3178-333 (Fig. 2A). Furthermore, the Nop the RNA where C247 (28) engages in hydro-
opposed to pure RNA interaction domains found domain of hPrp3178-333 closely resembles the gen bonds to the bases of C41 and G43, rep-
in other proteins (26). corresponding domain of the archaeal Nop5p resenting the only sequence-specific contact of
On the basis of this architecture, failure of protein in the absence of a primary RNP (24) hPrp3178-333 to the RNA (Fig. 3D; contact region
hPrp31 to bind either 15.5K or the RNA alone (table S2). Therefore, binding of the hPrp31 Nop 2 in Fig. 2B).
(13, 14) can be attributed to 15.5K and the RNA, domain to 15.5K and the K turn resembles a Major structural changes in the RNA upon
each contributing about half of the hPrp31 in- lock-and-key–type interaction. hPrp3178-333 binds formation of the ternary complex are confined to
teraction surface, so that neither of the com- to one side of the bulged U31 of the K-turn via the RNA pentaloop (nucleotides 36 to 40), which
ponents alone is able to supply a sufficiently large water-mediated interactions, van der Waals becomes ordered on addition of hPrp3178-333
interface. In addition, 15.5K stabilizes the RNA contacts, and hydrogen bonds to the backbone (Fig. 2A). hPrp3178-333 stabilizes the pentaloop
K-turn region in a conformation favorable for (Fig. 3C and contact region 1 in Fig. 2B). This by direct contacts and by reinforcing intra-
hPrp31 binding and thus pays the entropic cost situation is reminiscent of protein-DNA intermolecular interactions. H270 (28), from a
for immobilizing part of the RNA structure actions, where the DNA-bound water structure flexible loop of hPrp3178-333, stacks on the pe-
(SOM text). provides important latching points for proteins nultimate residue of the pentaloop, A39, which in
turn stacks on the terminal base pair of stem II
and forms a hydrogen bond across the loop (Fig.
3E and contact region 3 in Fig. 2B). The apparent
malleability of the RNA pentaloop allows its
remaining part to wrap around and engage in
hydrogen bonds with H270 (Fig. 3E). The
terminal U40 of the pentaloop stacks on a
hydrophobic surface patch of the Nop domain
(Fig. 3F). Because the above contacts ensue
between flexible elements of the protein and the
RNA, we conclude that hPrp3178-333 recognizes
and stabilizes the RNA pentaloop by induced fit
interactions (SOM text).
Although the entire pentaloop of the U4 5′-SL
can be removed without completely disrupting
the binding of hPrp31, it becomes protected from
hydroxyl radical cleavage upon binding of
hPrp31 (21) or hPrp3178-333 (Fig. 4A). Our struc-
ture reconciles this apparent discrepancy. Even
though large portions of the pentaloop are ex-
posed, the C4' atoms, which are the primary sites
of hydroxyl radical attack (29), are partially or
entirely buried for residues 36, 39, and 40 (Fig.
4B). The flexible loop of hPrp3178-333 (residues
256 to 265) is suspended next to the C4' atoms
of residues 37 and 38 (Fig. 4B), where it can
scavenge radicals and protect the RNA even in
the absence of direct contacts.
hPrp31 recognizes complexes of 15.5K with
U4 or U4atac snRNA, whose sequences differ
markedly in the hPrp31 contact regions defined
by the crystal structure (Fig. 1A). In the U4
structure, hPrp3178-333 avoids sequence-specific
interactions with the RNA bases and instead
maintains water-mediated interactions, contacts
to the backbone, or stacking interactions with the
Fig. 2. (A) Overview of the hPrp3178-333–15.5K–U4 5′-SL complex (left). hPrp3178-333, blue; 15.5K, bases (Fig. 3, C to F). With the exception of a
red; RNA, gold. RNA elements not seen in the binary 15.5K–22-mer RNA complex with a shorter stem single hydrogen bond from C247 of hPrp3178-333
I [right (12); PDB ID 1E7K] are in green. Positions A194 and A216, at which missense mutations have
to C41 of the U4 snRNA, all contacts could be
been linked to the RP11 form of retinitis pigmentosa, are shown as space-filling models and colored
maintained in a complex containing the U4atac
cyan. Dashed line in hPrp3178-333, disordered loop. Dashed line in the binary complex, unstructured
pentaloop. Although induced-fit interactions are the hallmark of most RNA-protein complexes (32), 5′-SL. Consistent with a similar interaction, H270
the structuring of the RNA pentaloop upon hPrp3178-333 binding observed here is particularly of hPrp31 has been ultraviolet light (UV)–cross-
pronounced. The crystal structure contains two crystallographically independent ternary complexes linked to U44 of the U4atac snRNA (13, 30),
per asymmetric unit that are largely identical (table S2). (B) Close-up views of the complex from the implying an identical stacking arrangement as
back (left) and from the bottom (right). Main contact regions between hPrp3178-333 and 15.5K and seen with the corresponding A39 of U4 snRNA
between hPrp3178-333 and the RNA are indicated by connecting lines and are labeled by letters and (Fig. 3E).
numbers, respectively. Regions of the RNA are color-coded: distal portion of stem I, gray; K-turn The largely sequence-independent RNA
region, gold; distal portion of stem II, brown; and pentaloop, beige. The bulged-out U31 denotes the contacts of hPrp3178-333 suggest that structural
tip of the K turn and is shown in sticks. rather than sequence differences preclude bind-

REPORTS
ing to box C/D-like RNAs. In our structure, all
hPrp31 contacts with the RNA are mediated by
the Nop domain, suggesting that this motif
alone is able to discriminate against an extended
stem II. To verify this hypothesis, we conducted
gel mobility shift assays by using wild-type
(WT) and mutant U4 5′-SLs and a Nop-domain
fusion protein [maltose binding protein (MBP)–
hPrp31215-333]. Like full-length hPrp31, the Nop
domain did not bind to RNPs, in which stem II of
the RNA was extended by a noncanonical U-U
(Fig. 4C, lanes 7 to 9) or by a canonical C-G base
pair (lanes 10 to 12). These data confirm that the
Nop domain is both required and sufficient for
binding to the primary RNP and for decoding its
structural specificity determinants.
An elongated stem II would reposition the
pentaloop and thus the stacking platform for
H270 (Fig. 3E). Loss of hPrp31 affinity to 15.5K
complexes with elongated stem II RNAs could,
therefore, arise due to the disruption of H270-
A39 stacking. We tested this possibility by con-
verting H270 to an alanine or to a lysine (as
found in Nop56/58). Loss or alteration of the
H270 side chain resulted in reduced affinity of
hPrp31H270A and hPrp31H270K to the 15.5K-
RNA 5′-SL complexes (Fig. 4D, lanes 1 to 5).
However, the mutants retained significant
binding activity and discriminated strongly
against long stem II constructs (Fig. 4D, lanes 6
to 10), indicating that H270 is not required for
measuring the length of stem II.
Next, we modeled an extended stem II A-
form duplex into the present structure. The
additional base pair would lie in the stacking
level occupied by A39 in the WT complex.
Although A39 fits snugly next to helix a10 of
the Nop domain, the helical twist would lead to a
severe clash between an additional Watson-
Crick base pair and helix a10 (fig. S6). In
contrast, in a tetraloop RNA, to which hPrp31
(21) and hPrp31215-333 (Fig. 4C, lanes 4 to 6)
still bind, the nucleotides would be turned away
from helix a10. Thus, we conclude that helix
a10 acts as a ruler for measuring the length of
stem II by presenting a physical barrier to addi-
tional base pairs. A different recognition mech-
anism must be at work in Nop56/58, in which
the Nop domain is compatible with elongated Fig. 3. Detailed interactions in the ternary complex. Selected interface residues (sticks) are labeled and
stem II RNAs. color-coded by atom type (carbon and phosphorus, as the respective molecule; oxygen, red; nitrogen, blue;
The hPrp3178-333 fragment encompasses sulfur, yellow; bridging waters, cyan spheres). (A) Hydrogen bonds and salt bridges (dashed lines) involving
both A194 and A216 (28) that have been helices a2 and a3 of 15.5K and the hPrp3178-333 Nop domain (view from bottom of Fig. 2A). A network of
linked to the RP11 form of retinitis pigmentosa alternating residues from 15.5K and hPrp3178-333 extends from the backbone carbonyl of I65 (15.5K) over
(23). A194 maps to the second helix of the the side chain of R304 (hPrp3178-333) and the side chain of N40 (15.5K) to the backbone nitrogen of A246
(hPrp3178-333). (B) Hydrophobic contacts between the N terminus of helix a3 of 15.5K and the C terminus of
coiled coil, whereas A216 lies in a short loop
helix a12 of hPrp31 (view as in Fig. 2A). Our crystal structure and NMR analysis show that residues 74 to 77
connecting the coiled coil to the Nop domain
of 15.5K, which upon joint mutation inhibited the binding of hPrp31 (33), are not involved in contacts to
(Fig. 2A and fig. S7). Thus, neither of the two hPrp3178-333 or full-length hPrp31, respectively, and elicit their effect indirectly, for example, by influencing
residues interacts directly with 15.5K or the the structure of 15.5K. (C to F) Details of the interaction of hPrp3178-333 with the K turn (C), with the major
U4 5′-SL in the present structure (Fig. 2A). groove of stem II (D), with the 5′ portion of the pentaloop (E) and with the 3′ portion of the pentaloop (F). The
The Ala194→Glu194 (A194E) substitution most central lock-and-key interaction region around the K turn [(C) and (D)] is of paramount importance for the
likely disturbs the local structure and/or the stability of the ternary complex, because the RNA pentaloop can be removed without completely disrupting
surface properties of the coiled coil, because the the binding of hPrp31 (21). The induction of a stable structure in the RNA pentaloop by hPrp3178-333 [(E)
A194 side chain is embedded in a hydrophobic and (F)] is reminiscent of the way some primary binding ribosomal proteins induce novel binding sites for
environment (fig. S7A). The Ala216→Pro216 secondary binding proteins (10). Here, three of the five pentaloop bases are turned outward (E) and
(A216P) replacement (fig. S7A) potentially conceivably provide a binding platform for another spliceosomal component.

REPORTS
alters the structure and flexibility of a protein Whereas introduction of proline at position 216 forces the 15.5K-RNA interaction. The latter
loop, which may affect the relative orientation had no effect on the interaction with hPrp6, a interaction is crucial for the transition from the
of the Nop and the coiled-coil domains. Either glutamate at position 194 significantly weakened spliceosomal B complex to the C complex (11),
of the above mutations could influence other the interaction (fig. S7B). This result supports the during which spliceosome activation occurs.
interactions of hPrp31, for example, with hPrp6 idea that the hPrp31 coiled coil is an interaction Thus, on the one side hPrp31 may regulate the
of the U5 snRNP. To test this hypothesis, we site for hPrp6 and links a retinitis pigmentosa RNA-protein network at the U4 5′-SL and
conducted a targeted yeast two-hybrid analysis mutation in a spliceosomal protein to an aberrant thereby facilitate disruption of the U4/U6 snRNA
using pGADT7-hPrp6 as prey and pGBKT7- molecular communication. duplex. Our work also suggests that hPrp31
hPrp31, pGBKT7-hPrp31A194E, and pGBKT7- By interacting concomitantly with both stabilizes the U4/U6-U5 tri-snRNP by concom-
hPrp31A216P as bait under stringent conditions. 15.5K and the RNA, the Nop domain rein- itantly interacting with hPrp6 via a separate
coiled-coil domain. Intriguingly, hPrp6 links
hPrp31 to hBrr2 and hSnu114 (14), which have
been shown to be the DEAD-box protein and
regulatory guanosine triphosphatase (GTPase),
respectively, that are crucial for both spliceosome
activation and disassembly (31). Therefore, our
structural data are in line with the previous
hypothesis (14) that hPrp31 may represent one
of the ultimate targets of the helicase and GTPase
machinery of the U5 snRNP that acts during
spliceosome activation.

1. C. B. Burge, T. Tuschl, P. A. Sharp, in The RNA World,
R. F. Gesteland, T. Cech, J. F. Atkins, Eds. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1999),
pp. 525–560.
2. T. W. Nilsen, Bioessays 25, 1147 (2003).
3. C. L. Will, R. Lührmann, in The RNA World, R. F. Gesteland,
T. Cech, J. F. Atkins, Eds. (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 2006), pp. 369–400.
4. R. Reed, L. Palandjian, in Eukaryotic mRNA Processing,
A. R. Krainer, Ed. (IRL Press, Oxford, 1997),
pp. 103–129.
5. J. Gornemann, K. M. Kotovic, K. Hujer, K. M. Neugebauer,
Mol. Cell 19, 53 (2005).
6. J. P. Staley, C. Guthrie, Cell 92, 315 (1998).
7. D. A. Brow, Annu. Rev. Genet. 36, 333 (2002).
8. P. Bringmann et al., EMBO J. 3, 1357 (1984).
9. C. Hashimoto, J. A. Steitz, Nucleic Acids Res. 12, 3283
(1984).
10. S. C. Agalarov, G. S. Prasad, P. M. Funke, C. D. Stout,
J. R. Williamson, Science 288, 107 (2000).
11. S. Nottrott et al., EMBO J. 18, 6119 (1999).
12. I. Vidovic, S. Nottrott, K. Hartmuth, R. Lührmann,
R. Ficner, Mol. Cell 6, 1331 (2000).
13. S. Nottrott, H. Urlaub, R. Lührmann, EMBO J. 21, 5527
(2002).
14. S. Liu, R. Rauhut, H. P. Vornlocher, R. Lührmann, RNA
12, 1418 (2006).
15. O. V. Makarova, E. M. Makarov, S. Liu, H. P. Vornlocher,
R. Lührmann, EMBO J. 21, 1148 (2002).
16. C. Schneider, C. L. Will, O. V. Makarova, E. M. Makarov,
R. Lührmann, Mol. Cell. Biol. 22, 3219 (2002).
17. N. J. Watkins et al., Cell 103, 457 (2000).
18. L. B. Szewczak, J. S. Gabrielsen, S. J. Degregorio,
S. A. Strobel, J. A. Steitz, RNA 11, 1407 (2005).
19. N. J. Watkins, A. Dickmanns, R. Lührmann, Mol. Cell.
Biol. 22, 8342 (2002).
Fig. 4. (A) Hydroxyl radical footprinting of the U4 5′-SL in the absence of protein (lane 2), in the 20. T. Moore, Y. Zhang, M. O. Fenley, H. Li, Structure 12, 807
presence of only 15.5K (lane 3) and in the presence of 15.5K and increasing amounts of hPrp3178-333 (2004).
(lanes 4 to 6). Numbers indicate the protein concentration in mmol/l. Numbers on the right indicate 21. A. Schultz, S. Nottrott, K. Hartmuth, R. Lührmann, J. Biol.
positions in the U4 5′-SL. The location of the pentaloop is indicated. (B) Surface of the hPrp3178-333– Chem. 281, 28278 (2006).
22. T. Gautier, T. Berges, D. Tollervey, E. Hurt, Mol. Cell. Biol.
15.5K–U4 5′-SL complex (RNA, gray; pentaloop, gold). Sugar C4' atoms are highlighted in black and
17, 7088 (1997).
labeled for residues 37 to 39. Red dots, beginning (lower dot) and end (upper dot) of a flexible loop in 23. E. N. Vithana et al., Mol. Cell 8, 375 (2001).
the protein that is suspended next to the sugars of the pentaloop. (C) Gel mobility shift assays 24. M. Aittaleb et al., Nat. Struct. Biol. 10, 256 (2003).
monitoring the binding of a Nop domain fusion protein (MBP-hPrp31215-333) to U4 5′-SL constructs. 25. Materials and methods are available as supporting
Lanes 1 to 3, WT RNA sequence; lanes 4 to 6, replacement of the pentaloop by a UGAA tetraloop; material on Science Online.
lanes 7 to 9, addition of a U-U base pair to stem II following the sheared G-A pairs; and lanes 10 to 26. C. G. Burd, G. Dreyfuss, Science 265, 615 (1994).
27. Z. Shakked et al., Nature 368, 469 (1994).
12, addition of a C-G base pair at the terminus of stem II. (D) Gel mobility shift assays monitoring the 28. Single-letter abbreviations for the amino acid residues
effects of converting H270 into an alanine (A) or a lysine (K). Mutant hPrp31 proteins bind less are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
strongly to a WT U4 5′-SL (lanes 1 to 5) but still discriminate against RNAs with a longer stem II (lanes Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q,
6 to 10). Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

REPORTS
29. J. C. Wu, J. W. Kozarich, J. Stubbe, J. Biol. Chem. 258, spectrometric analyses, G. Bourenkov (European Molecular be released upon publication. The authors declare that
4694 (1983). Biology Laboratory, Hamburg, Germany) and the group of they have no competing financial interests.
30. E. Kühn-Hölsken, C. Lenz, B. Sander, R. Lührmann, H. Bartunik (beamline BW6, Deutsches Elektronen
H. Urlaub, RNA 11, 1915 (2005). Synchrotron, Hamburg, Germany) for beamline support,
31. E. C. Small, S. R. Leggett, A. A. Winans, J. P. Staley, and C. Will for fruitful discussions and critical reading of www.sciencemag.org/cgi/content/full/316/5821/115/DC1
Mol. Cell 23, 389 (2006). the manuscript. This work was supported by the Max- Materials and Methods
SOM Text
32. J. R. Williamson, Nat. Struct. Biol. 7, 834 (2000). Planck-Gesellschaft (to R.L., T.C., and M.C.W.), the
33. A. Schultz, S. Nottrott, N. J. Watkins, R. Lührmann, Deutsche Forschungsgemeinschaft (LU294/12-3), the Figs. S1 to S7
Tables S1 and S2
Mol. Cell. Biol. 26, 5146 (2006). Fonds der Chemischen Industrie (to R.L.), and the Ernst-
References
34. PyMOL, http://pymol.sourceforge.net/. Jung-Stiftung (to R.L.). Coordinates and structure factors
35. We thank S. Bhattacharya (University College London, UK) have been deposited with the RCSB Protein Data Bank 22 November 2006; accepted 1 March 2007
for supplying pTriEx-hPrp31, H. Urlaub for mass (www.rcsb.org/pdb/) under accession code 2OZB and will 10.1126/science.1137924
An ATP Gate Controls Tubulin Binding types of model require the microtubule lattice
for their operation, either to set a prohibitive
distance between binding sites or to apply
by the Tethered Head of Kinesin-1 strain to the kinesin heads. Here, we report an
ATP gate that operates independently of the
microtubule lattice.
Maria C. Alonso,1 Douglas R. Drummond,1 Susan Kain,1 Julia Hoeng,2 We have found that kinesin-1 binds to free
Linda Amos,2 Robert A. Cross1* tubulin heterodimers in solution, causing tubulin-
activated release of adenosine diphosphate
Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated (ADP). This shows that tubulin activation of
by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role the kinesin adenosine triphosphatase (ATPase)
for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing is not unique, as had previously been thought,
tubulin-activated release of adenosine diphosphate (ADP). With no added nucleotide, each to the depolymerizing kinesins (13). The degree
kinesin-1 dimer binds one tubulin heterodimer. In adenylyl-imidodiphosphate (AMP-PNP), a of activation of the kinesin-1 ATPase by un-
nonhydrolyzable ATP analog, each kinesin-1 dimer binds two tubulin heterodimers. The data polymerized tubulin varies according to the
reveal an ATP gate that operates independently of the microtubule lattice, by ATP-dependent source of kinesin-1 and tubulin, but clearly
release of a steric or allosteric block on the tubulin binding site of the tethered kinesin-ADP head. activation of the kinesin-1 ATPase does not
require the interheterodimer interfaces that arise
inesin-1 molecular motors are adeno- gating is ascribed to the effects of the resulting in the microtubule lattice. For a fungal kinesin-
K sine triphosphate (ATP)–driven walk-

ing machines that move in 8-nm steps
toward the plus ends of microtubules, turning
intramolecular strain, together with any external
strain, on nucleotide exchange (11, 12). These
two types of model are not mutually exclusive;
1 and a fungal tubulin, maximal activation by
unpolymerized tubulin heterodimers is equiv-
alent to that produced by assembled micro-
over one ATP molecule per step under a range the influential Rice et al. model (9), for tubules (Fig. 1A). For brain tubulin and brain
of loads (1–5). Even at very high backward example, proposes that the first step in each kinesin, tubulin activation of the kinesin
loads, when the motor can be forced to step run of steps uses the first type of gate and that ATPase is modest compared with microtubule
processively backward (6), stepping remains subsequent steps use the second type. Both activation (Fig. 1, B to D).
coupled to ATP binding (6, 7). Between steps,
the motor pauses stably in a dwell state. It is
clear that ATP binding triggers exit from this Fig. 1. Activation of kinesin dimers S. pombe tubulin
dwell state, but the structural mechanism is by tubulin and microtubules. The
microtubule- or tubulin heterodimer–
A Neurospora kinesin-1 B Rat kinesin-1
controversial (8). 50 20
Two general types of model for this ATP stimulated steady state ATPase activity
40
ATPase rate (s-1)
ATPase rate (s-1)
of kinesin was measured at 25°C with HD 15

gate have been proposed. In the first, kinesin MT
an enzyme-linked assay in 20 mM 30 MT
dimers are proposed to dwell between steps
Pipes, pH 6.9, 5 mM MgCl2, 1 mM 10
with only one head attached to the microtubule, dithiothreitol (22). Values for Vmax 20
whereas the other diffuses to some extent on its (the projected maximum ATPase rate) 5
10
tether but cannot access its next binding site and Km (the tubulin concentration giv- HD
along the microtubule because the site is too far ing half-maximal ATPase) were obtained 0 0
0 1 2 3 4 5 0 2 4 6 8 10
away. ATP binding to the microtubule-attached by least-squares fitting to plots of ATPase tubulin concentration (μM) tubulin concentration (μM)
head drives a conformational change that shifts versus tubulin heterodimer concentra-
the tethered head along the microtubule, tion, using Kaleidagraph 3.6.4 (Syn- brain tubulin
biasing and focusing its diffusional search for ergy Software, Reading, PA, USA).
its next binding site (9, 10). In the second type MT, microtubule; HD, heterodimer. (A) C Neurospora kinesin-1 D Rat kinesin-1
70 20
of model, kinesin is proposed to dwell with Vmax 38.1 s−1, Km 0.44 mM for HD, 60
MT MT
Vmax 36.4 s−1, Km 0.39 mM for MT. (B)
ATPase rate (s-1)
ATPase rate (s-1)
both heads attached to the microtubule (5), and 15

Vmax 0.9 s−1, Km 2.03 mM for HD, Vmax
50
12.2 s−1, Km 0.28 mM for MT. (C) 40

HD 10
Vmax 35.9 s−1, Km 1.29 mM for HD,
1 30
Molecular Motors Group, Marie Curie Research Institute,
The Chart, Oxted, Surrey RH8 0TL, UK. 2Medical Research Vmax 71.1 s−1, Km 0.62 mM for MT. 20 5 HD
Council Laboratory of Molecular Biology, Hills Road,
Cambridge CB2 2QH, UK.
(D) Vmax 4.0 s−1, Km 0.86 mM for HD, 10
Vmax 17.0 s−1, Km 0.49 mM for MT. 0 0
*To whom correspondence should be addressed. E-mail: 0 2 4 6 8 10 0 2 4 6 8 10
[email protected] tubulin concentration (μM) tubulin concentration (μM)

REPORTS
Gel-filtration experiments (Fig. 2) revealed ATP “chase” (Fig. 3B and 3D). This shows that
that the stoichiometry and affinity for kinesin-1 only one head releases mantADP immediately
binding to unpolymerized tubulin depends on and that tubulin-activated mantADP release
the bound nucleotide. With no added nucleo- from the second kinesin head requires that
tide, kinesin dimers bind tightly to tubulin het- AMP-PNP or ATP bind to the first head. This
erodimers, and the two proteins elute together two-step tubulin-activated release of ADP from
from a fast protein liquid chromatography gel- kinesin is likely to be related to the half-site
filtration column as a complex in which each ADP release for kinesin binding to micro-
kinesin dimer binds 1.01 ± 0.06 SD (n = 4) tubules first reported by Hackney (14, 15), in
tubulin heterodimers (Fig. 2C). By contrast, in which microtubule-activated ADP release oc-
the presence of the nonhydrolyzable ATP ana- curs from only the microtubule-bound head,
log adenylyl-imidodiphosphate (AMP-PNP), whereas ADP release from the tethered head is
each kinesin dimer binds 1.83 ± 0.08 SD (n = 4) dependent on the binding of AMP-PNP or ATP
tubulin heterodimers (Fig. 2D). In the presence to the microtubule-bound head (16–18). A
of ADP, the binding is weakened, although similar structural mechanism may underlie both
some interaction is still apparent (Fig. 2E). behaviors.
Kinesin binding does not deplete the total amount How might ATP binding to the microtubule-
of tubulin included by the column, even in bound head convert the tethered head from a
AMP-PNP, which indicates that kinesin binding refractory state in which it traps ADP (19)
does not cause tubulin to aggregate. Further- and cannot bind tubulin into a state in which
more, addition of guanosine monophosphate– it binds tubulin and releases ADP? Strain-
carboxypiperazin-4-yl-propyl-1-phosphonic acid based mechanisms are ruled out, because ATP
(GMP-CPP) or taxol to AMP-PNP–kinesin- gating occurs with unpolymerized tubulin.
tubulin complex did not cause microtubule as- There are then two broad possibilities: a steric
sembly, as judged by video-enhanced differential mechanism, in which the tubulin binding site
interference contrast microscopy, suggesting in the tethered ADP head is physically masked,
that the two tubulin heterodimers in the AMP- or an allosteric mechanism, in which the tu-
PNP–kinesin-tubulin complex are held in an bulin binding site on the tethered ADP head
arrangement that prohibits their assembly into undergoes a conformational change that is
a microtubule. triggered by ATP binding to the tubulin-
Tubulin activation of the kinesin ATPase attached, nucleotide-free head. To try to dis-
occurs, as for microtubule activation, by tinguish these possibilities, we fitted 3KIN, the
acceleration of the ADP release step of the only available kinesin dimer crystal structure,
kinetic cycle (Fig. 3). Using kinesin in which into an existing cryogenic electron microscopy
both heads are primed with the fluorescent (cryo-EM) reconstruction of the complex of
analog 2′(3′)-O-(N-methylanthraniloyl)-ADP kinesin dimers with helical (15 protofilament)
(mantADP), we find that half of the fluores- microtubules (20, 21), obtained in the absence
cence signal corresponding to bound mantADP of added nucleotide. Fitting was only possi-
decays on initial mixing with an excess of ble by docking each head separately into the
tubulin heterodimers, whereas the remainder cryo-EM density. In the resulting fit, the sec-
decays only on addition of an AMP-PNP or ond kinesin head sits slightly ahead and to the
Fig. 3. Two-step tubulin-activated A B

Fluorescence intensity (a.u.)
30
ADP release from kinesin. (A and C) kinesinATP kinesin tubulin ATP
Fluorescence transients correspond-
ing to slow binding of 1 mM mantATP 20
to 1 mM rat kinesin, followed by slow
Fig. 2. Superose 12 column chromatography of release of mantADP from both 10
kinesin-tubulin complexes. (A) 6.5 mM rK430 rat kinesin heads induced by a chase
kinesin in ADP. (B) 13 mM pig-brain tubulin in of nonfluorescent 1 mM ATP or 0
AMP-PNP. (C) 13 mM tubulin + 6.5 mM kinesin; 1 mM AMP-PNP. (B and D) The
no added nucleotide. (D) 13 mM tubulin + 6.5 mM same experiment, but with 2 mM -10
kinesin in 0.2 mM AMP-PNP. (E) 13 mM tubulin + tubulin heterodimers added before 0 5 10 15 20 0 5 10 15 20
6.5 mM kinesin in 2 mM ADP. Y-axis marks are the addition of the chasing nu- C D
Fluorescence intensity (a.u.)
in mAU at 290 nm. X-axis marks are at intervals cleotide. Buffer 20 mM Pipes, pH 30
kinesin AMPPNP kinesin tubulin AMPPNP
of 1 ml. The included volume of the column was 6.9, 2 mM MgCl2.
20.0 ml, and the void volume was 8.1 ml. Sam- 20
ples (240 ml) were run at 0.5 ml min−1 in 50 mM
Pipes pH 6.9, 2 mM MgCl2, 1 mM EGTA with or 10
without 2 mM ADP or 0.2 mM AMP-PNP. The
gray vertical line indicates the tubulin elution 0
position. The elution profiles of tubulin alone
and kinesin alone were the same in ADP or -10
AMP-PNP. Binding stoichiometry was measured 0 5 10 15 20 0 5 10 15 20
for the shaded fractions. minutes minutes

REPORTS
left of the attached head (Fig. 4) (22), sug- 3KIN, there is already an asymmetry, but is steric or allosteric, our data identify an ATP
gesting that the tethered head is positioned so modeling indicates that both tubulin binding gate that operates independently of the micro-
as to mask its microtubule binding site. Fur- sites in 3KIN could be occupied without tubule lattice, by a mechanism that is not based
ther work will be required to test this prelim- producing a steric clash (22). Notwithstanding on the strain developed between two attached
inary interpretation. this issue, and whether the blocking mechanism heads, or on a step-change in the diffusional
Existing data indicates that ATP binding has distance to the next binding site along the
profound effects on the kinesin head, recon- microtubule. What role might this gate play in
figuring the active site and shifting the con- Wait-ATP state the kinesin walking mechanism?
formation of the microtubule binding loops L8 Our data show that in the absence of ATP,
and L12 and the neck linker. The neck linker is only one head binds tubulin and that ATP bind-
a 13-residue sequence that connects the coiled- MT-binding ing to this tubulin-attached head is required to
coil tail of kinesin-1 to the C terminus of the D blocked unblock the tubulin binding site on the other
alpha-6 helix in the head domain. Mutating the head. On this basis, we predict the scheme
neck linker or cross-linking it to the main part - + shown in Fig. 5, in which whenever the trail-
of the head inhibits kinesin-driven motility ing kinesin head cycles through to the K.ADP
ATP-Gate
(23–25). Rice and colleagues (9) first proposed intermediate, it detaches and reverts to a state
that ATP binding to the kinesin head drives an in which its tubulin binding site is blocked.
undocked-to-docked transition of its neck linker, Exit from this blocked state requires ATP bind-
and that this event drives stepping of two- ing and is expected to be rate limiting at low
D
headed molecules by shifting the leading head T ATP concentrations and/or high loads. At high
closer to its next binding site along the micro- ATP concentrations and low loads, ATP will
tubule. EM evidence from gold-labeled kinesin bind rapidly, and exit from the blocked state
monomers (9), electron paramagnetic reso- will be correspondingly fast. In these circum-
nance studies (9, 26), and a variety of subse- stances, it is possible that the two-heads-
quent evidence from fluorescently labeled kinesin attached configuration will have the longest
dimers (27–29) is consistent with ATP-dependent DP D lifetime in the cycle. Nonetheless, we empha-
neck-linker docking. Our own data show that size that the ATP gate will still operate, requir-
ATP binding to a tubulin-attached head releases ing that ATP must bind to sanction stepping,
the other head from a sterically or allosterically and fulfilling its function of checking and ad-
blocked state. Apparently, ATP binding drives justing the phasing of the kinetic cycles on the
both neck-linker docking and escape of the DP two heads.
tethered kinesin head from a blocked state. At least one current model proposes that the
Further work will be necessary to determine gate controlling kinesin’s first step is different
whether these two events are coupled. from that controlling subsequent steps (9). In
In the one-head-blocked kinesin dimer state our model, the same gate controls the first and
that we have identified, one head binds tubulin all subsequent steps. Our model is consistent
D
and the other has its tubulin binding site with recent single-molecule work showing that
blocked. It is not clear whether this functional ATP binding under load is necessary to escape
asymmetry is due to a preexisting structural a cycle of repeated futile back-stepping induced
asymmetry or whether blocking of one head by a slowly releasing phosphate analog (12)
results from tubulin binding to the other head. In and with earlier work showing that ATP
D binding is necessary for both foresteps and
backsteps (6, 7). Our model is, however, in-
consistent with proposals that in the ATP-
waiting dwell state, both kinesin heads attach
Wait-ATP state stably to the microtubule (30).
A key point of controversy in the kinesin
mechanism is the question of which biochemi-
D cal step or steps generate force. In our model,
ATP binding generates force, but indirectly, by
sanctioning binding of the ADP-containing lead
head to its next site, which in turn triggers
microtubule-activated ADP release from the
lead head and stabilizes it in a force-holding
Fig. 5. Gating scheme. The cycle begins with
state (6, 8). The requirement that ATP must bind
ATP-gated exit of the tethered head from a
refractory dwell state (dotted box) in which the to allow exit from this state then serves to co-
Fig. 4. Fitting of cryo-EM maps of the apo state tubulin binding site of the tethered head is ordinate lead-head attachment and trail-head
of kinesin dimers attached to microtubules. The blocked. After ATP binding to the attached detachment and to maintain tight coupling by
microtubule plus end is toward the top of the head, the block is released and the tethered ensuring a consistent phase lag between the
page. (Left) Cryo-EM map (20). (Right) Fitted head is then free to diffuse to its next site along kinetic cycles of the two heads (16). An emerg-
orientation of two heads of rat kinesin. One head the microtubule. Hydrolysis and phosphate re- ing general theme for mechanochemical nu-
(yellow) is attached to the underlying microtubule lease on the trailing head return it to a weak- cleotidases such as kinesin and myosin is the
protofilament, whereas the other head (orange) is binding K.ADP state, which then detaches, diffuses amplification of local, nucleotide-dependent
parked in a forward-biased position that masks its to a forward-biased position, and reverts to a conformational changes by causing them to gate
tubulin binding site. blocked state. larger-scale diffusional motions (8).

REPORTS
References and Notes 13. C. E. Walczak, Nat. Cell Biol. 8, 903 (2006). 28. K. Sugata, M. Nakamura, S. Ueki, P. G. Fajer, T. Arata,
1. J. Howard, A. J. Hudspeth, R. D. Vale, Nature 342, 154 14. D. D. Hackney, Proc. Natl. Acad. Sci. U.S.A. 91, 6865 (1994). Biochem. Biophys. Res. Commun. 314, 447 (2004).
(1989). 15. D. D. Hackney, Biochemistry 41, 4437 (2002). 29. A. B. Asenjo, Y. Weinberg, H. Sosa, Nat. Struct. Mol. Biol.
2. W. Hua, E. C. Young, M. L. Fleming, J. Gelles, Nature 16. Y. Z. Ma, E. W. Taylor, J. Biol. Chem. 272, 724 (1997). 13, 648 (2006).
388, 390 (1997). 17. S. P. Gilbert, M. L. Moyer, K. A. Johnson, Biochemistry 37, 30. A. Yildiz, P. R. Selvin, Trends Cell Biol. 15, 112
3. K. Kaseda, H. Higuchi, K. Hirose, Nat. Cell Biol. 5, 1079 792 (1998). (2005).
(2003). 18. I. Crevel, N. Carter, M. Schliwa, R. Cross, EMBO J. 18, 31. We thank our colleagues at the Medical Research Council
4. C. L. Asbury, A. N. Fehr, S. M. Block, Science 302, 2130 5863 (1999). (MRC) Laboratory of Molecular Biology and the Marie
(2003). 19. R. A. Cross, Trends Biochem. Sci. 29, 301 (2004). Curie Research Institute for discussions, the MRC for
5. A. Yildiz, M. Tomishige, R. D. Vale, P. R. Selvin, Science 20. K. Hirose, J. Lowe, M. Alonso, R. A. Cross, L. A. Amos, strategic funding for our S. pombe tubulin project, and
303, 676 (2004). Mol. Biol. Cell 10, 2063 (1999). the Marie Curie Foundation for continuing support. The
6. N. J. Carter, R. A. Cross, Nature 435, 308 (2005). 21. R. A. Cross et al., Philos. Trans. R. Soc. London B Biol. Sci. first author dedicates this paper to Graham Currie.
7. M. Nishiyama, H. Higuchi, T. Yanagida, Nat. Cell Biol. 4, 355, 459 (2000).
790 (2002). 22. Materials and methods are available as supporting
8. N. J. Carter, R. A. Cross, Curr. Opin. Cell Biol. 18, 61 material on Science Online. Supporting Online Material
(2006). 23. M. Tomishige, R. D. Vale, J. Cell Biol. 151, 1081 (2000).
9. S. Rice et al., Nature 402, 778 (1999). 24. R. B. Case, S. Rice, C. L. Hart, B. Ly, R. D. Vale, Curr. Biol.
10. W. H. Mather, R. F. Fox, Biophys. J. 91, 2416 (2006). 10, 157 (2000). SOM Text
Figs. S1 to S4
11. S. S. Rosenfeld, P. M. Fordyce, G. M. Jefferson, P. H. King, 25. K. Hahlen et al., J. Biol. Chem. 281, 18868 (2006).
S. M. Block, J. Biol. Chem. 278, 18550 (2003). 26. S. Rice et al., Biophys. J. 84, 1844 (2003). References
12. N. R. Guydosh, S. M. Block, Proc. Natl. Acad. Sci. U.S.A. 27. S. S. Rosenfeld, G. M. Jefferson, P. H. King, J. Biol. Chem. 31 October 2006; accepted 2 February 2007
103, 8054 (2006). 276, 40167 (2001). 10.1126/science.1136985

Career advice,
Protein Crystal Services
ProXyChem™ provides cost effective and
insight and tools.
timely crystallographic structure determi-
nation services using state-of-the-art Turn to the experts for the big picture.
synchrotron facilities and a highly
experienced team of scientists. Our ever- Visit www.ScienceCareers.org
expanding portfolio of catalog proteins is
one of the most extensive in the industry,
allowing for the rapid structure determina-
tion of ligands bound to their target
proteins.
Catalog Proteins include
AurA GSK3β PTP1b
CK2α KDR PDE4B, 4D, 5A
CDK4 JNK 1, 2, 3 PPARα,γ
Erk2 MAPKAP2 Cathepsin B
Future Catalog Proteins include
p38α,γ CDK5, 6, 7 MEK1, 2
cMET PDK1 Tie2
Other proteins available upon request
For information about our full portfolio of catalog
proteins visit our website at www.proxychem.com
Phone: 1-617-395-4266
Portland Press » publishing innovation

Your career is too important to leave to chance. So to find

the right job or get career advice, turn to the experts. At

ScienceCareers.org we know science. And we are committed to

helping take your career forward. Our knowledge is firmly

founded on the expertise of Science, the premier scientific

journal, and the long experience of AAAS in advancing science

around the world. Put yourself in the picture with the experts in
science. Visit www.ScienceCareers.org.

ALBERT EINSTEIN and related rights ™/© of
The Hebrew University of Jerusalem, used
under license. Represented by The Roger
Richman Agency, Inc., www.albert-einstein.net.

𰀟𰀘𰀕𰀎𰃛𰀰𰀊𰀘𰀎𰀢𰀊𰀎𰃛𰀲𰀎𰀊𰀗𰀢𰀤𰀟𰀤𰀖𰀘𰀎𰀰
𰀁𰀁𰀁𰀭𰃉𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰀉𰃒𰃄𰂅𰂖𰁦𰃄𰃄𰃊𰀡𰁲𰁲𰂅𰁓𰁦𰀓𰁦𰀺𰃍𰃒𰃀𰁦
𰀊𰀎𰀝𰀝𰀭𰀖𰀔𰀟𰀁𰀝𰀖𰀟𰀔
𰀪𰀕𰀡𰀭𰀪𰀕𰀡𰀬𰀷𰀝𰀁𰀮𰀖𰀡𰀟𰁚𰀜𰀖𰀟𰀁𰀭𰀎𰀭𰁚
𰀁𰀟𰀌𰀜𰀖𰀟𰀁𰀭𰀎𰀖𰀟𰀕𰀖𰀉𰀖𰀮𰀡𰀬𰀭
𰀊𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂅𰃄𰃊𰀺𰃊𰁊𰃀𰂜𰀺𰁝𰃊𰀺𰂖𰁝𰃊𰁲𰃀𰁦𰂵𰃒𰁦𰂖𰃍𰂎𰃛𰃊𰁓𰂜𰂖𰁲𰃒𰃄𰂅𰂖𰁹𰃊𰁳𰃊𰁦𰂎𰁝𰂮𰃊𰀮𰂂𰂅𰃄𰃊𰂜𰃘𰁦𰃀𰃘𰂅𰁦𰃙𰃊𰁝𰁦𰃄𰁓𰃀𰂅𰁊𰁦𰃄𰃊
𰃄𰂜𰂓𰁦𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰁦𰃚𰁓𰂅𰃍𰂅𰂖𰁹𰃊𰂖𰁦𰃙𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰂅𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰂨𰃀𰂜𰂓𰂅𰃄𰁦𰃊𰃍𰂜𰃊𰁝𰃀𰂅𰃘𰁦𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰃊𰃘𰂅𰀺𰃊𰂓𰂜𰃀𰁦𰃊
𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰁚𰃊𰃒𰃄𰁦𰃀𰂄𰁲𰃀𰂅𰁦𰂖𰁝𰂎𰃛𰃊𰀺𰃄𰃄𰀺𰃛𰃄𰁚𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰁦𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰃊𰂜𰁲𰃊𰂖𰁦𰃙𰃊
𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰂮𰃊𰀉𰃛𰃊𰀝𰃛𰂖𰂖𰁦𰃊𰀝𰁦𰁝𰁦𰃀𰂓𰀺𰂖
𰀯
𰂖𰁝𰁦𰃀𰃄𰃍𰀺𰂖𰁝𰂅𰂖𰁹𰃊𰃍𰂂𰁦𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰂖𰁦𰃍𰃙𰂜𰃀𰂍𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰂜𰂖𰃄𰃊
𰃍𰂂𰀺𰃍𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰃀𰁦𰂵𰃒𰂅𰃀𰁦𰃊𰃍𰂜𰃊𰁲𰃒𰂖𰁓𰃍𰂅𰂜𰂖𰃊𰂅𰃄𰃊𰀺𰃊𰂓𰀺𰂌𰂜𰃀𰃊𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰁳𰃊𰁓𰃊𰁓𰂂𰀺𰂎𰂎𰁦𰂖𰁹𰁦𰂮𰃊𰀜𰂅𰂖𰀺𰃄𰁦𰃄𰃊
𰀺𰃀𰁦𰃊𰂅𰂖𰃘𰂜𰂎𰃘𰁦𰁝𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰂓𰀺𰂖𰃛𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰃒𰃄𰁦𰃊𰁲𰂜𰃀𰃊𰁹𰃀𰂜𰃙𰃍𰂂𰃊
𰂨𰃀𰂜𰂎𰂅𰁲𰁦𰃀𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰁝𰂅𰁲𰁲𰁦𰃀𰁦𰂖𰃍𰂅𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰀺𰂖𰁝𰃊𰁝𰁦𰀺𰃍𰂂𰂮𰃊𰀁𰃄𰃄𰀺𰃛𰃄𰃊𰃍𰂜𰃊𰁦𰂎𰃒𰁓𰂅𰁝𰀺𰃍𰁦𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰂄
𰃀𰁦𰂎𰀺𰃍𰁦𰁝𰃊𰁓𰂜𰂓𰂨𰂜𰂖𰁦𰂖𰃍𰃄𰃊𰂜𰁲𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰂓𰀺𰃛𰃊𰃀𰁦𰂎𰃛𰃊𰂜𰂖𰃊𰂺𰁓𰂎𰀺𰃄𰃄𰂅𰁓𰂻𰃊𰂓𰁦𰃍𰂂𰂜𰁝𰃄𰃊𰃒𰃄𰂅𰂖𰁹𰃊
𰃀𰀺𰁝𰂅𰂜𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰁦𰃄𰃊𰃍𰂜𰃊𰁝𰁦𰃍𰁦𰁓𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰀺𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰃊𰂪𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰂫𰁚𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂅𰂜𰂖𰁚𰃊
𰂜𰃀𰃊𰁝𰁦𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰂜𰃀𰃊𰃍𰂂𰁦𰃛𰃊𰂓𰀺𰃛𰃊𰃀𰁦𰂎𰃛𰃊𰂜𰂖𰃊𰂓𰂜𰃀𰁦𰃊𰃀𰁦𰁓𰁦𰂖𰃍𰂎𰃛𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰁦𰁝𰃊𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰃍𰃊
𰂜𰃀𰃊𰁓𰂜𰂎𰂜𰃀𰂅𰂓𰁦𰃍𰃀𰂅𰁓𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰂅𰁦𰃄𰂮𰃊𰀁𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰃍𰂜𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰁚𰃊
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃄𰁦𰃀𰂅𰂖𰁦𰁚𰃊𰀺𰂖𰁝𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰂂𰃀𰁦𰂜𰂖𰂅𰂖𰁦𰃊𰀺𰂖𰁝𰃊𰃍𰂜𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃄𰃊
𰀺𰃀𰁦𰃊𰀺𰂎𰃄𰂜𰃊𰃒𰃄𰁦𰁲𰃒𰂎𰃊𰁲𰂜𰃀𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰂮𰃊𰀊𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰃊𰃙𰂅𰃍𰂂𰃊𰀺𰂖𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊
𰂅𰂖𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃊𰀺𰃊𰃘𰀺𰃀𰂅𰁦𰃍𰃛𰃊𰂜𰁲𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰃀𰁦𰃊𰁝𰃀𰂅𰃘𰂅𰂖𰁹𰃊𰁊𰀺𰃄𰂅𰁓𰃊
𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁚𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰂅𰂖𰁹𰃊𰁦𰂖𰃞𰃛𰂓𰁦𰃄𰁚𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰁚𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰁚𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰃊
𰂜𰁲𰁲𰂄𰃍𰂂𰁦𰂄𰃄𰂂𰁦𰂎𰁲𰃊𰂜𰃀𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰂍𰂅𰃍𰃄𰂮𰃊
𰀜𰁦𰁦𰂨𰂅𰂖𰁹𰃊𰀮𰃀𰀺𰁓𰂍
𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰂅𰃍𰁦𰃊𰂅𰃄𰃊𰀺𰃊𰃒𰂖𰂅𰂵𰃒𰁦𰃊𰂜𰂖𰂎𰂅𰂖𰁦𰃊𰃀𰁦𰃄𰂜𰃒𰃀𰁓𰁦𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰁦𰁝𰃊𰀺𰃍𰃊𰀊𰁦𰂎𰂎𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊
𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰂪𰀊𰀭𰀮𰂫𰁚𰃊𰃍𰂜𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰀺𰂎𰂎𰃊𰂅𰂖𰃊𰃘𰂅𰃘𰂜𰃊𰂓𰀺𰂓𰂓𰀺𰂎𰂅𰀺𰂖𰃊
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰃄𰂅𰃍𰁦𰃄𰂮𰃊𰀓𰃒𰂖𰁝𰁦𰁝𰃊𰁊𰃛𰃊𰁊𰂜𰃍𰂂𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰃀𰁦𰁦𰃊𰁹𰃀𰀺𰂖𰃍𰃄𰃊𰁲𰃀𰂜𰂓𰃊𰃍𰂂𰁦𰃊
𰀯𰀭𰀟𰀺𰃍𰂅𰂜𰂖𰀺𰂎𰃊𰀖𰂖𰃄𰃍𰂅𰃍𰃒𰃍𰁦𰃄𰃊𰂜𰁲𰃊𰀕𰁦𰀺𰂎𰃍𰂂𰃊𰂪𰀟𰀖𰀕𰂫𰁚𰃊𰂅𰃍𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁦𰃄𰃊𰂜𰃘𰁦𰃀𰃊𰂣𰁴𰁚𰃟𰃟𰃟𰃊𰃒𰂖𰂅𰂵𰃒𰁦𰃊𰃄𰂅𰃍𰁦𰃄𰃊𰁲𰃀𰂜𰂓𰃊
𰃍𰂂𰁦𰃊𰂎𰂅𰃍𰁦𰃀𰀺𰃍𰃒𰃀𰁦𰃊𰀺𰂖𰁝𰃊𰂜𰃘𰁦𰃀𰁫𰁚𰃟𰃟𰃟𰃊𰂨𰃀𰁦𰃘𰂅𰂜𰃒𰃄𰂎𰃛𰃊𰃒𰂖𰂨𰃒𰁊𰂎𰂅𰃄𰂂𰁦𰁝𰃊𰃄𰂅𰃍𰁦𰃄𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰁦𰁝𰃊𰁊𰃛𰃊𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰃄𰃍𰃄𰃊
𰀺𰃍𰃊𰀊𰀭𰀮𰃊𰀺𰂖𰁝𰃊𰁦𰂎𰃄𰁦𰃙𰂂𰁦𰃀𰁦𰂮𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰂅𰃍𰁦𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁦𰃄𰃊𰃄𰂅𰃍𰁦𰃄𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰀺𰂖𰁝𰃊
𰂺
𰁓𰂜𰂖𰃍𰀺𰂅𰂖𰃄𰃊𰁦𰃚𰃍𰁦𰂖𰃄𰂅𰃘𰁦𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰁓𰃒𰃀𰀺𰃍𰁦𰁝𰃊𰁲𰃀𰂜𰂓𰃊𰃍𰂂𰁦𰃊𰂎𰂅𰃍𰁦𰃀𰀺𰃍𰃒𰃀𰁦𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰃍𰂂𰁦𰃊𰃀𰁦𰁹𰃒𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰀺𰂖𰁝𰃊
𰁊𰂅𰂜𰂎𰂜𰁹𰂅𰁓𰀺𰂎𰃊𰁓𰂜𰂖𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃄𰃊𰂜𰁲𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰂮𰃊𰀉𰃛𰃊𰂓𰂅𰁝𰃊𰃑𰃟𰃟𰃇𰁚𰃊𰀺𰂖𰃊𰁦𰂖𰂂𰀺𰂖𰁓𰁦𰁝𰃊𰀜𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰀺𰃀𰁦𰃊𰂅𰂖𰃘𰂜𰂎𰃘𰁦𰁝𰃊𰂅𰂖𰃊𰁦𰃘𰁦𰃀𰃛𰃊𰁓𰁦𰂎𰂎𰃊
𰃘𰁦𰃀𰃄𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰂅𰃍𰁦𰃊𰂅𰃄𰃊𰁦𰃚𰂨𰁦𰁓𰃍𰁦𰁝𰃊𰃍𰂜𰃊𰁊𰁦𰃊𰂎𰀺𰃒𰂖𰁓𰂂𰁦𰁝𰂮𰃊𰀬𰂜𰁊𰁦𰃀𰃍𰂜𰃊𰀌𰂮𰃊𰀪𰂜𰂎𰀺𰂍𰂅𰁦𰃙𰂅𰁓𰃞𰁚𰃊𰁓𰂂𰂅𰁦𰁲𰃊
𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰁳𰃊𰁓𰃊𰂜𰁲𰁳𰃊𰁓𰁦𰃀𰁚𰃊𰁦𰃚𰂨𰁦𰁓𰃍𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰃍𰂂𰁦𰃊𰃄𰂅𰃍𰁦𰃊𰃙𰂅𰂎𰂎𰃊𰁊𰁦𰃊𰁲𰃀𰁦𰁦𰂎𰃛𰃊𰀺𰁓𰁓𰁦𰃄𰃄𰂅𰁊𰂎𰁦𰃊𰁊𰃛𰃊𰃍𰂂𰁦𰃊𰀺𰁓𰀺𰁝𰁦𰂓𰂅𰁓𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃊𰃛𰂜𰃒𰃊𰁓𰀺𰂖𰃊𰃍𰂂𰂅𰂖𰂍𰃊𰂜𰁲𰁚𰃊
𰃙𰂜𰃀𰂎𰁝𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰀺𰃍𰃊𰃍𰂂𰁦𰃀𰁦𰃊𰃙𰂅𰂎𰂎𰃊𰂨𰃀𰂜𰁊𰀺𰁊𰂎𰃛𰃊𰁊𰁦𰃊𰀺𰃊𰃄𰃒𰁊𰃄𰁓𰃀𰂅𰁊𰁦𰃀𰂽𰃄𰃊𰁲𰁦𰁦𰃊𰁲𰂜𰃀𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰃒𰃄𰁦𰃀𰃄𰂮𰃊𰂺𰀵𰁦𰃊𰂖𰁦𰁦𰁝𰃊
𰃍𰂂𰁦𰃊𰁲𰃒𰂖𰁝𰃄𰃊𰃍𰂜𰃊𰂍𰁦𰁦𰂨𰃊𰀺𰁝𰁝𰂅𰂖𰁹𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰁚𰂻𰃊𰀪𰂜𰂎𰀺𰂍𰂅𰁦𰃙𰂅𰁓𰃞𰃊𰃄𰀺𰃛𰃄𰁚𰃊𰂺𰃄𰂜𰃊𰃍𰂂𰁦𰃊𰁝𰀺𰃍𰀺𰁊𰀺𰃄𰁦𰃊𰃙𰂜𰂖𰂽𰃍𰃊𰀺𰂖𰁝𰁚𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰃊𰂜𰃀𰃊𰂅𰂖𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰁚𰃊𰂅𰂖𰃊𰂓𰂜𰃄𰃍𰃊
𰁊𰁦𰁓𰂜𰂓𰁦𰃊𰂜𰁊𰃄𰂜𰂎𰁦𰃍𰁦𰂮𰂻
𰀕𰃒𰁝𰀺𰃊𰀭𰂂𰃒𰁊𰁦𰂅𰃍𰀺𰁚𰃊𰃍𰁦𰁓𰂂𰂖𰂅𰁓𰀺𰂎𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃊𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰃄𰃍𰁚𰃊𰀎𰀞𰀌𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰁚𰃊𰀺𰂖𰃊𰀺𰁲𰁳𰃊𰂎𰂅𰀺𰃍𰁦𰃊
𰂜𰁲𰃊𰀞𰁦𰃀𰁓𰂍𰃊𰀜𰀔𰀺𰀁𰁚𰃊𰃙𰂂𰂅𰁓𰂂𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃄𰃊𰃍𰂂𰁦𰃊𰀊𰀺𰂎𰁊𰂅𰂜𰁓𰂂𰁦𰂓𰃊𰁊𰃀𰀺𰂖𰁝𰁚𰃊𰂖𰂜𰃍𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰃊𰁊𰂅𰁹𰃊𰁲𰂜𰁓𰃒𰃄𰃊𰂜𰁲𰃊
𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰃊𰂅𰃄𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰃊𰃍𰃀𰀺𰂖𰃄𰁝𰃒𰁓𰃍𰂅𰂜𰂖𰂮𰃊𰀊𰀺𰂎𰁊𰂅𰂜𰁓𰂂𰁦𰂓𰂽𰃄𰃊𰀖𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰀪𰀺𰃍𰂂𰃙𰀺𰃛𰃊𰀬𰁦𰃄𰂜𰃒𰃀𰁓𰁦𰃊
𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰃊𰂨𰃀𰂜𰁓𰁦𰃄𰃄𰁦𰃄𰂮
𰂻
𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃄𰃊𰂜𰃘𰁦𰃀𰃑𰃟𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰂅𰂖𰁹𰃊𰀁𰂍𰃍𰃊𰀺𰂖𰁝𰃊𰀪𰀖𰃎𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰁚𰃊
𰀺𰂖𰁝𰃊𰂓𰀮𰀡𰀬𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰂮𰃊𰀮𰂂𰁦𰃀𰁦𰃊𰀺𰃀𰁦𰃊𰀺𰂎𰃄𰂜𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰂄𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰂅𰂖𰁹𰃊𰂜𰂖𰁦𰃄𰃊
𰀝𰂜𰂜𰂍𰃊𰁲𰂜𰃀𰃊𰃍𰂂𰁦𰃄𰁦𰃊𰀯𰂨𰁓𰂜𰂓𰂅𰂖𰁹𰀁𰃀𰃍𰂅𰁓𰂎𰁦𰃄
𰁲𰂜𰃀𰃊𰁝𰂅𰀺𰁊𰁦𰃍𰁦𰃄𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰁵𰃊𰀺𰂓𰂓𰀺𰃍𰂅𰂜𰂖𰂮𰃊𰀯𰃄𰁦𰃀𰃄𰃊𰃄𰁦𰂎𰁦𰁓𰃍𰃊𰀺𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰃙𰂅𰃍𰂂𰂅𰂖𰃊𰀺𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃊𰃍𰂜𰃊𰂜𰁊𰃍𰀺𰂅𰂖𰃊𰀭𰃍𰁦𰂓𰃊𰀊𰁦𰂎𰂎𰃄𰃊𰁭𰀁𰂨𰃀𰂅𰂎𰃊𰃑𰃟
𰂎𰂅𰃄𰃍𰃄𰃊𰂜𰁲𰃊𰀺𰃘𰀺𰂅𰂎𰀺𰁊𰂎𰁦𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰂍𰂅𰃍𰃄𰁚𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰁚𰃊𰁦𰂖𰃞𰃛𰂓𰁦𰃄𰁚𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰁚𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰀬𰀟𰀁𰂅𰃊𰁭𰀛𰃒𰂖𰁦𰃊𰂣
𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰂮𰃊𰂺𰀜𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰀺𰃀𰁦𰃊𰂅𰂖𰃘𰂜𰂎𰃘𰁦𰁝𰃊𰂅𰂖𰃊𰁦𰃘𰁦𰃀𰃛𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃊𰃛𰂜𰃒𰃊𰁓𰀺𰂖𰃊𰃍𰂂𰂅𰂖𰂍𰃊𰀊𰁦𰂎𰂎𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰃑𰁭𰀛𰃒𰂖𰁦𰃊𰃑𰃑
𰂜𰁲𰁚𰃊𰀺𰂖𰁝𰁚𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰃊𰂜𰃀𰃊𰂅𰂖𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰁚𰃊𰂅𰂖𰃊𰂓𰂜𰃄𰃍𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰃊𰂨𰃀𰂜𰁓𰁦𰃄𰃄𰁦𰃄𰁚𰂻𰃊𰀭𰂂𰃒𰁊𰁦𰂅𰃍𰀺𰃊𰃄𰀺𰃛𰃄𰂮𰃊𰂺𰀮𰂂𰁦𰃊𰀞𰂅𰁓𰃀𰂜𰀺𰃀𰃀𰀺𰃛𰃊𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰂅𰁦𰃄𰃊𰁭𰀁𰃒𰁹𰃒𰃄𰃍𰃊𰃑𰁷
𰁝𰂅𰁲𰁲𰁦𰃀𰁦𰂖𰃍𰂅𰀺𰂎𰃊𰃀𰁦𰁹𰃒𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰁦𰃄𰃊𰁝𰂅𰁓𰃍𰀺𰃍𰁦𰃄𰃊𰃍𰂂𰁦𰃊𰁓𰂂𰀺𰃀𰀺𰁓𰃍𰁦𰃀𰂅𰃄𰃍𰂅𰁓𰃄𰃊𰂜𰁲𰃊𰁓𰁦𰂎𰂎𰃄𰁚𰃊𰃙𰂂𰁦𰃍𰂂𰁦𰃀𰃊
𰃍𰂂𰁦𰃛𰃊𰃀𰁦𰂓𰀺𰂅𰂖𰃊𰂖𰂜𰃀𰂓𰀺𰂎𰃊𰂜𰃀𰃊𰁊𰁦𰁓𰂜𰂓𰁦𰃊𰂨𰀺𰃍𰂂𰂜𰂎𰂜𰁹𰂅𰁓𰀺𰂎𰂮𰂻
𰀁𰂓𰁊𰂅𰂜𰂖𰁚𰃊𰀺𰂖𰃊𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰀉𰂅𰂜𰃄𰃛𰃄𰃍𰁦𰂓𰃄𰃊𰁊𰃒𰃄𰂅𰂖𰁦𰃄𰃄𰁚𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰃙𰁦𰁊𰃄𰂅𰃍𰁦𰃊𰃙𰂂𰁦𰃀𰁦𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰁦𰃀𰃄𰃊𰃙𰂅𰂎𰂎𰃊𰀖𰂖𰁓𰂎𰃒𰃄𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰃊𰂅𰂖𰃊𰃍𰂂𰂅𰃄𰃊𰀺𰃀𰃍𰂅𰁓𰂎𰁦𰃊𰁝𰂜𰁦𰃄𰃊𰂖𰂜𰃍𰃊𰂅𰂖𰁝𰂅𰁓𰀺𰃍𰁦𰃊
𰁦𰂖𰁝𰂜𰃀𰃄𰁦𰂓𰁦𰂖𰃍𰃊𰁊𰃛𰃊𰁦𰂅𰃍𰂂𰁦𰃀𰃊𰀁𰀁𰀁𰀭𰃊𰂜𰃀𰃊𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰁚𰃊𰂖𰂜𰃀𰃊𰂅𰃄𰃊𰂅𰃍𰃊𰂓𰁦𰀺𰂖𰃍𰃊𰃍𰂜𰃊
𰁊𰁦𰃊𰀺𰁊𰂎𰁦𰃊𰃍𰂜𰃊𰃄𰁦𰀺𰃀𰁓𰂂𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰃎𰃟𰃟𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰁲𰂜𰃀𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰂜𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊𰃍𰂜𰃊𰃄𰁦𰁦𰃊𰃙𰂂𰀺𰃍𰃊𰂅𰃄𰃊𰂅𰂓𰂨𰂎𰃛𰃊𰃍𰂂𰀺𰃍𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰃊𰂜𰃀𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃄𰃊𰀺𰃀𰁦𰃊𰃄𰃒𰂨𰁦𰃀𰂅𰂜𰃀𰃊𰃍𰂜𰃊𰃍𰂂𰂜𰃄𰁦𰃊𰂜𰁲
𰃒𰂨𰂄𰃊𰀺𰂖𰁝𰃊𰁝𰂜𰃙𰂖𰃄𰃍𰃀𰁦𰀺𰂓𰃊𰀺𰂖𰁝𰃊𰃙𰂂𰁦𰃍𰂂𰁦𰃀𰃊𰂅𰃍𰃄𰃊𰀺𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰃊𰂅𰃄𰃊𰀺𰃄𰃄𰂜𰁓𰂅𰀺𰃍𰁦𰁝𰃊𰃙𰂅𰃍𰂂𰃊𰃍𰂂𰁦𰃊𰁓𰁦𰂎𰂎𰃊𰁓𰂜𰂖𰃍𰂅𰂖𰃒𰁦𰁝𰃊𰂁𰂜𰃍𰂂𰁦𰃀𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰂮
𰂰𰃥𰁽
𰀟𰀘𰀕𰀎𰃛𰀰𰀊𰀘𰀎𰀢𰀊𰀎𰃛𰀲𰀎𰀊𰀗𰀢𰀤𰀟𰀤𰀖𰀘𰀎𰀰𰀁𰀁𰀁𰀭𰃉𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰀉𰃒𰃄𰂅𰂖𰁦𰃄𰃄𰃊𰀡𰁲𰁲𰂅𰁓𰁦𰃊𰀓𰁦𰀺𰃍𰃒𰃀𰁦
𰀊𰁦𰂎𰂎𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹
𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰃍𰃊𰃀𰁦𰃄𰂜𰂖𰀺𰂖𰃍𰃊𰁦𰂖𰁦𰃀𰁹𰃛𰃊𰃍𰃀𰀺𰂖𰃄𰁲𰁦𰃀𰂫𰁝𰁦𰃍𰁦𰁓𰃍𰂅𰂜𰂖𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰃊𰂅𰃄𰃊𰂂𰁦𰂎𰂨𰂅𰂖𰁹𰃊
𰂺𰀖𰂖𰃊𰀺𰂖𰃊𰂅𰁝𰁦𰀺𰂎𰃊𰃄𰂅𰃍𰃒𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰂨𰀺𰃍𰂅𰁦𰂖𰃍𰃄𰂽𰁝𰃀𰂅𰃘𰁦𰃊𰁝𰃀𰃒𰁹𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰃊𰂅𰂖𰃊𰁓𰀺𰂖𰁓𰁦𰃀𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁚𰃊𰀺𰂓𰂜𰂖𰁹𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰀺𰃀𰁦𰀺𰃄𰂮𰃊𰀖𰀞𰀁𰀪
𰃒𰃄𰁦𰃄𰃊𰀺𰃊𰂂𰂅𰁹𰂂𰂄𰀺𰁲𰁳𰃊𰂖𰂅𰃍𰃛𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰁚𰃊𰃍𰃀𰂅𰃘𰀺𰂎𰁦𰂖𰃍𰃊𰂓𰁦𰃍𰀺𰂎𰃊𰁓𰂜𰂓𰂨𰂎𰁦𰃚𰃊𰂅𰂓𰂓𰂜𰁊𰂅𰂎𰂅𰃞𰁦𰁝
𰁊𰂎𰂜𰂜𰁝𰃊𰁓𰂜𰃒𰂎𰁝𰁊𰁦𰃊𰂓𰂜𰂖𰂅𰃍𰂜𰃀𰁦𰁝𰂜𰂖𰃊𰂖𰀺𰂖𰂜𰂨𰀺𰃀𰃍𰂅𰁓𰂎𰁦𰃄𰃊𰁊𰂜𰃒𰂖𰁝𰃘𰂅𰀺𰃊𰀺𰃊𰂎𰂅𰂖𰂍𰁦𰃀𰃊𰃍𰂜𰃊𰀺𰃊𰃍𰁦𰃀𰁊𰂅𰃒𰂓𰃊𰂪𰀮𰁊𰂫𰁝𰂜𰂖𰂜𰃀𰁚𰃊𰃍𰂜𰃊𰁊𰂅𰂖𰁝
𰁲𰂜𰃀𰃊𰃍𰂂𰁦𰃊𰁝𰃀𰃒𰁹𰂽𰃄𰃊𰁦𰁲𰁲𰁦𰁓𰃍𰃊𰂜𰂖𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰁦𰂮𰃊𰀵𰂂𰁦𰂖𰃊𰀺𰃊𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰃍𰃊𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃊𰁓𰂜𰂖𰃍𰀺𰂅𰂖𰂅𰂖𰁹𰃊𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰁚𰃊
𰃄𰁦𰃀𰂅𰂖𰁦𰁚𰃊𰂜𰃀𰃊𰃍𰂂𰃀𰁦𰂜𰂖𰂅𰂖𰁦𰃊𰂅𰃄𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰁦𰁝𰁊𰃛𰃊𰀺𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰁚𰃊𰃍𰂂𰁦𰃊𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰃍𰃊
𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰂮𰂻𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃊𰁊𰂅𰂖𰁝𰃄𰃊𰃍𰂜𰃊𰃍𰂂𰁦𰃊𰀖𰀞𰀁𰀪𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰁚𰃊𰁊𰃀𰂅𰂖𰁹𰂅𰂖𰁹𰃊𰂅𰃍𰃊𰁓𰂎𰂜𰃄𰁦𰃀𰃊
𰃍𰂜𰃊𰃍𰂂𰁦𰃊𰀮𰁊𰂄𰁝𰂜𰂖𰂜𰃀𰃊𰁓𰂜𰂓𰂨𰂎𰁦𰃚𰁚𰃊𰃀𰁦𰃄𰃒𰂎𰃍𰂅𰂖𰁹𰃊𰂅𰂖𰃊𰃀𰁦𰃄𰂜𰂖𰀺𰂖𰁓𰁦𰃊𰁦𰂖𰁦𰃀𰁹𰃛𰃊𰃍𰃀𰀺𰂖𰃄𰁲𰁦𰃀𰃊𰁲𰃀𰂜𰂓𰃊
𰃍𰂂𰁦𰃊𰁝𰂜𰂖𰂜𰃀𰃊𰃍𰂜𰃊𰃍𰂂𰁦𰃊𰀺𰁓𰁓𰁦𰂨𰃍𰂜𰃀𰃊𰂪𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰂫𰃊𰀺𰂖𰁝𰃊𰁦𰂓𰂅𰃄𰃄𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰀺𰃊
𰃄𰃒𰃀𰁲𰀺𰁓𰁦𰁚𰃊𰂖𰃒𰁓𰂎𰁦𰃒𰃄𰁚𰃊𰂜𰃀𰃊𰁓𰃛𰃍𰂜𰂨𰂎𰀺𰃄𰂓𰂮𰃊𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰂽𰃄𰃊𰂜𰃙𰂖𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰃊𰀮𰃀𰀺𰂖𰃄𰁝𰃒𰁓𰃍𰂅𰂜𰂖𰃊𰃄𰃍𰀺𰁊𰂎𰁦𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰃊𰀺𰃄𰃊𰂓𰁦𰀺𰃄𰃒𰃀𰀺𰁊𰂎𰁦𰃊𰂎𰂅𰁹𰂂𰃍𰂮𰃊𰀖𰂖𰃊𰀺𰁝𰁝𰂅𰃍𰂅𰂜𰂖𰃊𰃍𰂜𰃊𰀮𰀬𰂄𰀓𰀬𰀎𰀮𰁚𰃊𰀖𰀞𰀁𰀪𰃊𰁓𰀺𰂖𰃊𰁊𰁦𰃊
𰀜𰂖𰂜𰃙𰂎𰁦𰁝𰁹𰁦𰃊𰀎𰂖𰃘𰂅𰃀𰂜𰂖𰂓𰁦𰂖𰃍𰂪𰀭𰀮𰀜𰀎𰂫𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃄𰃊𰃍𰂜𰂜𰂎𰃄𰃊𰀺𰂖𰁝𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰁦𰃄𰃊𰃍𰂜𰃊𰂨𰁦𰃀𰁲𰂜𰃀𰂓𰁦𰁝𰃊𰃒𰃄𰂅𰂖𰁹𰃊𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰁓𰁦𰃊𰂨𰂜𰂎𰀺𰃀𰂅𰃞𰀺𰃍𰂅𰂜𰂖𰃊𰂪𰀓𰀪𰂫𰃊𰃀𰁦𰀺𰁝𰂜𰃒𰃍𰂮𰃊𰀮𰂂𰂅𰃄𰃊𰀺𰃄𰃄𰀺𰃛𰃊
𰀺𰂎𰂎𰂜𰃙𰃊𰃒𰃄𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰀺𰁓𰂵𰃒𰂅𰃀𰁦𰁚𰃊𰃒𰂖𰁝𰁦𰃀𰃄𰃍𰀺𰂖𰁝𰁚𰃊𰀺𰂖𰁝𰃊𰃒𰃄𰁦𰃊𰃍𰂂𰁦𰃊𰃘𰀺𰃄𰃍𰃊𰀺𰃀𰃀𰀺𰃛𰃊𰂜𰁲𰃊𰁓𰁦𰂎𰂎𰃊𰁓𰂅𰃀𰁓𰃒𰂓𰃘𰁦𰂖𰃍𰃄𰃊𰃍𰂂𰁦𰃊𰂖𰁦𰁦𰁝𰃊𰁲𰂜𰃀𰃊𰂂𰂅𰁹𰂂𰂎𰃛𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰀺𰃄𰃊𰃙𰁦𰂎𰂎𰃊𰀺𰃄𰃊𰃍𰂂𰁦𰃊
𰃀𰁦𰁹𰃒𰂎𰀺𰃍𰂜𰃀𰃛𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰃍𰂂𰀺𰃍𰃊𰂅𰃄𰃊𰀺𰃘𰀺𰂅𰂎𰀺𰁊𰂎𰁦𰁚𰃊𰃍𰂂𰃀𰂜𰃒𰁹𰂂𰃊𰀺𰃊𰁝𰀺𰃍𰀺𰁊𰀺𰃄𰁦𰁚𰃊𰃘𰂅𰃀𰃍𰃒𰀺𰂎𰃊𰁦𰁲𰁲𰂜𰃀𰃍𰃊𰀺𰂖𰁝𰃊𰁓𰂜𰃄𰃍𰃊𰃀𰁦𰂵𰃒𰂅𰃀𰁦𰁝𰃊𰃍𰂜𰃊𰁹𰁦𰂖𰁦𰃀𰀺𰃍𰁦𰃊𰃍𰂂𰁦𰂓𰂮
𰂌𰂜𰃒𰃀𰂖𰀺𰂎𰃊𰁝𰂅𰃄𰁓𰃒𰃄𰃄𰂅𰂜𰂖𰃊𰁹𰃀𰂜𰃒𰂨𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰁲𰂜𰃀𰂓𰀺𰃍𰃄𰂮𰀁𰂖𰂜𰃍𰂂𰁦𰃀𰃊𰀺𰁝𰃘𰀺𰂖𰃍𰀺𰁹𰁦𰃊𰂜𰁲𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰃛𰃊𰂅𰂖𰁝𰁦𰂨𰁦𰂖𰁝𰁦𰂖𰁓𰁦𰃊𰂅𰃄𰃊𰃍𰂂𰁦𰃊𰀺𰁊𰂅𰂎𰂅𰃍𰃛𰃊𰃍𰂜𰃊𰃍𰁦𰃄𰃍𰃊
𰂎𰂅𰂨𰂅𰁝𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰁝𰂅𰁦𰃄𰃍𰁦𰃀𰀺𰃄𰁦𰃄𰂮𰃊𰂺𰀖𰀞𰀁𰀪𰁳𰃊𰃍𰃄𰃘𰁦𰃀𰃛𰃊𰃙𰁦𰂎𰂎𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰁓𰁦𰂎𰂎𰃊
𰀁𰂖𰃍𰂅𰁊𰂜𰁝𰃛𰂄𰀉𰀺𰃄𰁦𰁝𰃊𰀮𰂜𰂜𰂎𰃄𰃊𰂅𰂖𰃊𰀊𰂜𰂓𰁊𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰂅𰁦𰃄𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰁳𰃊𰁦𰂎𰁝𰂮𰃊𰀵𰁦𰃊𰂂𰀺𰃘𰁦𰃊𰀺𰃊𰂨𰂜𰃀𰃍𰁲𰂜𰂎𰂅𰂜𰃊𰂜𰁲𰃊𰂜𰃘𰁦𰃀𰃊𰂣𰁴𰃟𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰃍𰂎𰃛𰃊𰂎𰀺𰁊𰁦𰂎𰁦𰁝
𰀪𰂜𰂎𰀺𰂍𰂅𰁦𰃙𰂅𰁓𰃞𰁊𰁦𰂎𰂅𰁦𰃘𰁦𰃄𰃊𰀊𰁦𰂎𰂎𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰂺𰂎𰁦𰀺𰁝𰃄𰃊𰃍𰂂𰁦𰃊𰂨𰀺𰁓𰂍𰃊𰂅𰂖𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰃊𰁲𰂜𰃀𰃊𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰁚𰃊𰃄𰁦𰃀𰂅𰂖𰁦𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰃀𰁦𰂜𰂖𰂅𰂖𰁦𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰂮𰃊𰀡𰃒𰃀𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰃊
𰃍𰁦𰃀𰂓𰃄𰃊𰂜𰁲𰃊𰃍𰂜𰂜𰂎𰃄𰃊𰃍𰂜𰃊𰃄𰃍𰃒𰁝𰃛𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰃊𰃍𰃀𰀺𰂖𰃄𰁝𰃒𰁓𰃍𰂅𰂜𰂖𰁚𰃊𰂂𰀺𰃘𰂅𰂖𰁹𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰁦𰁝𰁏𰀺𰃄𰃊𰂨𰀺𰃀𰃍𰃊𰀺𰃀𰁦𰃊𰁹𰁦𰀺𰃀𰁦𰁝𰃊𰃍𰂜𰃙𰀺𰃀𰁝𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰃙𰂂𰁦𰃀𰁦𰃊𰃍𰂂𰁦𰃀𰁦𰃊𰂅𰃄𰃊𰂨𰃀𰁦𰃄𰃄𰃒𰃀𰁦𰃊𰃍𰂜𰃊𰂵𰃒𰂅𰁓𰂍𰂎𰃛𰃊𰃘𰀺𰂎𰂅𰁝𰀺𰃍𰁦𰃊
𰂜𰁲𰃊𰀟𰁦𰃙𰀎𰂖𰁹𰂎𰀺𰂖𰁝𰃊𰀉𰂅𰂜𰂎𰀺𰁊𰃄𰁐𰃊𰃍𰂂𰁦𰃊𰁳𰃊𰃀𰃄𰃍𰃊𰁓𰂜𰂓𰂓𰁦𰃀𰁓𰂅𰀺𰂎𰂎𰃛𰃊𰀺𰃘𰀺𰂅𰂎𰀺𰁊𰂎𰁦𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰀺𰃊𰂂𰂅𰁹𰂂𰂵𰃒𰀺𰂎𰂅𰃍𰃛𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰁲𰂜𰃀𰃊𰂂𰂅𰁹𰂂𰃊𰃍𰂂𰃀𰂜𰃒𰁹𰂂𰂨𰃒𰃍𰃊𰃄𰁓𰃀𰁦𰁦𰂖𰂅𰂖𰁹𰂮𰂻𰃊𰀉𰂜𰁹𰁦𰃊𰃄𰀺𰃛𰃄𰃊𰃍𰂂𰁦𰃊
𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰀺𰂎𰂎𰃛𰃊𰃍𰂜𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰀺𰁓𰃍𰂅𰃘𰀺𰃍𰂅𰂜𰂖𰃊𰃄𰃍𰀺𰃍𰁦𰂮𰃊𰀞𰂜𰃀𰁦𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰀺𰂎𰃄𰂜𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊𰃍𰁦𰃄𰃍𰂅𰂖𰁹𰃊𰃍𰂂𰁦𰃊𰀺𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰃊𰂜𰁲𰃊𰂨𰂜𰃍𰁦𰂖𰃍𰂅𰀺𰂎𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰂮𰃊
𰃀𰁦𰁓𰁦𰂖𰃍𰂎𰃛𰁚𰂻𰂂𰁦𰃊𰃄𰀺𰃛𰃄𰁚𰃊𰂺𰃙𰁦𰃊𰂂𰀺𰃘𰁦𰃊𰁊𰁦𰁦𰂖𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂅𰂖𰁹𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰁓𰀺𰂨𰀺𰁊𰂎𰁦𰃊𰂜𰁲𰃊𰀞𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰀌𰁦𰃘𰂅𰁓𰁦𰃄𰃊𰃄𰁦𰂎𰂎𰃄𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰁚𰃊𰁊𰃒𰁲𰁲𰁦𰃀𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰁦𰃊𰀖𰀞𰀁𰀪𰁊𰂅𰂖𰁝𰂅𰂖𰁹𰃊
𰁝𰁦𰃍𰁦𰁓𰃍𰂅𰂖𰁹𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂖𰃍𰁦𰃚𰃍𰃊𰂜𰁲𰃊𰁳𰃚𰁦𰁝𰃊𰀺𰂓𰂅𰂖𰂜𰃊𰀺𰁓𰂅𰁝𰃊𰃀𰁦𰃄𰂅𰁝𰃒𰁦𰃄𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰃊𰂨𰃀𰂅𰂓𰀺𰃀𰂅𰂎𰃛𰃊𰃍𰂜𰃊𰃒𰃄𰁦𰃀𰃄𰃊𰃙𰂂𰂜𰃊𰂺𰁝𰂜𰂖𰂽𰃍𰃊𰂖𰁦𰁦𰁝𰃊𰃒𰃄𰃊𰃍𰂜𰃊𰃍𰁦𰂎𰂎𰃊𰃍𰂂𰁦𰂓𰃊𰂂𰂜𰃙𰃊𰃍𰂜
𰁓𰂜𰃀𰃀𰁦𰃄𰂨𰂜𰂖𰁝𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃀𰁦𰁓𰂜𰁹𰂖𰂅𰃍𰂅𰂜𰂖𰃊𰂓𰂜𰃍𰂅𰁲𰃄𰁚𰃊𰃙𰂂𰂅𰁓𰂂𰂂𰁦𰂎𰂨𰃊𰃍𰂜𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰃊𰁝𰁦𰃄𰂅𰁹𰂖𰃊𰀺𰂖𰃊𰀺𰃄𰃄𰀺𰃛𰂮𰃊𰀵𰁦𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃊𰀺𰃊𰂓𰂜𰁝𰃒𰂎𰀺𰃀𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰃙𰂅𰃍𰂂𰁝𰁦𰁝𰂅𰁓𰀺𰃍𰁦𰁝
𰀺𰂖𰁝𰁝𰁦𰃍𰁦𰁓𰃍𰃊𰂓𰃒𰂎𰃍𰂅𰂨𰂎𰁦𰃊𰂖𰂜𰃘𰁦𰂎𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰂅𰂖𰃊𰀺𰃊𰁊𰂅𰂜𰂎𰂜𰁹𰂅𰁓𰀺𰂎𰃊𰃄𰃒𰂨𰂨𰂜𰃀𰃍𰁚𰂻𰃊𰀉𰂜𰁹𰁦𰃊𰂖𰂜𰃍𰁦𰃄𰂮𰃊
𰃄𰀺𰂓𰂨𰂎𰁦𰂮𰂻𰀮𰂂𰁦𰃄𰁦𰃊𰀺𰂖𰁝𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰃍𰂜𰂜𰂎𰃄𰃊𰂎𰂅𰂍𰁦𰃊𰁹𰁦𰂖𰁦𰃀𰀺𰂎𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊
𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰁚𰃊𰀪𰂜𰂎𰀺𰂍𰂅𰁦𰃙𰂅𰁓𰃞𰃊𰂜𰁊𰃄𰁦𰃀𰃘𰁦𰃄𰁚𰃊𰀺𰃀𰁦𰃊𰁊𰁦𰁓𰂜𰂓𰂅𰂖𰁹𰃊𰃒𰃄𰁦𰁲𰃒𰂎𰃊𰃍𰂜𰃊𰃄𰃍𰃒𰁝𰃛𰃊𰃍𰂂𰁦𰃊𰀖𰂖𰂂𰂅𰁊𰂅𰃍𰃊𰀺𰂖𰁝𰃊𰀪𰃀𰂜𰃍𰁦𰁓𰃍
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰃒𰃄𰂅𰂖𰁹𰃊𰀊𰀭𰀮𰂽𰃄𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰁓𰀺𰂖𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰂮𰂻𰂺𰀁𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃊𰃙𰁦𰂽𰃀𰁦𰃊𰃀𰁦𰀺𰂎𰂎𰃛𰃊𰁦𰃚𰁓𰂅𰃍𰁦𰁝𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰁲𰂜𰃀𰃊𰃑𰃟𰃟𰃇𰃊𰂅𰃄𰀪𰂂𰂜𰃄𰀭𰀮𰀡𰀪𰁚𰂻𰃊𰃄𰀺𰃛𰃄𰃊
𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰁓𰀺𰂖𰃊𰁓𰀺𰂖𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰃊𰂂𰃒𰂖𰁝𰃀𰁦𰁝𰃄𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊𰃄𰂅𰃍𰁦𰃄𰃊𰀛𰁦𰁲𰁲𰃀𰁦𰃛𰃊𰀎𰂓𰁓𰂂𰁚𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃊𰂓𰀺𰂖𰀺𰁹𰁦𰃀𰁚𰃊𰀬𰂜𰁓𰂂𰁦𰃊𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰃊𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰂮𰃊𰂺𰀮𰂂𰁦𰃄𰁦𰃊
𰂅𰂖𰃊𰀺𰃊𰃄𰂅𰂖𰁹𰂎𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰂮𰃊𰀖𰃍𰃊𰃒𰃄𰁦𰃄𰃊𰀺𰂖𰃊𰂅𰂓𰂓𰃒𰂖𰂜𰀺𰁲𰁳𰃊𰂖𰂅𰃍𰃛𰃊𰃄𰃍𰁦𰂨𰃊𰃍𰂜𰃊𰂅𰃄𰂜𰂎𰀺𰃍𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰁓𰂜𰁓𰂍𰃍𰀺𰂅𰂎𰃊𰃍𰀺𰁊𰂎𰁦𰃍𰃄𰃊𰁹𰃀𰁦𰀺𰃍𰂎𰃛𰃊𰂅𰂖𰁓𰃀𰁦𰀺𰃄𰁦𰃊𰁓𰂜𰂖𰃘𰁦𰂖𰂅𰁦𰂖𰁓𰁦𰁚𰃊
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃄𰃊𰁲𰃀𰂜𰂓𰃊𰂨𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰂄𰁝𰂅𰁹𰁦𰃄𰃍𰁦𰁝𰃊𰁓𰁦𰂎𰂎𰃒𰂎𰀺𰃀𰃊𰂜𰃀𰃊𰃍𰂅𰃄𰃄𰃒𰁦𰃊𰁦𰃚𰃍𰃀𰀺𰁓𰃍𰃄𰂮𰃊𰃄𰃍𰀺𰁊𰂅𰂎𰂅𰃍𰃛𰁚𰃊𰀺𰂖𰁝𰃊𰃀𰁦𰂎𰂅𰀺𰁊𰂅𰂎𰂅𰃍𰃛𰁚𰃊𰀺𰂎𰂎𰂜𰃙𰂅𰂖𰁹𰃊𰃒𰃄𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰂓𰀺𰂍𰁦𰃊𰀺𰃊𰂨𰃀𰁦𰂜𰂨𰃍𰂅𰂓𰂅𰃞𰁦𰁝
𰀮𰂂𰁦𰃊𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃄𰃊𰀺𰃀𰁦𰃊𰃍𰂂𰁦𰂖𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁦𰁝𰃊𰃒𰃄𰂅𰂖𰁹𰃊𰃍𰀺𰂖𰁝𰁦𰂓𰃊𰂓𰀺𰃄𰃄𰃊𰃄𰂨𰁦𰁓𰃍𰃀𰂜𰂓𰁦𰃍𰃀𰃛𰂮𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰃄𰂜𰂎𰃒𰃍𰂅𰂜𰂖𰃊𰀺𰃄𰃊𰂖𰁦𰁦𰁝𰁦𰁝𰂮𰂻𰀪𰂂𰂜𰃄𰀭𰀮𰀡𰀪𰃊𰂅𰃄𰁝𰁦𰃄𰂅𰁹𰂖𰁦𰁝𰃊𰃍𰂜𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰃊𰃍𰂂𰁦𰃊
𰂺𰀖𰁲𰃊𰃛𰂜𰃒𰃊𰃙𰀺𰂖𰃍𰃊𰃍𰂜𰃊𰁹𰂎𰂜𰁊𰀺𰂎𰂎𰃛𰃊𰂅𰂖𰃍𰁦𰃀𰃀𰂜𰁹𰀺𰃍𰁦𰃊𰃍𰂂𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂜𰂓𰁦𰃊𰂅𰂖𰃊𰁊𰃀𰂜𰀺𰁝𰃊𰃄𰂨𰁦𰁓𰃍𰃀𰃒𰂓𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰃀𰁦𰃊𰃀𰁦𰂎𰁦𰀺𰃄𰁦𰁝𰃙𰂂𰁦𰂖𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰀺𰃀𰁦𰃊
𰀺𰃊𰁓𰁦𰂎𰂎𰃊𰃍𰃛𰂨𰁦𰃊𰂜𰃀𰃊𰃍𰂅𰃄𰃄𰃒𰁦𰃊𰂅𰂖𰃊𰂜𰂖𰁦𰃊𰃄𰂂𰂜𰃍𰁚𰃊𰂅𰃍𰂽𰃄𰃊𰀺𰃊𰃀𰂜𰁊𰃒𰃄𰃍𰃊𰀺𰂖𰁝𰃊𰂨𰂜𰃙𰁦𰃀𰁲𰃒𰂎𰃊𰂓𰁦𰃍𰂂𰂜𰁝𰁚𰂻𰂎𰃛𰃄𰁦𰁝𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂜𰃊𰂨𰃀𰂜𰃍𰁦𰁓𰃍𰃊𰃍𰂂𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰃄𰃍𰀺𰃍𰁦𰃊𰂜𰁲𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰁲𰃀𰂜𰂓𰃊𰁊𰁦𰂅𰂖𰁹𰃊
𰀪𰂜𰂎𰀺𰂍𰂅𰁦𰃙𰂅𰁓𰃞𰃊𰁓𰂜𰂖𰁓𰂎𰃒𰁝𰁦𰃄𰂮𰀺𰂎𰃍𰁦𰃀𰁦𰁝𰁚𰃊𰃙𰂂𰂅𰁓𰂂𰃊𰁓𰀺𰂖𰃊𰀺𰁲𰁲𰁦𰁓𰃍𰃊𰃍𰂂𰁦𰃊𰁝𰁦𰃍𰁦𰁓𰃍𰂅𰂜𰂖𰃊𰀺𰂖𰁝𰃊𰂅𰃄𰂜𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰁲𰃒𰂖𰁓𰃍𰂅𰂜𰂖𰀺𰂎𰃊
𰀁𰁓𰁓𰂜𰃀𰁝𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰀬𰂜𰁊𰁦𰃀𰃍𰂜𰃊𰀊𰀺𰂓𰂨𰂜𰃄𰂄𰀔𰂜𰂖𰃞𰀺𰂎𰁦𰃞𰁚𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂜𰃀𰁚𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰂜𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰂮𰃊𰀮𰂂𰂅𰃄𰃊𰃄𰂂𰂜𰃒𰂎𰁝𰃊𰀺𰂎𰂎𰂜𰃙𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰁓𰀺𰂨𰃍𰃒𰃀𰁦𰃊𰂓𰂜𰃀𰁦𰃊
𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁚𰃊𰀉𰀌𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰁚𰃊𰂺𰀡𰂖𰁦𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰂓𰂜𰃄𰃍𰃊𰁦𰃚𰁓𰂅𰃍𰂅𰂖𰁹𰃊𰃀𰁦𰁓𰁦𰂖𰃍𰃊𰀺𰁓𰁓𰃒𰃀𰀺𰃍𰁦𰂎𰃛𰃊𰃍𰂂𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰃄𰃍𰀺𰃍𰁦𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰀺𰃍𰃊𰀺𰃊𰂨𰀺𰃀𰃍𰂅𰁓𰃒𰂎𰀺𰃀𰃊
𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰃄𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰁳𰃊𰁦𰂎𰁝𰃊𰁲𰂜𰃀𰃊𰀉𰀌𰃊𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰃊𰂅𰃄𰃊𰂓𰂜𰂓𰁦𰂖𰃍𰃊𰂅𰂖𰃊𰃍𰂅𰂓𰁦𰂮𰃊
𰃍𰂂𰁦𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰃊𰂜𰁲𰃊𰀉𰀌𰀪𰂂𰂜𰃄𰁵𰃊𰂜𰃙𰁚𰃊𰀺𰃊𰃒𰂖𰂅𰂵𰃒𰁦𰃊𰃙𰀺𰃛𰃊𰃍𰂜𰃊𰂓𰂜𰂖𰂅𰃍𰂜𰃀𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰃄𰃍𰀺𰃍𰁦𰃄𰃊𰂅𰂖𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰂮𰂻𰀮𰂂𰁦𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰀁𰃍𰃊𰀷𰂜𰃒𰃀𰃊𰀭𰁦𰃀𰃘𰂅𰁓𰁦
𰂅𰃄𰃊𰁊𰀺𰃄𰁦𰁝𰃊𰂜𰂖𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰂄𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰁚𰁵𰃊𰃒𰂜𰃀𰂜𰁓𰂂𰃀𰂜𰂓𰁦𰂄𰂎𰀺𰁊𰁦𰂎𰁦𰁝𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰀮𰂂𰂜𰃄𰁦𰃊𰃙𰂂𰂜𰃊𰁝𰂜𰂖𰂽𰃍𰃊𰂂𰀺𰃘𰁦𰃊𰃍𰂂𰁦𰃊𰃍𰂅𰂓𰁦𰁚𰃊𰃀𰁦𰃄𰂜𰃒𰃀𰁓𰁦𰃄𰁚𰃊𰂜𰃀𰃊𰂅𰂖𰁓𰂎𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰃍𰂜𰃊𰂨𰁦𰃀𰁲𰂜𰃀𰂓𰃊
𰃍𰂂𰀺𰃍𰃊𰀺𰃀𰁦𰃊𰁝𰁦𰃍𰁦𰁓𰃍𰁦𰁝𰃊𰃒𰃄𰂅𰂖𰁹𰃊𰁵𰃊𰂜𰃙𰃊𰁓𰃛𰃍𰂜𰂓𰁦𰃍𰃀𰃛𰂮𰃊𰂺𰀖𰃍𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰃒𰂖𰂅𰂵𰃒𰁦𰃊𰀺𰂨𰂨𰁦𰀺𰂎𰃊𰃍𰂜𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰂜𰃙𰂖𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰀺𰃄𰃄𰀺𰃛𰃄𰃊𰁓𰀺𰂖𰃊𰃍𰃒𰃀𰂖𰃊𰃍𰂜𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰃊𰃙𰂂𰂜𰃊𰃙𰂅𰂎𰂎𰃊𰂨𰁦𰃀𰁲𰂜𰃀𰂓𰃊
𰃍𰂂𰁦𰃊𰂨𰂂𰀺𰃀𰂓𰀺𰁓𰁦𰃒𰃍𰂅𰁓𰀺𰂎𰃊𰃄𰁦𰁹𰂓𰁦𰂖𰃍𰃊𰃙𰂂𰁦𰃀𰁦𰃊𰂅𰃍𰃊𰁓𰂜𰃒𰂎𰁝𰁊𰁦𰃊𰃒𰃄𰁦𰁝𰃊𰃍𰂜𰃊𰂓𰂜𰂖𰂅𰃍𰂜𰃀𰃊𰃍𰂂𰁦𰃊𰃍𰂂𰁦𰂓𰃊𰀺𰃄𰃊𰀺𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰂮𰃊𰀛𰁦𰁲𰁲𰃊𰀮𰂅𰂎𰂎𰁚𰃊𰁹𰃀𰂜𰃒𰂨𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃊𰂓𰀺𰂖𰀺𰁹𰁦𰃀𰁚𰃊𰁝𰃀𰃒𰁹𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰁚𰃊
𰁦𰁲𰁲𰁦𰁓𰃍𰂅𰃘𰁦𰂖𰁦𰃄𰃄𰃊𰂜𰁲𰃊𰀺𰃊𰁓𰂜𰂓𰂨𰂜𰃒𰂖𰁝𰃊𰂅𰂖𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰃍𰃀𰂅𰀺𰂎𰃄𰁚𰂻𰂂𰁦𰃊𰂜𰁊𰃄𰁦𰃀𰃘𰁦𰃄𰂮𰃊𰂺𰀖𰂖𰃊𰀺𰂖𰃊𰀞𰂅𰂎𰂎𰂅𰂨𰂜𰃀𰁦𰁚𰃊𰂜𰁊𰃄𰁦𰃀𰃘𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰁚𰃊𰀺𰃄𰃊𰀯𰂨𰃄𰃍𰀺𰃍𰁦𰁚𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰃊𰃙𰀺𰃄𰃊𰃍𰂂𰁦𰃊𰁳𰃊𰃀𰃄𰃍𰃊𰃍𰂜𰃊𰃄𰁦𰂎𰂎𰃊
𰂅𰁝𰁦𰀺𰂎𰃊𰃄𰂅𰃍𰃒𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰂨𰀺𰃍𰂅𰁦𰂖𰃍𰃄𰂽𰁊𰂎𰂜𰂜𰁝𰃊𰁓𰂜𰃒𰂎𰁝𰁊𰁦𰃊𰂓𰂜𰂖𰂅𰃍𰂜𰃀𰁦𰁝𰃊𰁲𰂜𰃀𰃊𰃍𰂂𰁦𰃊𰁝𰃀𰃒𰁹𰂽𰃄𰃊𰃀𰁦𰁓𰂜𰂓𰁊𰂅𰂖𰀺𰂖𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂜𰁝𰀺𰃛𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰀺𰃀𰁦𰃊𰃄𰃍𰂅𰂎𰂎𰃊𰁊𰂅𰁹𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃄𰃊𰂅𰂖𰃊𰁓𰀺𰂖𰁓𰁦𰃀𰃊
𰁦𰁲𰁲𰁦𰁓𰃍𰃊𰂜𰂖𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰁲𰂜𰃀𰃊𰁦𰃚𰀺𰂓𰂨𰂎𰁦𰁚𰃊𰃍𰂂𰁦𰃊𰀁𰂍𰃍𰃊𰂜𰃀𰃊𰀞𰀁𰀪𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃍𰂂𰁦𰃀𰀺𰂨𰁦𰃒𰃍𰂅𰁓𰃄𰂮𰃊𰀞𰂅𰂎𰂎𰂅𰂨𰂜𰃀𰁦𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰂨𰂜𰃀𰃍𰁲𰂜𰂎𰂅𰂜𰃊𰂜𰁲𰃊𰂜𰃘𰁦𰃀𰃊𰃑𰁴𰃟𰃊𰃀𰁦𰁓𰂜𰂓𰁊𰂅𰂖𰀺𰂖𰃍𰁚𰃊
𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰂅𰂖𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰁓𰁦𰂎𰂎𰃊𰃍𰃛𰂨𰁦𰃄𰃊𰁊𰁦𰁓𰀺𰃒𰃄𰁦𰃊𰁵𰃊𰂜𰃙𰃊𰁓𰃛𰃍𰂜𰂓𰁦𰃍𰃀𰃛𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰂄𰃀𰁦𰂎𰁦𰃘𰀺𰂖𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰃙𰂂𰂅𰁓𰂂𰃊𰁲𰂜𰃀𰂓𰃊𰃍𰂂𰁦𰃊𰁊𰀺𰃄𰂅𰃄𰃊𰂜𰁲𰃊𰀜𰂅𰂖𰀺𰃄𰁦𰀪𰃀𰂜𰁳𰃊𰂎𰁦𰃀𰁚𰃊𰀺𰃊
𰁝𰂅𰃄𰁓𰃀𰂅𰂓𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰁊𰁦𰃍𰃙𰁦𰁦𰂖𰃊𰁝𰂅𰁲𰁲𰁦𰃀𰁦𰂖𰃍𰃊𰁓𰁦𰂎𰂎𰃊𰂅𰂓𰂓𰃒𰂖𰂜𰂨𰂂𰁦𰂖𰂜𰃍𰃛𰂨𰁦𰃄𰁚𰃊𰃄𰃒𰁓𰂂𰃊𰀺𰃄𰁝𰂅𰃀𰁦𰁓𰃍𰃊𰃀𰀺𰁝𰂅𰂜𰂓𰁦𰃍𰃀𰂅𰁓𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰂨𰃀𰂜𰁳𰃊𰂎𰂅𰂖𰁹𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃊𰁲𰂜𰃀𰃊𰃍𰂂𰁦𰃊𰁦𰃘𰀺𰂎𰃒𰀺𰃍𰂅𰂜𰂖𰃊
𰁝𰂅𰁲𰁲𰁦𰃀𰁦𰂖𰃍𰃊𰃄𰃒𰁊𰃍𰃛𰂨𰁦𰃄𰃊𰂜𰁲𰃊𰀉𰃊𰂜𰃀𰃊𰀮𰃊𰁓𰁦𰂎𰂎𰃄𰂮𰂻𰀎𰃚𰂨𰁦𰃀𰂅𰂓𰁦𰂖𰃍𰃄𰃊𰂂𰀺𰃘𰁦𰃊𰁊𰁦𰁦𰂖𰃊𰁝𰂜𰂖𰁦𰃊𰃙𰂅𰃍𰂂𰂜𰁲𰃊𰁓𰀺𰂖𰁝𰂅𰁝𰀺𰃍𰁦𰃊𰂨𰂂𰀺𰃀𰂓𰀺𰁓𰁦𰃒𰃍𰂅𰁓𰀺𰂎𰃊𰁓𰂜𰂓𰂨𰂜𰃒𰂖𰁝𰃄𰂮𰃊𰀮𰂅𰂎𰂎𰃊𰃀𰁦𰁲𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰃍𰂂𰁦𰃊𰀺𰃄𰃄𰀺𰃛𰁚𰃊
𰃒𰂨𰃊𰃍𰂜𰃊𰂣𰂣𰁝𰂅𰁲𰁲𰁦𰃀𰁦𰂖𰃍𰃊𰁓𰂜𰂎𰂜𰃀𰃄𰃊𰂜𰁲𰃊𰂎𰀺𰁊𰁦𰂎𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰀊𰀺𰂓𰂨𰂜𰃄𰃊𰁊𰁦𰂎𰂅𰁦𰃘𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰃍𰂂𰂅𰃄𰃊𰃙𰂂𰂅𰁓𰂂𰃙𰀺𰃄𰃊𰂅𰂖𰃍𰃀𰂜𰁝𰃒𰁓𰁦𰁝𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰁳𰃘𰁦𰃊𰃛𰁦𰀺𰃀𰃄𰃊𰀺𰁹𰂜𰁚𰃊𰀺𰃄𰃊𰃍𰂂𰁦𰃊𰂺𰃍𰃀𰂅𰁦𰁝𰃊𰀺𰂖𰁝𰃊𰃍𰃀𰃒𰁦𰃊
𰂅𰃄𰃊𰂖𰂜𰃍𰃊𰃍𰂂𰁦𰃊𰃒𰂨𰂨𰁦𰃀𰃊𰂎𰂅𰂓𰂅𰃍𰂮𰃊𰂺𰀖𰃍𰂽𰃄𰃊𰀺𰃊𰂨𰂜𰃙𰁦𰃀𰁲𰃒𰂎𰃊𰃙𰀺𰃛𰃊𰃍𰂜𰃊𰂓𰂜𰂖𰂅𰃍𰂜𰃀𰃊𰂅𰂖𰃊𰃘𰂅𰃘𰂜𰃊𰁹𰂜𰂎𰁝𰃊𰃄𰃍𰀺𰂖𰁝𰀺𰃀𰁝𰂮𰂻𰀮𰂂𰁦𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰁚𰃊𰂨𰁦𰃀𰁲𰂜𰃀𰂓𰁦𰁝𰃊𰂅𰂖𰀌𰃒𰂖𰁝𰁦𰁦𰁚𰃊𰀭𰁓𰂜𰃍𰂎𰀺𰂖𰁝𰁚𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊
𰃄𰃍𰃒𰁝𰂅𰁦𰃄𰃊𰃙𰂂𰁦𰃀𰁦𰃊𰂨𰃀𰂅𰂓𰀺𰃀𰃛𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰀺𰃀𰁦𰃊𰂖𰂜𰃍𰃊𰀺𰃄𰃊𰀺𰁊𰃒𰂖𰁝𰀺𰂖𰃍𰃊𰀺𰃄𰃊𰀺𰃀𰁦𰃊𰁓𰁦𰂎𰂎𰃊𰂎𰂅𰂖𰁦𰃄𰃊𰁓𰂎𰂅𰁦𰂖𰃍𰃄𰃊𰃍𰂜𰃊𰀺𰃘𰂜𰂅𰁝𰃊𰃍𰂂𰁦𰃊𰃒𰃄𰁦𰃊𰂜𰁲𰃊𰃀𰀺𰁝𰂅𰂜𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰂅𰃄𰂜𰃍𰂜𰂨𰁦𰃄𰂮𰃊𰀮𰂅𰂎𰂎𰃊𰃄𰀺𰃛𰃄𰁚𰃊𰂺𰀞𰂜𰃄𰃍𰃊𰂜𰁲𰃊
𰃒𰃄𰁦𰁝𰃊𰁲𰂜𰃀𰃊𰂅𰂖𰃊𰃘𰂅𰃍𰃀𰂜𰃊𰃄𰃍𰃒𰁝𰂅𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰀺𰃀𰁦𰃊𰂨𰃀𰁦𰃄𰁦𰂖𰃍𰃊𰂅𰂖𰃊𰂓𰂅𰃚𰁦𰁝𰃊𰁓𰁦𰂎𰂎𰁮𰃍𰃛𰂨𰁦𰃊𰂜𰃒𰃀𰃊𰁓𰂎𰂅𰁦𰂖𰃍𰃄𰃊𰀺𰃀𰁦𰃊𰂎𰂜𰂜𰂍𰂅𰂖𰁹𰃊𰁲𰂜𰃀𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰁚𰃊𰂨𰃀𰂅𰂓𰀺𰃀𰂅𰂎𰃛𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰂜𰂖𰁓𰂜𰂎𰂜𰁹𰃛𰃊
𰂨𰂜𰂨𰃒𰂎𰀺𰃍𰂅𰂜𰂖𰃄𰁚𰂻𰂂𰁦𰃊𰃄𰀺𰃛𰃄𰂮𰃊𰃄𰁦𰃍𰃍𰂅𰂖𰁹𰂮𰂻𰀮𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰃊𰀺𰂎𰃄𰂜𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰂅𰃍𰃛𰃊𰃍𰁦𰃄𰃍𰂅𰂖𰁹𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃊𰁲𰂜𰃀𰃊𰀪𰀖𰃎𰂄
𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰁚𰃊𰁓𰀺𰂎𰂎𰁦𰁝𰀪𰀖𰀪𰃀𰂜𰁳𰃊𰂎𰁦𰃀𰂮𰃊𰀉𰁦𰁓𰀺𰃒𰃄𰁦𰃊𰀪𰀖𰃎𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰃄𰃊𰀺𰃊𰂎𰂅𰂨𰂅𰁝𰂍𰂅𰂖𰀺𰃄𰁦𰁚𰃊𰂅𰃍𰃊𰂅𰃄𰃊
𰀊𰂅𰃀𰁓𰃒𰂓𰃘𰁦𰂖𰃍𰂅𰂖𰁹𰃊𰃍𰂂𰁦𰃊𰀯𰃄𰁦𰃊𰂜𰁲𰀁𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰂖𰂜𰃍𰃊𰀺𰃄𰃊𰀺𰂓𰁦𰂖𰀺𰁊𰂎𰁦𰃊𰃍𰂜𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰃊𰃀𰀺𰁝𰂅𰂜𰂓𰁦𰃍𰃀𰂅𰁓𰂄𰁲𰂜𰃀𰂓𰀺𰃍𰃍𰁦𰁝𰃊𰃍𰁦𰃄𰃍𰂅𰂖𰁹𰃊𰀺𰃄𰃊𰀺𰃀𰁦𰃊𰃍𰂂𰁦𰃊
𰀁𰁓𰁓𰂜𰃀𰁝𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰀁𰂖𰂖𰁦𰁹𰃀𰁦𰃍𰃊𰀉𰂜𰁹𰁦𰁚𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂜𰃀𰃊𰁲𰂜𰃀𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃊𰀺𰂖𰁝𰃊𰀺𰃄𰃄𰀺𰃛𰃊𰀬𰁁𰀌𰁚𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰂮𰃊𰀪𰀖𰀪𰃀𰂜𰁳𰃊𰂎𰁦𰃀𰃊𰃒𰃄𰁦𰃄𰃊𰀕𰀮𰀬𰀓𰃊𰂪𰂂𰂜𰂓𰂜𰁹𰁦𰂖𰁦𰂜𰃒𰃄𰃊𰃍𰂅𰂓𰁦𰂄𰃀𰁦𰃄𰂜𰂎𰃘𰁦𰁝
𰀞𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰀌𰁦𰃘𰂅𰁓𰁦𰃄𰁚𰃊𰂂𰁦𰃀𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰂽𰃄𰃊𰀖𰀞𰀁𰀪𰀮𰀬𰂄𰀓𰀬𰀎𰀮𰂪𰃍𰂅𰂓𰁦𰂄𰃀𰁦𰃄𰂜𰂎𰃘𰁦𰁝𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰁓𰁦𰂫𰁚𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰂽𰃄𰃊𰁓𰂜𰂨𰃛𰃀𰂅𰁹𰂂𰃍𰁦𰁝𰃊𰂖𰀺𰂓𰁦𰃊𰁲𰂜𰃀𰃊𰂅𰃍𰃄𰃊𰁓𰂜𰂖𰃍𰂅𰂖𰃒𰁦𰁝𰂁
𰂰𰃥𰃙
𰀁𰀁𰀁𰀭𰃉𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰀉𰃒𰃄𰂅𰂖𰁦𰃄𰃄𰃊𰀡𰁲𰁲𰂅𰁓𰁦𰃊𰀓𰁦𰀺𰃍𰃒𰃀𰁦
𰀟𰀘𰀕𰀎𰃛𰀰𰀊𰀘𰀎𰀢𰀊𰀎𰃛𰀲𰀎𰀊𰀗𰀢𰀤𰀟𰀤𰀖𰀘𰀎𰀰
𰀊𰁦𰂎𰂎𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹
𰀮𰀬𰂄𰀓𰀬𰀎𰀮𰂄𰁊𰀺𰃄𰁦𰁝𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰁚𰃊𰁲𰂜𰃀𰃊𰂎𰁦𰀺𰁝𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁓𰀺𰃍𰂅𰂜𰂖𰃊𰀺𰂖𰁝𰃊𰁦𰃘𰀺𰂎𰃒𰀺𰃍𰂅𰂜𰂖𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰃀𰁦𰂎𰀺𰃍𰂅𰂜𰂖𰃄𰂂𰂅𰂨𰃊𰃍𰂜𰂨𰀺𰃍𰂅𰁦𰂖𰃍𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰁊𰁦𰂖𰁦𰁳𰃊𰃍𰃊𰁲𰃀𰂜𰂓𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰁦𰁝
𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰃍𰂂𰁦𰃊𰂂𰃒𰂓𰀺𰂖𰃊𰀪𰀖𰃎𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰁊𰁦𰃍𰀺𰁚𰃊𰁹𰀺𰂓𰂓𰀺𰁚𰃊𰀺𰂖𰁝𰁝𰁦𰂎𰃍𰀺𰂮𰃊𰃍𰂂𰁦𰃀𰀺𰂨𰂅𰁦𰃄𰁚𰂻𰂂𰁦𰃊𰃄𰀺𰃛𰃄𰂮𰃊𰀮𰂂𰂅𰃄𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰁓𰂜𰃒𰂎𰁝𰃊𰁊𰁦𰃊𰃒𰃄𰁦𰁝𰃊𰃍𰂜𰃊𰀺𰂖𰀺𰂎𰃛𰃞𰁦𰃊
𰀉𰂅𰂜𰂓𰂜𰂎𰀖𰂖𰃍𰁦𰃀𰂖𰀺𰃍𰂅𰂜𰂖𰀺𰂎𰁚𰃊𰂅𰂖𰂨𰀺𰃀𰃍𰂖𰁦𰃀𰃄𰂂𰂅𰂨𰃊𰃙𰂅𰃍𰂂𰀖𰂖𰂖𰂜𰃘𰀺𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰁚𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰃍𰃀𰂅𰀺𰂎𰃊𰃀𰁦𰃄𰃒𰂎𰃍𰃄𰃊𰃀𰁦𰃍𰃀𰂜𰃄𰂨𰁦𰁓𰃍𰂅𰃘𰁦𰂎𰃛𰃊𰃍𰂜𰁝𰁦𰁳𰃊𰂖𰁦𰃊𰃍𰂂𰁦𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰂨𰃀𰂜𰁳𰃊𰂎𰁦𰃄𰃊
𰂎𰀺𰃒𰂖𰁓𰂂𰁦𰁝𰀭𰂨𰁦𰁦𰁝𰀝𰁦𰀺𰁝𰂄𰀪𰃊𰂅𰂖𰀛𰃒𰂎𰃛𰃑𰃟𰃟𰁴𰁚𰃊𰀺𰁓𰁓𰂜𰃀𰁝𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰀞𰀺𰃀𰂍𰀎𰂖𰁹𰂎𰁦𰂍𰀺𰁚𰂜𰁲𰃊𰂨𰀺𰃍𰂅𰁦𰂖𰃍𰃄𰃊𰃙𰂂𰂜𰃄𰁦𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰃊𰃙𰂜𰃒𰂎𰁝𰃊𰃀𰁦𰃄𰂨𰂜𰂖𰁝𰃊𰁲𰀺𰃘𰂜𰃀𰀺𰁊𰂎𰃛𰃊𰃍𰂜𰃊𰀺𰃊𰃍𰃀𰁦𰀺𰃍𰂓𰁦𰂖𰃍𰃆
𰃍𰁦𰁓𰂂𰂖𰂅𰁓𰀺𰂎𰃊𰂓𰀺𰃀𰂍𰁦𰃍𰂅𰂖𰁹𰃊𰃄𰂨𰁦𰁓𰂅𰀺𰂎𰂅𰃄𰃍𰂮𰃊𰀭𰂨𰁦𰁦𰁝𰀝𰁦𰀺𰁝𰂄𰀪𰃊𰂅𰃄𰃊𰀺𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃊𰁲𰂜𰃀𰃊𰃄𰁦𰂎𰁦𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰂅𰃍𰃊𰁓𰂜𰃒𰂎𰁝𰃊𰁦𰃘𰁦𰂖𰃍𰃒𰀺𰂎𰂎𰃛𰃊𰁊𰁦𰃊𰀺𰂨𰂨𰂎𰂅𰁦𰁝𰃊𰀺𰃄𰃊𰀺𰃊𰂨𰃀𰂜𰃄𰂨𰁦𰁓𰃍𰂅𰃘𰁦𰃊𰁝𰂅𰀺𰁹𰂖𰂜𰃄𰃍𰂅𰁓𰂮𰃊𰂺𰀁𰂖𰂜𰃍𰂂𰁦𰃀𰃊
𰃄𰁓𰃀𰁦𰁦𰂖𰂅𰂖𰁹𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰀺𰃊𰂨𰀺𰂖𰁦𰂎𰃊𰂜𰁲𰃊𰂣𰂘𰂂𰂅𰁹𰂂𰂎𰃛𰃊𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰂮𰃊𰀮𰂂𰁦𰃊𰀺𰃀𰁦𰀺𰃊𰂜𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊𰂅𰃄𰃊𰃍𰂜𰃊𰁊𰁦𰃍𰃍𰁦𰃀𰃊𰁝𰁦𰁳𰃊𰂖𰁦𰃊𰃍𰂂𰁦𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰁦𰃘𰁦𰂖𰃍𰃄𰃊𰁝𰂜𰃙𰂖𰃄𰃍𰃀𰁦𰀺𰂓𰃊
𰃍𰃒𰃀𰂖𰀺𰃀𰂜𰃒𰂖𰁝𰃊𰂅𰃄𰃊𰃍𰃙𰂜𰃊𰃙𰁦𰁦𰂍𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰁓𰂜𰂓𰂨𰂜𰃒𰂖𰁝𰃄𰃊𰁓𰀺𰂖𰃊𰁊𰁦𰃊𰃄𰁓𰃀𰁦𰁦𰂖𰁦𰁝𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰂜𰁲𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰁚𰃊𰃒𰃍𰂅𰂎𰂅𰃞𰂅𰂖𰁹𰃊𰁹𰁦𰂖𰂜𰂓𰂅𰁓𰃊𰀺𰂖𰁝𰂨𰃀𰂜𰃍𰁦𰂜𰂓𰂅𰁓𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰁦𰃄𰃊
𰃍𰂂𰁦𰃊𰁦𰂖𰃍𰂅𰃀𰁦𰃊𰂨𰀺𰂖𰁦𰂎𰃊𰂜𰃀𰃊𰀺𰃊𰃄𰃒𰁊𰃄𰁦𰃍𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰁦𰂖𰃞𰃛𰂓𰁦𰃄𰃊𰁲𰂜𰃀𰃊𰂨𰁦𰃀𰁓𰁦𰂖𰃍𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂅𰂜𰂖𰃊𰂅𰂖𰃊𰂖𰂜𰂖𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰂓𰂜𰁝𰁦𰂎𰃄𰃊𰃍𰂜𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰃊𰁊𰂅𰂜𰂓𰀺𰃀𰂍𰁦𰃀𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰃀𰁦𰃊𰂨𰃀𰁦𰁝𰂅𰁓𰃍𰂅𰃘𰁦𰃊𰂜𰁲𰃊
𰂜𰃀𰀖𰀊𰁴𰃟𰂮𰃊𰂺𰀡𰃒𰃀𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰁦𰃊𰃍𰂂𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂅𰂜𰂖𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰃀𰁦𰃄𰂨𰂜𰂖𰃄𰁦𰁚𰃊𰀺𰂖𰁝𰃊𰃒𰂎𰃍𰂅𰂓𰀺𰃍𰁦𰂎𰃛𰃊𰃍𰂜𰃊𰀺𰂨𰂨𰂎𰃛𰃊𰃍𰂂𰁦𰃊𰂓𰂜𰃄𰃍𰃊𰃀𰂜𰁊𰃒𰃄𰃍𰃊𰁊𰂅𰂜𰂓𰀺𰃀𰂍𰁦𰃀𰃊
𰂨𰃀𰂜𰁳𰃊𰂎𰁦𰃊𰂜𰁲𰃊𰀺𰃊𰁹𰂅𰃘𰁦𰂖𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰁦𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰀺𰃊𰂨𰀺𰂖𰁦𰂎𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰃊𰃙𰂅𰃍𰂂𰂜𰃒𰃍𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰁦𰃄𰃊𰁲𰂜𰃀𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰃒𰃄𰁦𰁚𰂻𰃊𰀊𰂂𰃀𰂅𰃄𰃍𰁦𰂖𰃄𰁦𰂖𰃊𰃄𰀺𰃛𰃄𰂮𰃊
𰂂𰀺𰃘𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰁓𰂎𰂜𰂖𰁦𰃊𰀺𰂖𰁝𰃊𰁦𰃚𰂨𰃀𰁦𰃄𰃄𰃊𰂓𰀺𰂖𰃛𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰃊𰀺𰃄𰃄𰀺𰃛𰃄𰃊
𰂅𰂖𰃊𰂂𰂜𰃒𰃄𰁦𰁚𰂻𰃊𰃄𰀺𰃛𰃄𰃊𰀎𰂖𰁹𰂎𰁦𰂍𰀺𰂮𰃊𰀉𰂅𰂜𰂓𰂜𰂎𰃊𰀺𰂎𰃄𰂜𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰂎𰀺𰃀𰁹𰁦𰃊𰁓𰂜𰂎𰂎𰁦𰁓𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰀺𰃄𰃄𰀺𰃛𰀎𰃚𰂨𰃀𰁦𰃄𰃄𰀺𰂖𰁝𰀭𰂅𰂎𰁦𰂖𰁓𰁦
𰂍𰂅𰃍𰃄𰁚𰃊𰃀𰁦𰁓𰂜𰂓𰁊𰂅𰂖𰀺𰂖𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰁚𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰁚𰀁𰂖𰂜𰃍𰂂𰁦𰃀𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰃊𰃍𰂜𰁝𰂅𰃄𰃄𰁦𰁓𰃍𰂅𰂖𰁹𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰂅𰃄𰃊𰃍𰂜𰃊𰃒𰃄𰁦𰃊
𰂅𰂖𰁓𰂎𰃒𰁝𰂅𰂖𰁹𰃊𰀺𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰂎𰂅𰁊𰃀𰀺𰃀𰃛𰁚𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰃄𰂓𰀺𰂎𰂎𰃊𰂜𰃀𰃊𰃄𰂂𰂜𰃀𰃍𰃊𰂅𰂖𰃍𰁦𰃀𰁲𰁦𰃀𰂅𰂖𰁹𰃊𰀬𰀟𰀁𰂪𰃄𰂅𰀬𰀟𰀁𰂫𰃊𰃍𰂜𰃊𰃄𰂅𰂎𰁦𰂖𰁓𰁦𰃊𰃍𰂂𰁦𰃊𰁦𰃚𰂨𰃀𰁦𰃄𰃄𰂅𰂜𰂖𰃊𰂜𰁲𰃊
𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰂮𰃊𰁹𰁦𰂖𰁦𰃄𰃊𰂜𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊𰂅𰂖𰃊𰃘𰂅𰀺𰁊𰂎𰁦𰃊𰁓𰁦𰂎𰂎𰃄𰂮𰃊𰀁𰃊𰂖𰃒𰂓𰁊𰁦𰃀𰃊𰂜𰁲𰃊𰃙𰁦𰂎𰂎𰂄𰂍𰂖𰂜𰃙𰂖𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰃊
𰂜𰁲𰁲𰁦𰃀𰃊𰂍𰂅𰃍𰃄𰃊𰀺𰂖𰁝𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃄𰃊𰁲𰂜𰃀𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰂄𰃀𰁦𰂎𰀺𰃍𰁦𰁝𰃊𰁹𰁦𰂖𰁦𰃊𰃄𰂅𰂎𰁦𰂖𰁓𰂅𰂖𰁹𰃊𰃄𰃍𰃒𰁝𰂅𰁦𰃄𰃊
𰀌𰂅𰃄𰁦𰀺𰃄𰁦𰂄𰀭𰂨𰁦𰁓𰂅𰁳𰁓𰃊𰀁𰂨𰂨𰂎𰂅𰁓𰀺𰃍𰂅𰂜𰂖𰃄𰃀𰁦𰂎𰃛𰂅𰂖𰁹𰃊𰂜𰂖𰃊𰀬𰀟𰀁𰃊𰂅𰂖𰃍𰁦𰃀𰁲𰁦𰃀𰁦𰂖𰁓𰁦𰃊𰂪𰀬𰀟𰀁𰂅𰂫𰂮𰃊𰀁𰂓𰁊𰂅𰂜𰂖𰃊𰀭𰂅𰂎𰁦𰂖𰁓𰁦𰃀𰃊𰃄𰂅𰀬𰀟𰀁𰃊𰂎𰂅𰁊𰃀𰀺𰃀𰂅𰁦𰃄𰃊
𰀁𰃍𰃊𰀪𰁳𰃞𰁦𰃀𰂽𰃄𰀬𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰀊𰁦𰂖𰃍𰁦𰃀𰁚𰃊𰃍𰂂𰁦𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂨𰂎𰀺𰃍𰁲𰂜𰃀𰂓𰃊𰂂𰀺𰃄𰃊𰂂𰀺𰃘𰁦𰃊𰃍𰂂𰃀𰁦𰁦𰃊𰂅𰂖𰁝𰂅𰃘𰂅𰁝𰃒𰀺𰂎𰃊𰃄𰂅𰀬𰀟𰀁𰃄𰃊𰂨𰁦𰃀𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰁚𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃊𰂂𰃒𰂓𰀺𰂖𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰁚
𰀺𰃊𰂓𰀺𰂖𰁝𰀺𰃍𰁦𰃊𰃍𰂜𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰃊𰂓𰁦𰂓𰁊𰁦𰃀𰃄𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰁹𰁦𰂖𰁦𰃊𰁲𰀺𰂓𰂅𰂎𰃛𰃊𰀺𰃄𰁝𰃀𰃒𰁹𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰁚𰃊𰀺𰂖𰁝𰁝𰃀𰃒𰁹𰁹𰀺𰁊𰂎𰁦𰃊𰁹𰁦𰂖𰂜𰂓𰁦𰃊𰂎𰂅𰁊𰃀𰀺𰃀𰂅𰁦𰃄𰁚𰃊𰃙𰂂𰂅𰁓𰂂𰃊𰁓𰂜𰂓𰂨𰃀𰂅𰃄𰁦𰃊
𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃄𰁚𰃊𰀺𰁓𰁓𰂜𰃀𰁝𰂅𰂖𰁹𰃊𰃍𰂜𰃊𰀛𰁦𰃄𰃄𰂅𰁦𰃊𰀞𰂮𰃊𰀎𰂖𰁹𰂎𰂅𰃄𰂂𰁚𰁝𰂅𰃀𰁦𰁓𰃍𰂜𰃀𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰃍𰃀𰀺𰂖𰃄𰁓𰃀𰂅𰂨𰃍𰂅𰂜𰂖𰃊𰁲𰀺𰁓𰃍𰂜𰃀𰃄𰃊𰀺𰂖𰁝𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰂓𰀺𰃛𰃊𰁊𰁦𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃀𰀺𰂨𰁦𰃒𰃍𰂅𰁓𰃊
𰀜𰂅𰂖𰀺𰃄𰁦𰃊𰀊𰁦𰂖𰃍𰁦𰃀𰃊𰂜𰁲𰃊𰀎𰂓𰂨𰂂𰀺𰃄𰂅𰃄𰂮𰃊𰀮𰂂𰁦𰃊𰂨𰂎𰀺𰃍𰁲𰂜𰃀𰂓𰃊𰂨𰀺𰃀𰃍𰂖𰁦𰃀𰃄𰃊𰃙𰂅𰃍𰂂𰀪𰁳𰃞𰁦𰃀𰃊𰃀𰁦𰂎𰁦𰃘𰀺𰂖𰁓𰁦𰂮𰃊𰀜𰀺𰃍𰂂𰃛𰀝𰀺𰃍𰂂𰀺𰂓𰁚𰀁𰂓𰁊𰂅𰂜𰂖𰂽𰃄𰃊𰃄𰁦𰂖𰂅𰂜𰃀𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃊𰂓𰀺𰂖𰀺𰁹𰁦𰃀𰁚𰀬𰀟𰀁𰂅𰁚
𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰃄𰃍𰃄𰁚𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰂅𰂖𰁹𰃊𰃙𰂂𰁦𰃀𰁦𰃊𰃀𰂜𰀺𰁝𰁊𰂎𰂜𰁓𰂍𰃄𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰂨𰃀𰂜𰁓𰁦𰃄𰃄𰃊𰃄𰀺𰃛𰃄𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰃊𰀺𰂎𰃄𰂜𰃊𰂓𰀺𰂍𰁦𰃄𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰃊𰃄𰂅𰀬𰀟𰀁𰃊𰂎𰂅𰁊𰃀𰀺𰃀𰂅𰁦𰃄𰂮𰃊𰂺𰀬𰀟𰀁𰂅𰃊𰀺𰃄𰃊𰀺𰃊
𰂓𰀺𰃛𰃊𰂜𰁓𰁓𰃒𰃀𰁚𰃊𰀺𰂖𰁝𰃊𰂎𰂜𰂜𰂍𰂅𰂖𰁹𰃊𰁲𰂜𰃀𰃊𰀺𰂨𰂨𰃀𰂜𰀺𰁓𰂂𰁦𰃄𰃊𰃍𰂜𰃊𰀺𰁝𰁝𰃀𰁦𰃄𰃄𰃊𰂖𰁦𰁦𰁝𰃄𰃊𰃙𰂅𰃍𰂂𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰂂𰀺𰃄𰃊𰃀𰁦𰃘𰂜𰂎𰃒𰃍𰂅𰂜𰂖𰂅𰃞𰁦𰁝𰂂𰂜𰃙𰃊𰂨𰁦𰂜𰂨𰂎𰁦𰃊𰃍𰂂𰂅𰂖𰂍𰃊𰂜𰁲𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰃘𰀺𰂎𰂅𰁝𰀺𰃍𰂅𰂜𰂖𰂮𰃊
𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰂮𰃊𰂺𰀪𰁳𰃞𰁦𰃀𰃊𰂅𰃄𰃊𰀺𰃊𰁝𰃀𰃒𰁹𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰁚𰂻𰀎𰂖𰁹𰂎𰂅𰃄𰂂𰃊𰃄𰀺𰃛𰃄𰂮𰃊𰂺𰀡𰃒𰃀𰃊𰀖𰂖𰃄𰃍𰁦𰀺𰁝𰃊𰂜𰁲𰁳𰃊𰂖𰁝𰂅𰂖𰁹𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰃊𰃍𰂜𰃊𰁳𰃊𰁹𰃒𰃀𰁦𰃊𰂜𰃒𰃍𰃊𰃍𰂂𰁦𰃊𰃀𰂜𰂎𰁦𰃊𰂜𰁲𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰃊𰂅𰂖𰃊𰀺𰃊
𰂨𰃀𰂅𰂓𰀺𰃀𰃛𰃊𰁹𰂜𰀺𰂎𰃊𰂅𰃄𰃊𰃍𰂜𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰃊𰂖𰂜𰃘𰁦𰂎𰁚𰃊𰁦𰁲𰁲𰁦𰁓𰃍𰂅𰃘𰁦𰁚𰃊𰀺𰂖𰁝𰃊𰃄𰀺𰁲𰁦𰃊𰃍𰂂𰁦𰃀𰀺𰂨𰁦𰃒𰃍𰂅𰁓𰃄𰃊𰁲𰂜𰃀𰃊𰂨𰀺𰃀𰃍𰂅𰁓𰃒𰂎𰀺𰃀𰃊𰁊𰂅𰂜𰂎𰂜𰁹𰂅𰁓𰃊𰁦𰃘𰁦𰂖𰃍𰁚𰀬𰀟𰀁𰂅𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊𰃛𰂜𰃒𰃊𰃍𰂜𰃊𰂍𰂖𰂜𰁓𰂍𰁝𰂜𰃙𰂖𰃊𰁦𰃚𰂨𰃀𰁦𰃄𰃄𰂅𰂜𰂖𰃊
𰂂𰃒𰂓𰀺𰂖𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰂮𰃊𰀮𰂂𰁦𰃀𰁦𰃊𰂅𰃄𰃊𰀺𰃊𰃄𰃒𰁊𰃄𰃍𰀺𰂖𰃍𰂅𰀺𰂎𰃊𰂖𰁦𰁦𰁝𰃊𰃍𰂜𰃊𰁊𰁦𰃍𰃍𰁦𰃀𰃊𰃒𰂖𰁝𰁦𰃀𰃄𰃍𰀺𰂖𰁝𰂅𰁲𰃊𰃛𰂜𰃒𰃊𰂍𰂖𰂜𰃙𰃊𰃍𰂂𰁦𰃊𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰁹𰁦𰂖𰁦𰁚𰂻𰃊𰃄𰂂𰁦𰃊𰂜𰁊𰃄𰁦𰃀𰃘𰁦𰃄𰂮𰃊𰀌𰀺𰃘𰂅𰁝𰀌𰂜𰃀𰃀𰂅𰃄𰁚
𰃍𰂂𰁦𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰁦𰃘𰁦𰂖𰃍𰃄𰃊𰃒𰂖𰁝𰁦𰃀𰂎𰃛𰂅𰂖𰁹𰃊𰁦𰁲𰁳𰃊𰁓𰀺𰁓𰃛𰃊𰀺𰂖𰁝𰃊𰃄𰀺𰁲𰁦𰃍𰃛𰃊𰀺𰃄𰃊𰃙𰁦𰂎𰂎𰃊𰀺𰃄𰃊𰂓𰂜𰃀𰁦𰃊𰃄𰁦𰂖𰂅𰂜𰃀𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂜𰃀𰃊𰀺𰂖𰁝𰃊𰁹𰁦𰂖𰁦𰃀𰀺𰂎𰃊𰂓𰀺𰂖𰀺𰁹𰁦𰃀𰁚𰃊𰁓𰁦𰂎𰂎𰃊𰁊𰂅𰂜𰂎𰂜𰁹𰃛𰃊𰀺𰂖𰁝𰀬𰀟𰀁𰂅𰁚𰃊𰀺𰁹𰃀𰁦𰁦𰃄𰂮𰃊
𰁓𰂎𰁦𰀺𰃀𰂎𰃛𰁝𰁦𰂎𰂅𰂖𰁦𰀺𰃍𰁦𰃊𰃍𰂂𰁦𰃊𰂅𰂓𰂨𰂎𰂅𰁓𰀺𰃍𰂅𰂜𰂖𰃄𰃊𰂜𰁲𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂅𰂖𰁹𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃄𰂮𰃊𰂺𰀖𰃍𰂽𰃄𰃊𰀺𰃊𰁹𰃀𰁦𰀺𰃍𰃊𰃍𰂜𰂜𰂎𰂮𰃊𰀪𰂂𰀺𰃀𰂓𰀺𰁓𰁦𰃒𰃍𰂅𰁓𰀺𰂎𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰂅𰁦𰃄𰃊𰂂𰀺𰃘𰁦𰃊𰀺𰁝𰂜𰂨𰃍𰁦𰁝𰃊𰃍𰂂𰁦𰃊
𰀮𰂂𰁦𰃀𰁦𰃊𰃄𰂜𰂓𰁦𰃍𰂅𰂓𰁦𰃄𰃊𰂅𰃄𰃊𰀺𰃊𰁹𰀺𰂨𰃊𰁊𰁦𰃍𰃙𰁦𰁦𰂖𰃊𰃙𰂂𰀺𰃍𰃊𰂅𰃄𰃊𰃄𰁦𰁦𰂖𰃊𰂅𰂖𰃊𰂅𰂖𰃊𰃘𰂅𰃍𰃀𰂜𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰂓𰂜𰃄𰃍𰃊𰂵𰃒𰂅𰁓𰂍𰂎𰃛𰁚𰂨𰀺𰃀𰃍𰂅𰁓𰃒𰂎𰀺𰃀𰂎𰃛𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰁳𰃊𰁦𰂎𰁝𰂮𰃊𰀬𰀟𰀁𰂅𰃊𰁓𰀺𰂖𰃊
𰀺𰃄𰃄𰀺𰃛𰃄𰃊𰀺𰂖𰁝𰃊𰃙𰂂𰀺𰃍𰃊𰂂𰀺𰂨𰂨𰁦𰂖𰃄𰃊𰂅𰂖𰃊𰁓𰁦𰂎𰂎𰃄𰂮𰂻𰁊𰁦𰃊𰃒𰃄𰁦𰁝𰃊𰃍𰂜𰃊𰃄𰁦𰁦𰃊𰂂𰂜𰃙𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰃍𰂂𰁦𰃊𰂓𰁦𰁓𰂂𰀺𰂖𰂅𰃄𰂓𰃊𰂜𰁲𰃊𰀺𰁓𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰀺𰃊𰁝𰃀𰃒𰁹𰃊𰂅𰃄𰂮𰃊
𰀛𰀺𰂓𰁦𰃄𰃊𰀊𰂂𰃀𰂅𰃄𰃍𰁦𰂖𰃄𰁦𰂖𰁚𰃊𰀺𰃄𰃄𰂜𰁓𰂅𰀺𰃍𰁦𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰃊𰁲𰁦𰂎𰂎𰂜𰃙𰁚𰀪𰀔𰀬𰀌𰂪𰀪𰁳𰃞𰁦𰃀𰃊𰀔𰂎𰂜𰁊𰀺𰂎𰃊𰀬𰀟𰀁𰂅𰃊𰁓𰀺𰂖𰃊𰂍𰂖𰂜𰁓𰂍𰁝𰂜𰃙𰂖𰃊𰀺𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰂅𰂖𰃊𰀺𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰃊𰃍𰃀𰀺𰂖𰃄𰁝𰃒𰁓𰃍𰂅𰂜𰂖𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰁚𰃊𰀺𰂖𰁝
𰀬𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰃊𰀺𰂖𰁝𰀌𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰂫𰁚𰃊𰂅𰃄𰃊𰃙𰂜𰃀𰂍𰂅𰂖𰁹𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰂜𰂖𰁓𰂜𰂎𰂜𰁹𰃛𰃊𰃍𰂂𰁦𰃀𰀺𰂨𰁦𰃒𰃍𰂅𰁓𰃊𰃍𰂂𰁦𰃊𰁦𰁲𰁲𰁦𰁓𰃍𰃄𰃊𰂜𰂖𰃊𰃍𰂂𰁦𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃊𰃒𰂨𰂄𰃊𰀺𰂖𰁝𰁝𰂜𰃙𰂖𰃄𰃍𰃀𰁦𰀺𰂓𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰁓𰀺𰂖𰃊𰁊𰁦𰃊
𰀺𰃀𰁦𰀺𰃊𰂅𰂖𰃊𰁝𰃀𰃒𰁹𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰃊𰀺𰂖𰁝𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰂮𰃊𰀕𰂅𰃄𰃊𰂨𰃀𰂜𰂌𰁦𰁓𰃍𰃄𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃊𰃄𰁦𰁦𰂖𰂮𰂻𰀁𰃄𰃊𰂨𰀺𰃀𰃍𰃊𰂜𰁲𰃊𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰃊𰀉𰂅𰂜𰃄𰃛𰃄𰃍𰁦𰂓𰃄𰁚𰃊𰃍𰂂𰁦𰃊𰁓𰂜𰂓𰂨𰀺𰂖𰃛𰂂𰀺𰃄𰃊𰁳𰃊𰁦𰂎𰁝𰂄𰁊𰀺𰃄𰁦𰁝
𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰂅𰂖𰁹𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰃊𰂅𰂖𰃊𰁦𰀺𰃀𰂎𰃛𰃊𰁓𰂎𰂅𰂖𰂅𰁓𰀺𰂎𰃊𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰂮𰃊𰀡𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰃄𰃍𰃄𰃊𰃙𰂜𰃀𰂎𰁝𰃙𰂅𰁝𰁦𰁚𰃊𰃙𰂂𰂜𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃊𰃍𰁦𰁓𰂂𰂖𰂅𰁓𰀺𰂎𰃊𰃄𰃒𰂨𰂨𰂜𰃀𰃍𰂮
𰂅𰃄𰃊𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰂅𰂖𰁹𰃊𰃍𰂂𰁦𰃊𰃄𰃒𰁊𰃍𰃛𰂨𰁦𰃄𰃊𰂜𰁲𰃊𰂨𰀺𰃍𰂅𰁦𰂖𰃍𰃄𰃊𰃙𰂂𰂜𰃄𰁦𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰃊𰃙𰂅𰂎𰂎𰃊𰁊𰁦𰃊𰂓𰂜𰃄𰃍𰃊𰀭𰂅𰁹𰂓𰀺𰂄𰀁𰂎𰁝𰃀𰂅𰁓𰂂𰂂𰀺𰃄𰃊𰁝𰁦𰃄𰂅𰁹𰂖𰃊𰃄𰁦𰃀𰃘𰂅𰁓𰁦𰃄𰃊𰁲𰂜𰃀𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰂅𰃞𰁦𰁝𰃊𰃄𰂅𰀬𰀟𰀁
𰂎𰂅𰂍𰁦𰂎𰃛𰃊𰃍𰂜𰃊𰃀𰁦𰃄𰂨𰂜𰂖𰁝𰃊𰃍𰂜𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰂮𰃊𰂺𰀵𰁦𰃊𰀺𰃀𰁦𰃊𰂎𰂜𰂜𰂍𰂅𰂖𰁹𰃊𰀺𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂜𰂎𰂅𰁹𰂜𰂖𰃒𰁓𰂎𰁦𰂜𰃍𰂅𰁝𰁦𰃊𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰃀𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃞𰁦𰁝𰃊𰃍𰂜𰃊𰃀𰁦𰁝𰃒𰁓𰁦𰃊𰂖𰂜𰂖𰃍𰀺𰃀𰁹𰁦𰃍𰃊
𰂨𰂂𰀺𰃀𰂓𰀺𰁓𰂜𰁹𰁦𰂖𰂜𰂓𰂅𰁓𰃄𰁭𰃍𰀺𰃀𰁹𰁦𰃍𰂅𰂖𰁹𰃊𰂨𰂜𰂎𰃛𰂓𰂜𰃀𰂨𰂂𰂅𰃄𰂓𰃄𰁚𰃊𰁹𰁦𰂖𰁦𰃊𰀺𰂓𰂨𰂎𰂅𰁳𰃊𰁓𰀺𰃍𰂅𰂜𰂖𰃄𰁚𰁊𰂅𰂖𰁝𰂅𰂖𰁹𰂮𰃊𰀖𰃍𰃊𰀺𰂎𰃄𰂜𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃄𰃊𰀺𰃊𰃙𰂅𰁝𰁦𰃊𰃀𰀺𰂖𰁹𰁦𰃊𰂜𰁲𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰀺𰂖𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃊
𰂓𰃒𰃍𰀺𰃍𰂅𰂜𰂖𰃄𰃊𰀺𰂖𰁝𰃊𰂜𰃍𰂂𰁦𰃀𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰂨𰃀𰂜𰁳𰃊𰂎𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰂅𰂖𰁹𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃄𰃊𰀺𰂖𰁝𰂍𰂅𰃍𰃄𰃊𰀺𰃄𰃊𰃙𰁦𰂎𰂎𰃊𰀺𰃄𰃊𰃍𰂂𰁦𰃊𰂅𰂖𰁝𰂅𰃘𰂅𰁝𰃒𰀺𰂎𰃊𰁦𰂖𰃞𰃛𰂓𰁦𰃄𰁚𰃊𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰁚
𰀺𰁓𰃍𰂅𰃘𰀺𰃍𰂜𰃀𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰂮𰃊𰀭𰂅𰂓𰂅𰂎𰀺𰃀𰂎𰃛𰁚𰀫𰂅𰀺𰁹𰁦𰂖𰃊𰂜𰁲𰁲𰁦𰃀𰃄𰃊𰀺𰃊𰂨𰂜𰃀𰃍𰁲𰂜𰂎𰂅𰂜𰃊𰂜𰁲𰃊𰂂𰂅𰁹𰂂
𰀓𰁦𰀺𰃍𰃒𰃀𰁦𰁝𰀪𰀺𰃀𰃍𰂅𰁓𰂅𰂨𰀺𰂖𰃍𰃄𰃍𰂂𰃀𰂜𰃒𰁹𰂂𰂨𰃒𰃍𰁮𰃍𰁦𰃄𰃍𰁦𰁝𰃊𰃄𰂅𰀬𰀟𰀁𰃊𰃄𰁦𰃍𰃄𰃊𰂅𰂖𰃊𰀺𰁝𰁝𰂅𰃍𰂅𰂜𰂖𰃊𰃍𰂜𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰂅𰂖𰁹𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰃊𰃄𰂅𰀬𰀟𰀁𰃄𰃊
𰀺𰂖𰁝𰃊𰁓𰂜𰂖𰃍𰃀𰂜𰂎𰃄𰂮𰃊𰀖𰂖𰃊𰀺𰁝𰁝𰂅𰃍𰂅𰂜𰂖𰃊𰃍𰂜𰃊𰂂𰃒𰂓𰀺𰂖𰃊𰀺𰂖𰁝𰃊𰂓𰂜𰃒𰃄𰁦𰃊𰃄𰁦𰃍𰃄𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰃍𰂂𰁦𰃊𰃙𰂂𰂜𰂎𰁦𰃊
𰀁𰂓𰁊𰂅𰂜𰂖𰃊𰀞𰂅𰂎𰂎𰂅𰂨𰂜𰃀𰁦𰀊𰂜𰃀𰂨𰂜𰃀𰀺𰃍𰂅𰂜𰂖𰁹𰁦𰂖𰂜𰂓𰁦𰃄𰁚𰁝𰃀𰃒𰁹𰁹𰀺𰁊𰂎𰁦𰃊𰁹𰁦𰂖𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃊𰀺𰂖𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰂄
𰃙𰃙𰃙𰂮𰀺𰂓𰁊𰂅𰂜𰂖𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰂓𰂅𰂎𰂎𰂅𰂨𰂜𰃀𰁦𰂮𰁓𰂜𰂓
𰀺𰃄𰃄𰂜𰁓𰂅𰀺𰃍𰁦𰁝𰃊𰁹𰁦𰂖𰁦𰃄𰃊𰀺𰂖𰁝𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰁚𰀫𰂅𰀺𰁹𰁦𰂖𰂽𰃄𰃊𰂖𰁦𰃙𰁦𰃄𰃍𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃊
𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰀉𰂅𰂜𰃄𰃛𰃄𰃍𰁦𰂓𰃄𰀞𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰀌𰁦𰃘𰂅𰁓𰁦𰃄𰀊𰂜𰃀𰂨𰂜𰃀𰀺𰃍𰂅𰂜𰂖𰃄𰂅𰀬𰀟𰀁𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰃀𰀺𰃍𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃄𰁚𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰂄
𰃙𰃙𰃙𰂮𰀺𰂨𰂨𰂎𰂅𰁦𰁝𰁊𰂅𰂜𰃄𰃛𰃄𰃍𰁦𰂓𰃄𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰁝𰁦𰃘𰂅𰁓𰁦𰃄𰂮𰁓𰂜𰂓
𰀺𰃄𰃄𰂜𰁓𰂅𰀺𰃍𰁦𰁝𰃊𰁹𰁦𰂖𰁦𰃄𰂮𰃊𰀖𰃍𰃄𰃊𰂖𰁦𰃙𰃊𰀓𰂎𰁦𰃚𰂅𰀪𰂎𰀺𰃍𰁦𰃊𰃄𰂅𰀬𰀟𰀁𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰃊𰀺𰂎𰂎𰂜𰃙𰃄𰃊𰁓𰃒𰃄𰃍𰂜𰂓𰃊
𰀉𰀌𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰀪𰁳𰃞𰁦𰃀𰃄𰁦𰂎𰁦𰁓𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰃄𰂅𰀬𰀟𰀁𰃄𰃊𰃍𰀺𰃀𰁹𰁦𰃍𰂅𰂖𰁹𰃊𰂂𰃒𰂓𰀺𰂖𰃊𰀺𰂖𰁝𰃊𰂓𰂜𰃒𰃄𰁦𰃊𰁹𰁦𰂖𰁦𰃄𰃊𰀺𰂎𰂜𰂖𰁹𰃊𰃙𰂅𰃍𰂂
𰃙𰃙𰃙𰂮𰁊𰁝𰁊𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰂨𰁳𰃞𰁦𰃀𰂮𰁓𰂜𰂓
𰁓𰂜𰂖𰃍𰃀𰂜𰂎𰃄𰁚𰃊𰃄𰁓𰀺𰂎𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰂨𰂎𰀺𰃍𰁦𰃊𰂎𰀺𰃛𰂜𰃒𰃍𰃊𰁲𰂜𰃀𰃊𰁦𰁲𰁳𰃊𰁓𰂅𰁦𰂖𰃍𰁚𰃊𰁦𰁓𰂜𰂖𰂜𰂓𰂅𰁓𰀺𰂎𰃊𰃄𰁓𰃀𰁦𰁦𰂖𰂅𰂖𰁹𰃊
𰀉𰂅𰂜𰂓𰂜𰂎𰀖𰂖𰃍𰁦𰃀𰂖𰀺𰃍𰂅𰂜𰂖𰀺𰂎𰀫𰂅𰀺𰁹𰁦𰂖𰃙𰂅𰃍𰂂𰃊𰂓𰂅𰂖𰂅𰂓𰃒𰂓𰃊𰂜𰁲𰁲𰂄𰃍𰀺𰃀𰁹𰁦𰃍𰃊𰁦𰁲𰁲𰁦𰁓𰃍𰃄𰃊𰀺𰂖𰁝𰃊𰂓𰀺𰃚𰂅𰂓𰀺𰂎𰃊𰂍𰂖𰂜𰁓𰂍𰁝𰂜𰃙𰂖𰃊𰁦𰁲𰁳𰃊𰁓𰂅𰁦𰂖𰁓𰃛𰂮
𰃙𰃙𰃙𰂮𰁊𰂅𰂜𰂓𰂜𰂎𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰂵𰂅𰀺𰁹𰁦𰂖𰂮𰁓𰂜𰂓
𰀊𰁦𰂎𰂎𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰀬𰂜𰁓𰂂𰁦𰃊𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰀌𰃀𰂅𰃘𰂅𰂖𰁹𰀓𰂜𰃀𰁓𰁦𰃄
𰃙𰃙𰃙𰂮𰁓𰁦𰂎𰂎𰃄𰂅𰁹𰂖𰀺𰂎𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰃀𰂜𰁓𰂂𰁦𰂄𰀺𰂨𰂨𰂎𰂅𰁦𰁝𰂄𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰂮𰁓𰂜𰂓𰀁𰃄𰃊𰂓𰂜𰃀𰁦𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰀺𰁓𰁓𰃒𰂓𰃒𰂎𰀺𰃍𰁦𰃄𰃊𰀺𰁊𰂜𰃒𰃍𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰁚𰃊𰂅𰃍
𰀎𰀞𰀌𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰀭𰂅𰁹𰂓𰀺𰂄𰀁𰂎𰁝𰃀𰂅𰁓𰂂𰃙𰂅𰂎𰂎𰃊𰁊𰁦𰃊𰁓𰃀𰃒𰁓𰂅𰀺𰂎𰃊𰁲𰂜𰃀𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁦𰃀𰃄𰃊𰃍𰂜𰃊𰁊𰁦𰃊𰀺𰁊𰂎𰁦𰃊𰃍𰂜𰃊𰀺𰁓𰁓𰁦𰃄𰃄𰃊𰁝𰀺𰃍𰀺𰁊𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝
𰃙𰃙𰃙𰂮𰁦𰂓𰁝𰁊𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰂮𰁓𰂜𰂓𰃙𰃙𰃙𰂮𰃄𰂅𰁹𰂓𰀺𰀺𰂎𰁝𰃀𰂅𰁓𰂂𰂮𰁓𰂜𰂓𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰃘𰁦𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰃍𰂜𰃊𰃄𰁦𰁦𰃊𰃙𰂂𰁦𰃀𰁦𰃊𰃍𰂂𰁦𰂅𰃀𰃊𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂜𰁲𰃊𰂅𰂖𰃍𰁦𰃀𰁦𰃄𰃍𰃊𰁳𰃊𰃍𰃄𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊
𰀖𰂖𰂖𰂜𰃘𰀺𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰀭𰂅𰁹𰂖𰀺𰂎𰀮𰃀𰀺𰂖𰃄𰁝𰃒𰁓𰃍𰂅𰂜𰂖𰃊𰀜𰂖𰂜𰃙𰂎𰁦𰁝𰁹𰁦𰁊𰂅𰁹𰃊𰂨𰂅𰁓𰃍𰃒𰃀𰁦𰂮𰃊𰀊𰂜𰂓𰁊𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰂅𰁦𰃄𰁚𰃊𰃍𰂜𰂜𰁚𰃊𰃙𰂅𰂎𰂎𰃊𰁓𰂜𰂖𰃍𰂅𰂖𰃒𰁦𰃊𰃍𰂜𰃊𰁝𰃀𰂅𰃘𰁦𰃊
𰃙𰃙𰃙𰂮𰂅𰂖𰂖𰂜𰃘𰀺𰁊𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄𰂮𰁓𰂜𰂓𰀎𰂖𰃘𰂅𰃀𰂜𰂖𰂓𰁦𰂖𰃍𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰃊𰀺𰂖𰁝𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰂮
𰀞𰁦𰃀𰁓𰂍𰀜𰀔𰀺𰀁𰃙𰃙𰃙𰂮𰃄𰃍𰂍𰁦𰂮𰂜𰃀𰁹
𰃙𰃙𰃙𰂮𰂓𰁦𰃀𰁓𰂍𰂮𰁝𰁦𰀝𰃛𰂖𰂖𰁦𰃊𰀝𰁦𰁝𰁦𰃀𰂓𰀺𰂖𰁚𰃊𰀪𰂂𰂮𰀌𰂮𰁚𰃊𰂅𰃄𰃊𰀺𰃊𰁲𰃀𰁦𰁦𰂎𰀺𰂖𰁓𰁦𰃊𰂓𰁦𰁝𰂅𰁓𰀺𰂎𰃊𰀺𰂖𰁝𰃊𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰃙𰃀𰂅𰃍𰁦𰃀𰃊𰁊𰀺𰃄𰁦𰁝𰃊𰂅𰂖𰃊
𰀟𰁦𰃙𰃊𰀷𰂜𰃀𰂍𰂮𰃊
𰂰𰃥𰃘
𰀟𰀘𰀕𰀎𰃛𰀰𰀊𰀘𰀎𰀢𰀊𰀎𰃛𰀲𰀎𰀊𰀗𰀢𰀤𰀟𰀤𰀖𰀘𰀎𰀰𰀁𰀁𰀁𰀭𰃉𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰀉𰃒𰃄𰂅𰂖𰁦𰃄𰃄𰃊𰀡𰁲𰁲𰂅𰁓𰁦𰃊𰀓𰁦𰀺𰃍𰃒𰃀𰁦
𰀟𰁦𰃙𰀪𰃀𰂜𰁝𰃒𰁓𰃍𰃄
𰀟𰁦𰃚𰃍𰂄𰀔𰁦𰂖𰁦𰃀𰀺𰃍𰂅𰂜𰂖𰃊𰀊𰂜𰃒𰂎𰃍𰁦𰃀𰃊𰀊𰂜𰃒𰂖𰃍𰁦𰃀
𰀮𰂂𰁦𰃊𰀞𰃒𰂎𰃍𰂅𰃄𰂅𰃞𰁦𰃀𰃊𰁷𰀊𰂜𰃒𰂎𰃍𰁦𰃀𰃊𰀊𰂜𰃒𰂖𰃍𰁦𰃀𰁚𰃊𰃍𰂂𰁦𰃊𰂎𰀺𰃍𰁦𰃄𰃍𰃊𰀺𰁝𰃘𰀺𰂖𰁓𰁦𰂓𰁦𰂖𰃍𰃊𰂅𰂖𰃊𰀺𰃊𰂎𰂜𰂖𰁹𰂎𰂅𰂖𰁦𰃊𰂜𰁲𰃊𰂨𰀺𰃀𰃍𰂅𰁓𰂎𰁦𰃊𰁓𰂜𰃒𰂖𰃍𰂅𰂖𰁹𰃊𰀺𰂖𰁝
𰃄𰂅𰃞𰂅𰂖𰁹𰃊𰂅𰂖𰃄𰃍𰃀𰃒𰂓𰁦𰂖𰃍𰃄𰁚𰃊𰂅𰃄𰁝𰁦𰃄𰂅𰁹𰂖𰁦𰁝𰃊𰃍𰂜𰁝𰁦𰂎𰂅𰃘𰁦𰃀𰃊𰀺𰂖𰃊𰂅𰂖𰁓𰃀𰁦𰀺𰃄𰁦𰁝𰁝𰃛𰂖𰀺𰂓𰂅𰁓𰃊𰃀𰀺𰂖𰁹𰁦𰂮𰀖𰃍𰃄𰃊𰃒𰂖𰂅𰂵𰃒𰁦𰃊𰁝𰂅𰁹𰂅𰃍𰀺𰂎𰂨𰃒𰂎𰃄𰁦𰃊
𰂨𰃀𰂜𰁓𰁦𰃄𰃄𰂅𰂖𰁹𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃄𰃊𰃄𰂅𰃞𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰃊𰃀𰁦𰃄𰃒𰂎𰃍𰃄𰃊𰂅𰂖𰃊𰃀𰁦𰀺𰂎𰃊𰃍𰂅𰂓𰁦𰂮𰃊𰀡𰃀𰂅𰁹𰂅𰂖𰀺𰂎𰂎𰃛𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰁦𰁝𰃊𰃍𰂜𰃊𰁓𰂜𰃒𰂖𰃍𰃊𰁊𰂎𰂜𰂜𰁝𰃊𰁓𰁦𰂎𰂎𰃄𰁚𰃊𰃍𰂂𰁦𰃊
𰀊𰂜𰃒𰂎𰃍𰁦𰃀𰃊𰀊𰂜𰃒𰂖𰃍𰁦𰃀𰃊𰂂𰀺𰃄𰃊𰀺𰃊𰁊𰃀𰂜𰀺𰁝𰃊𰃀𰀺𰂖𰁹𰁦𰃊𰂜𰁲𰃊𰀺𰂨𰂨𰂎𰂅𰁓𰀺𰃍𰂅𰂜𰂖𰃄𰁚𰃊𰁲𰃀𰂜𰂓𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰀺𰂖𰁝𰁊𰀺𰁓𰃍𰁦𰃀𰂅𰀺𰃊𰃍𰂜𰃊𰁲𰂜𰂜𰁝𰃊𰀺𰂖𰁝𰂂𰃛𰁝𰃀𰀺𰃒𰂎𰂅𰁓𰃊
𰁵𰃊𰃒𰂅𰁝𰃄𰂮𰀮𰂂𰁦𰃊𰀞𰃒𰂎𰃍𰂅𰃄𰂅𰃞𰁦𰃀𰃊𰁷𰃊𰁲𰁦𰀺𰃍𰃒𰃀𰁦𰃄𰃊𰂖𰁦𰃙𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃊𰂓𰀺𰂖𰀺𰁹𰁦𰂓𰁦𰂖𰃍𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰃍𰂜𰃊𰁦𰂖𰃄𰃒𰃀𰁦𰃊𰃀𰁦𰂨𰃀𰂜𰁝𰃒𰁓𰂅𰁊𰂅𰂎𰂅𰃍𰃛𰂮
𰀮𰂂𰁦𰃊𰀎𰀹𰀁𰁓𰁓𰁦𰃄𰃄𰃊𰁵𰃊𰃒𰂅𰁝𰂓𰀺𰂖𰀺𰁹𰁦𰂓𰁦𰂖𰃍𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰁚𰃊𰃄𰂜𰁲𰃍𰃙𰀺𰃀𰁦𰃊𰃙𰂅𰃞𰀺𰃀𰁝𰃄𰁚𰃊𰀺𰂖𰁝𰃊𰀺𰃒𰃍𰂜𰂓𰀺𰃍𰁦𰁝𰃊𰁲𰃒𰂖𰁓𰃍𰂅𰂜𰂖𰃄𰃊𰂅𰂓𰂨𰃀𰂜𰃘𰁦𰃊
𰁦𰀺𰃄𰁦𰃊𰂜𰁲𰃊𰃒𰃄𰁦𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰁓𰃀𰁦𰀺𰃄𰁦𰃊𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰂮𰀭𰂜𰁲𰃍𰃙𰀺𰃀𰁦𰃊𰁲𰃒𰂖𰁓𰃍𰂅𰂜𰂖𰃄𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃊𰀺𰂖𰃊𰀺𰃒𰃍𰂜𰂓𰀺𰃍𰁦𰁝𰃊𰃍𰂅𰂓𰁦𰃊𰃄𰃍𰀺𰂓𰂨𰃊𰁲𰂜𰃀𰃊
𰃀𰁦𰀺𰂎𰂄𰃍𰂅𰂓𰁦𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃊𰃍𰃀𰀺𰁓𰂍𰂅𰂖𰁹𰃊𰀺𰂖𰁝𰃊𰁦𰂎𰁦𰁓𰃍𰃀𰂜𰂖𰂅𰁓𰃊𰁊𰂎𰂜𰁓𰂍𰀺𰁹𰁦𰃊𰁝𰁦𰃍𰁦𰁓𰃍𰂅𰂜𰂖𰂮𰀮𰂂𰁦𰃊𰁝𰂅𰁹𰂅𰃍𰀺𰂎𰂨𰃒𰂎𰃄𰁦𰃊𰂨𰃀𰂜𰁓𰁦𰃄𰃄𰂅𰂖𰁹𰃊𰃄𰁓𰀺𰂖𰃄𰃊
𰀺𰂖𰁝𰃊𰃄𰃍𰂜𰃀𰁦𰃄𰁝𰀺𰃍𰀺𰃊𰁲𰂜𰃀𰃊𰀺𰁝𰁝𰂅𰃍𰂅𰂜𰂖𰀺𰂎𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰃊𰃀𰁦𰂨𰂜𰃀𰃍𰂅𰂖𰁹𰂮
𰀉𰁦𰁓𰂍𰂓𰀺𰂖𰃊𰀊𰂜𰃒𰂎𰃍𰁦𰃀
𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰃇𰂣𰁷𰂄𰂘𰂘𰃎𰂄𰁫𰂘𰁴𰁴
𰃙𰃙𰃙𰂮𰁓𰂜𰃒𰂎𰃍𰁦𰃀𰁓𰂜𰃒𰂖𰃍𰁦𰃀𰂮𰁓𰂜𰂓
𰀊𰁦𰂎𰂎𰂄𰀉𰀺𰃄𰁦𰁝𰃊𰀎𰀬𰀜𰂣𰃉𰃑𰃊𰀎𰀝𰀖𰀭𰀁𰁝𰁦𰃘𰁦𰂎𰂜𰂨𰂓𰁦𰂖𰃍𰁚𰁊𰃒𰃍𰃊𰀺𰂎𰃄𰂜𰃊𰁦𰂖𰀺𰁊𰂎𰁦𰃄𰃊𰃍𰂂𰁦𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁓𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰁮

𰀮𰂂𰁦𰃊𰁝𰃒𰀺𰂎𰂄𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰁓𰁦𰃊𰀪𰂂𰂜𰃄𰀪𰂂𰂜𰂄𰀎𰀬𰀜𰂣𰃉𰃑𰀊𰁦𰂎𰂎𰂄𰀉𰀺𰃄𰁦𰁝𰀎𰀝𰀖𰀭𰀁𰂪𰁦𰂖𰃞𰃛𰂓𰁦𰂄𰁝𰁦𰂨𰁦𰂖𰁝𰁦𰂖𰃍𰃊𰁊𰂅𰂜𰃄𰂅𰁹𰂖𰀺𰃍𰃒𰃀𰁦𰃄𰃊𰁲𰂜𰃀𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰂮
𰂎𰂅𰂖𰂍𰁦𰁝𰃊𰂅𰂓𰂓𰃒𰂖𰂜𰃄𰂜𰃀𰁊𰁦𰂖𰃍𰃊𰀺𰃄𰃄𰀺𰃛𰂫𰃊𰂅𰃄𰃊𰃍𰂂𰁦𰃊𰁳𰃊𰃀𰃄𰃍𰃊𰃍𰂜𰂓𰁦𰀺𰃄𰃒𰃀𰁦𰃊𰃍𰂜𰃍𰀺𰂎𰃊𰀺𰂖𰁝𰀉𰀌𰃊𰀉𰂅𰂜𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃄
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰁦𰁝𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰃄𰂅𰂓𰃒𰂎𰃍𰀺𰂖𰁦𰂜𰃒𰃄𰂎𰃛𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰃄𰀺𰂓𰁦𰃊𰃙𰁦𰂎𰂎𰁚𰃙𰂅𰃍𰂂𰂜𰃒𰃍𰃊𰃍𰂂𰁦𰃊𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰁫𰃇𰃇𰂄𰃑𰃎𰃑𰂄𰁫𰂘𰂘𰁴
𰂖𰁦𰁦𰁝𰃊𰁲𰂜𰃀𰃊𰃄𰂨𰁦𰁓𰂅𰀺𰂎𰂅𰃞𰁦𰁝𰃊𰁦𰂵𰃒𰂅𰂨𰂓𰁦𰂖𰃍𰂮𰀭𰀺𰂓𰂨𰂎𰁦𰃊𰂨𰃀𰁦𰂨𰀺𰃀𰀺𰃍𰂅𰂜𰂖𰃊𰂅𰃄𰃊𰃄𰂅𰂓𰂨𰂎𰁦𰁙𰀊𰁦𰂎𰂎𰃄𰃊𰀺𰃀𰁦𰃊𰃙𰃙𰃙𰂮𰁊𰁝𰂨𰂂𰂜𰃄𰁵𰂜𰃙𰂮𰁓𰂜𰂓
𰁹𰃀𰂜𰃙𰂖𰁚𰂨𰁦𰃀𰂓𰁦𰀺𰁊𰂅𰂎𰂅𰃞𰁦𰁝𰁚𰃊𰀺𰂖𰁝𰁊𰂎𰂜𰁓𰂍𰁦𰁝𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰃄𰀺𰂓𰁦𰃊𰂘𰃈𰂄𰃙𰁦𰂎𰂎𰂨𰂎𰀺𰃍𰁦𰂮𰀪𰃀𰂅𰂓𰀺𰃀𰃛
𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰀺𰁹𰀺𰂅𰂖𰃄𰃍𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰁦𰁝𰀎𰀬𰀜𰂣𰃉𰃑𰂜𰃀𰃊𰃍𰂜𰃍𰀺𰂎𰀎𰀬𰀜𰂣𰃉𰃑𰁚𰃊𰁓𰂜𰂓𰁊𰂅𰂖𰁦𰁝𰀪𰃀𰂜𰃍𰁦𰂅𰂖𰀖𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰂜𰂖𰀁𰂖𰀺𰂎𰃛𰃄𰂅𰃄
𰃙𰂅𰃍𰂂𰃊𰃄𰂨𰁦𰁓𰂅𰁦𰃄𰂄𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰂂𰂜𰃀𰃄𰁦𰃀𰀺𰁝𰂅𰃄𰂂𰂨𰁦𰃀𰂜𰃚𰂅𰁝𰀺𰃄𰁦𰂄𰁓𰂜𰂖𰂌𰃒𰁹𰀺𰃍𰁦𰁝𰂜𰃀𰃊𰀺𰂎𰂍𰀺𰂎𰂅𰂖𰁦𰃊𰀉𰂅𰀺𰁓𰂜𰃀𰁦𰃊𰀶𰂣𰃟𰃟𰃊𰂅𰃄𰃊𰀺𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰃊𰁲𰂜𰃀𰃊𰂎𰀺𰁊𰁦𰂎𰂄𰁲𰃀𰁦𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰂜𰂖𰃊𰁲𰂜𰃀𰃊
𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰂄𰁓𰂜𰂖𰂌𰃒𰁹𰀺𰃍𰁦𰁝𰃊𰃄𰁦𰁓𰂜𰂖𰁝𰀺𰃀𰃛𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰃊𰀺𰂖𰁝𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰁵𰃊𰃒𰂜𰃀𰂜𰁹𰁦𰂖𰂅𰁓𰃊𰂓𰃒𰂎𰃍𰂅𰂨𰃀𰂜𰂌𰁦𰁓𰃍𰃊𰂎𰂅𰁲𰁦𰃊𰃄𰁓𰂅𰁦𰂖𰁓𰁦𰃊𰃀𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰂎𰀺𰁊𰂜𰃀𰀺𰃍𰂜𰃀𰂅𰁦𰃄𰂮𰀮𰂂𰂅𰃄𰃊𰁹𰁦𰂖𰁦𰃀𰀺𰂎𰂨𰃒𰃀𰂨𰂜𰃄𰁦𰃊
𰃄𰃒𰁊𰃄𰃍𰃀𰀺𰃍𰁦𰃄𰃊𰀺𰂎𰂎𰂜𰃙𰃊𰁲𰂜𰃀𰃊𰂓𰁦𰀺𰃄𰃒𰃀𰁦𰂓𰁦𰂖𰃍𰃊𰃒𰃄𰂅𰂖𰁹𰃊𰀺𰃊𰃄𰃍𰀺𰂖𰁝𰀺𰃀𰁝𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰁓𰁦𰃊𰂨𰂎𰀺𰃍𰁦𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰁚𰁝𰁦𰃄𰂅𰁹𰂖𰁦𰁝𰃊𰃍𰂜𰁊𰁦𰃊𰃀𰁦𰀺𰁝𰂅𰂎𰃛𰃊𰀺𰁓𰁓𰁦𰃄𰃄𰂅𰁊𰂎𰁦𰃊𰃍𰂜𰂓𰃒𰂎𰃍𰂅𰂨𰂎𰁦𰃊𰃒𰃄𰁦𰃀𰃄𰁚𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃄𰃊
𰃀𰁦𰀺𰁝𰁦𰃀𰂮𰀟𰂜𰃀𰂓𰀺𰂎𰂅𰃞𰂅𰂖𰁹𰃊𰃍𰂂𰁦𰃊𰁵𰃊𰃒𰂜𰃀𰁦𰃄𰁓𰁦𰂖𰁓𰁦𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰁦𰁝𰀎𰀬𰀜𰂣𰃉𰃑𰃊𰃍𰂜𰃊𰃍𰂜𰃍𰀺𰂎𰃄𰃍𰀺𰃍𰁦𰂄𰂜𰁲𰂄𰃍𰂂𰁦𰂄𰀺𰃀𰃍𰃊𰃄𰂜𰁲𰃍𰃙𰀺𰃀𰁦𰃊𰃍𰂂𰀺𰃍𰃊𰂎𰁦𰀺𰁝𰃄𰃊𰃍𰂂𰁦𰃊𰃒𰃄𰁦𰃀𰃊𰁲𰃀𰂜𰂓𰂨𰃀𰁦𰂨𰀺𰃀𰀺𰃍𰂅𰂜𰂖𰃊𰃍𰂜𰃊𰃀𰁦𰃄𰃒𰂎𰃍𰃊
𰀎𰀬𰀜𰂣𰃉𰃑𰂓𰀺𰂍𰁦𰃄𰃊𰂅𰃍𰃊𰁦𰀺𰃄𰃛𰃊𰃍𰂜𰃊𰀺𰁓𰁓𰂜𰃒𰂖𰃍𰃊𰁲𰂜𰃀𰃊𰃙𰁦𰂎𰂎𰂄𰃍𰂜𰂄𰃙𰁦𰂎𰂎𰃘𰀺𰃀𰂅𰀺𰃍𰂅𰂜𰂖𰃄𰃊𰃄𰃒𰁓𰂂𰃊𰀺𰃄𰃊𰁓𰁦𰂎𰂎𰃙𰂅𰃍𰂂𰂅𰂖𰃊𰀺𰃊𰁝𰀺𰃛𰂮𰀮𰂂𰁦𰃊𰀉𰂅𰀺𰁓𰂜𰃀𰁦𰃊𰀶𰂣𰃟𰃟𰃊𰁦𰂖𰀺𰁊𰂎𰁦𰃄𰃊𰃄𰁓𰂅𰁦𰂖𰃍𰂅𰃄𰃍𰃄𰃊𰃍𰂜𰁝𰃀𰀺𰃙𰃊𰁓𰂜𰂖𰁓𰂎𰃒𰃄𰂅𰂜𰂖𰃄𰃊
𰂖𰃒𰂓𰁊𰁦𰃀𰂮𰁊𰀺𰃄𰁦𰁝𰂜𰂖𰃊𰁓𰂜𰂓𰂨𰃀𰁦𰂂𰁦𰂖𰃄𰂅𰃘𰁦𰃊𰁓𰂂𰀺𰃀𰀺𰁓𰃍𰁦𰃀𰂅𰃞𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰂂𰂜𰃙𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰃊𰃙𰂅𰃍𰂂
𰀬𰁁𰀌𰀭𰃛𰃄𰃍𰁦𰂓𰃄𰂜𰃍𰂂𰁦𰃀𰃊𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰁦𰃄𰃊𰂅𰂖𰃊𰃀𰁦𰀺𰂎𰃊𰃍𰂅𰂓𰁦𰂮𰀮𰂂𰂅𰃄𰃊𰃄𰂅𰂖𰁹𰂎𰁦𰃊𰂅𰂖𰃄𰃍𰃀𰃒𰂓𰁦𰂖𰃍𰃊𰁓𰀺𰂖𰃊𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰁦𰃊𰀺𰁲𰁳𰃊𰂖𰂅𰃍𰃛
𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰁫𰃟𰃟𰂄𰃎𰁷𰃎𰂄𰃇𰁷𰃇𰁴𰀺𰂖𰁝𰃊𰃀𰀺𰃍𰁦𰃊𰁓𰂜𰂖𰃄𰃍𰀺𰂖𰃍𰃄𰁚𰁊𰂅𰂖𰁝𰂅𰂖𰁹𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰂅𰃍𰃛𰁚𰃊𰁓𰂜𰂖𰁓𰁦𰂖𰃍𰃀𰀺𰃍𰂅𰂜𰂖𰁚𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰁦𰃀𰂓𰂜𰁝𰃛𰂖𰀺𰂓𰂅𰁓𰃊
𰃙𰃙𰃙𰂮𰀬𰂖𰀌𰀭𰃛𰃄𰃍𰁦𰂓𰃄𰂮𰁓𰂜𰂓𰃉𰁹𰂜𰃉𰀊𰁦𰂎𰂎𰀉𰀺𰃄𰁦𰁝𰀎𰀝𰀖𰀭𰀁𰂨𰀺𰃀𰀺𰂓𰁦𰃍𰁦𰃀𰃄𰃊𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰂜𰂖𰂮𰀖𰃍𰃊𰂨𰃀𰂜𰃘𰂅𰁝𰁦𰃄𰃊𰁝𰁦𰁦𰂨𰁦𰃀𰃊𰃒𰂖𰁝𰁦𰃀𰃄𰃍𰀺𰂖𰁝𰂅𰂖𰁹𰂜𰁲𰃊
𰂓𰂜𰂎𰁦𰁓𰃒𰂎𰀺𰃀𰃊𰂓𰁦𰁓𰂂𰀺𰂖𰂅𰃄𰂓𰃄𰃊𰀺𰂖𰁝𰃊𰂅𰂖𰃍𰁦𰃀𰀺𰁓𰃍𰂅𰂜𰂖𰃊𰂨𰀺𰃍𰂂𰃙𰀺𰃛𰃄𰃊𰃍𰂜𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊
𰀪𰃀𰂜𰃍𰁦𰁓𰃍𰂅𰂜𰂖𰁲𰃀𰂜𰂓𰀪𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰃊𰀪𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰃄𰁲𰃒𰂖𰁓𰃍𰂅𰂜𰂖𰀺𰂎𰂅𰃍𰃛𰃊𰀺𰂖𰁝𰃊𰁦𰂎𰃒𰁓𰂅𰁝𰀺𰃍𰁦𰃊𰁝𰂅𰃄𰁦𰀺𰃄𰁦𰃊𰂓𰁦𰁓𰂂𰀺𰂖𰂅𰃄𰂓𰃄𰂮
𰀮𰂂𰁦𰃊𰁓𰂜𰂓𰁊𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰂜𰁲𰃊𰀪𰂂𰂜𰃄𰀭𰀮𰀡𰀪𰀪𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃊𰀖𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰀊𰂜𰁓𰂍𰃍𰀺𰂅𰂎𰀮𰀺𰁊𰂎𰁦𰃍𰃄𰃊𰀉𰂅𰀺𰁓𰂜𰃀𰁦𰃉𰀔𰀎𰃊𰀕𰁦𰀺𰂎𰃍𰂂𰁓𰀺𰃀𰁦
𰀺𰂖𰁝𰃊𰁓𰀡𰂓𰂨𰂎𰁦𰃍𰁦𰃊𰀪𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰃊𰀖𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰀮𰀺𰁊𰂎𰁦𰃍𰃄𰃊𰁓𰀺𰂖𰃊𰂨𰃀𰂜𰃍𰁦𰁓𰃍𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰁲𰃀𰂜𰂓𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰂲𰁷𰁷𰃊𰂪𰃟𰂫𰃊𰃇𰂘𰃊𰃇𰂣𰃟𰃊𰁫𰃎𰂣𰂣
𰁊𰂜𰃍𰂂𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰃊𰀺𰂖𰁝𰂨𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰃄𰂮𰀪𰂂𰂜𰃄𰀭𰀮𰀡𰀪𰃊𰂅𰃄𰃊𰀺𰃊𰂨𰃀𰂜𰂨𰃀𰂅𰁦𰃍𰀺𰃀𰃛𰁊𰂎𰁦𰂖𰁝𰃙𰃙𰃙𰂮𰁊𰂅𰀺𰁓𰂜𰃀𰁦𰂮𰁓𰂜𰂓
𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃄𰃊𰃍𰂂𰀺𰃍𰃊𰀺𰁓𰃍𰃄𰃊𰂜𰂖𰃊𰀺𰃊𰁊𰃀𰂜𰀺𰁝𰃊𰃄𰂨𰁦𰁓𰃍𰃀𰃒𰂓𰂜𰁲𰃊𰀺𰁓𰂅𰁝
𰀺𰂖𰁝𰃊𰀺𰂎𰂍𰀺𰂎𰂅𰂖𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰁚𰃊𰃄𰁦𰃀𰂅𰂖𰁦𰃉𰃍𰂂𰃀𰁦𰂜𰂖𰂅𰂖𰁦𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰁚𰃊𰀺𰂖𰁝𰀮𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰀌𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰀮𰂜𰂜𰂎
𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰀺𰃍𰀺𰃄𰁦𰃄𰂮𰀮𰂂𰁦𰃊𰁓𰀡𰂓𰂨𰂎𰁦𰃍𰁦𰃊𰀪𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰃊𰀖𰂖𰂂𰂅𰁊𰂅𰃍𰂜𰃀𰃊𰀮𰂂𰁦𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰁓𰀺𰂖𰃊𰂪𰀪𰂄𰀮𰃛𰃀𰂄𰂣𰃟𰃟𰂫𰀜𰂅𰃍𰃊𰁲𰁦𰀺𰃍𰃒𰃀𰁦𰃄𰃊𰂨𰀺𰃍𰁦𰂖𰃍𰁦𰁝𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛
𰀮𰀺𰁊𰂎𰁦𰃍𰃄𰃊𰂅𰂖𰂂𰂅𰁊𰂅𰃍𰃊𰃄𰁦𰃀𰂅𰂖𰁦𰁚𰃊𰁓𰃛𰃄𰃍𰁦𰂅𰂖𰁦𰁚𰃊𰀺𰂖𰁝𰂓𰁦𰃍𰀺𰂎𰂎𰂜𰂨𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰃊𰀺𰁓𰃍𰂅𰃘𰂅𰃍𰃛𰁚𰃊𰀺𰂖𰁝𰃊𰀺𰃀𰁦𰃊𰃍𰂂𰀺𰃍𰃊𰁦𰂖𰀺𰁊𰂎𰁦𰃄𰃊𰂅𰂖𰃘𰁦𰃄𰃍𰂅𰁹𰀺𰃍𰂜𰃀𰃄𰃊𰃍𰂜𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁲𰃛𰂂𰃒𰂖𰁝𰃀𰁦𰁝𰃄𰃊𰂜𰁲𰃊𰃍𰂂𰂜𰃒𰃄𰀺𰂖𰁝𰃄𰃊𰂜𰁲𰃊
𰀺𰃘𰀺𰂅𰂎𰀺𰁊𰂎𰁦𰃊𰂅𰂖𰃊𰃍𰀺𰁊𰂎𰁦𰃍𰃄𰃊𰁲𰂜𰃀𰃊𰁦𰂅𰃍𰂂𰁦𰃀𰃊𰂣𰃟𰂓𰂎𰂜𰃀𰃊𰁴𰃟𰂓𰂎𰂜𰁲𰃊𰂎𰃛𰃄𰀺𰃍𰁦𰁚𰃙𰂅𰃍𰂂𰂜𰃀𰃊𰃙𰂅𰃍𰂂𰂜𰃒𰃍𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰁦𰁝𰃊𰃄𰁦𰂵𰃒𰁦𰂖𰁓𰁦𰃄𰃊𰀺𰂖𰁝𰂜𰁊𰃄𰁦𰃀𰃘𰁦𰃊𰃍𰂂𰁦𰃊𰃄𰃍𰀺𰃍𰁦𰃊𰂜𰁲𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊
𰁦𰃍𰂂𰃛𰂎𰁦𰂖𰁦𰁝𰂅𰀺𰂓𰂅𰂖𰁦𰃍𰁦𰃍𰃀𰀺𰀺𰁓𰁦𰃍𰂅𰁓𰃊𰀺𰁓𰂅𰁝𰂪𰀎𰀌𰀮𰀁𰂫𰂮𰃊𰀉𰂜𰃍𰂂𰂨𰃀𰂜𰁝𰃒𰁓𰃍𰃄𰃊𰀺𰃀𰁦𰃊𰂖𰂜𰂖𰃍𰂜𰃚𰂅𰁓𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂅𰂖𰃊𰁓𰁦𰂎𰂎𰃄𰃊𰀺𰂖𰁝𰃊𰃍𰂅𰃄𰃄𰃒𰁦𰃄𰂮𰀮𰂂𰁦𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰁓𰀺𰂖𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛
𰀺𰂖𰁝𰃊𰁦𰁲𰁲𰁦𰁓𰃍𰂅𰃘𰁦𰃊𰀺𰁓𰃀𰂜𰃄𰃄𰃊𰀺𰃊𰃙𰂅𰁝𰁦𰃊𰃀𰀺𰂖𰁹𰁦𰃊𰂜𰁲𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃊𰂓𰀺𰃍𰁦𰃀𰂅𰀺𰂎𰃄𰁚𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰂅𰂖𰁹𰃙𰀺𰃄𰃊𰃀𰁦𰁓𰁦𰂖𰃍𰂎𰃛𰃊𰃒𰃄𰁦𰁝𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰂜𰁲𰃊𰀺𰃊𰂖𰂜𰃘𰁦𰂎𰃊𰀺𰁓𰃍𰂅𰃘𰀺𰃍𰂅𰂖𰁹𰂓𰃒𰃍𰀺𰃍𰂅𰂜𰂖𰃊𰂅𰂖𰃊𰃍𰂂𰁦𰃊
𰀺𰂖𰂅𰂓𰀺𰂎𰃄𰁚𰂨𰂎𰀺𰂖𰃍𰃄𰁚𰃛𰁦𰀺𰃄𰃍𰁚𰃊𰀺𰂖𰁝𰁊𰀺𰁓𰃍𰁦𰃀𰂅𰀺𰂮𰀛𰀁𰀜𰃎𰂍𰂅𰂖𰀺𰃄𰁦𰃊𰂅𰂖𰃊𰀺𰁓𰃒𰃍𰁦𰃊𰂓𰃛𰁦𰂎𰂜𰂅𰁝𰂎𰁦𰃒𰂍𰁦𰂓𰂅𰀺𰃊𰁓𰁦𰂎𰂎𰃄𰂮𰀮𰂂𰁦𰃊𰂓𰁦𰃍𰂂𰂜𰁝𰃊𰃒𰂖𰁝𰁦𰃀𰂎𰃛𰂅𰂖𰁹
𰀬𰂜𰁓𰂂𰁦𰃊𰀁𰂨𰂨𰂎𰂅𰁦𰁝𰃊𰀭𰁓𰂅𰁦𰂖𰁓𰁦𰃍𰂂𰁦𰃊𰂍𰂅𰃍𰃊𰂅𰂖𰃘𰂜𰂎𰃘𰁦𰃄𰃊𰃍𰂂𰁦𰃊𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰁦𰂖𰃀𰂅𰁓𰂂𰂓𰁦𰂖𰃍𰃊𰂜𰁲𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰂄𰁓𰂜𰂖𰃍𰀺𰂅𰂖𰂅𰂖𰁹
𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰃎𰂣𰃇𰂄𰁴𰃑𰂣𰂄𰃑𰃟𰃟𰃟𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃄𰃊𰁲𰃀𰂜𰂓𰂨𰃀𰂜𰃍𰁦𰀺𰃄𰁦𰂄𰁝𰂅𰁹𰁦𰃄𰃍𰁦𰁝𰃊𰁓𰁦𰂎𰂎𰃊𰁦𰃚𰃍𰃀𰀺𰁓𰃍𰃄𰃊𰃒𰃄𰂅𰂖𰁹𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰂄𰀮𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊
𰃙𰃙𰃙𰂮𰃀𰂜𰁓𰂂𰁦𰂄𰁝𰂅𰀺𰁹𰂖𰂜𰃄𰃍𰂅𰁓𰃄𰂮𰃒𰃄𰀞𰂜𰃒𰃄𰁦𰃊𰂓𰀁𰁊𰂪𰀪𰂄𰀮𰃛𰃀𰂄𰂣𰃟𰃟𰂫𰂛𰂘𰁷𰂣𰂣𰃊𰁓𰂜𰃒𰂨𰂎𰁦𰁝𰃊𰃍𰂜𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃊𰀔𰃊𰀺𰁹𰀺𰃀𰂜𰃄𰁦𰃊𰁊𰁦𰀺𰁝𰃄𰂮
𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰂨𰁦𰂨𰃍𰂅𰁝𰁦𰃄𰃊𰁦𰂎𰃒𰃍𰁦𰁝𰃊𰁲𰃀𰂜𰂓𰃊𰃍𰂂𰁦𰃊𰁊𰁦𰀺𰁝𰃄𰃊𰀺𰃀𰁦𰃊𰃄𰃒𰁊𰃄𰁦𰂵𰃒𰁦𰂖𰃍𰂎𰃛𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁦𰁝
𰁊𰃛𰂎𰂅𰂵𰃒𰂅𰁝𰃊𰁓𰂂𰃀𰂜𰂓𰀺𰃍𰂜𰁹𰃀𰀺𰂨𰂂𰃛𰃉𰃍𰀺𰂖𰁝𰁦𰂓𰂓𰀺𰃄𰃄𰃊𰃄𰂨𰁦𰁓𰃍𰃀𰂜𰂓𰁦𰃍𰃀𰃛𰂮𰀮𰃛𰂨𰂅𰁓𰀺𰂎𰂎𰃛𰁚
𰀞𰃒𰂎𰃍𰂅𰂨𰂎𰁦𰃚𰁦𰁝𰃊𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰀁𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰃊𰂅𰂖𰀭𰂅𰂖𰁹𰂎𰁦𰃊𰀊𰁦𰂎𰂎𰃄𰃄𰁦𰃘𰁦𰃀𰀺𰂎𰂂𰃒𰂖𰁝𰃀𰁦𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃍𰃀𰃛𰃀𰂜𰃄𰂅𰂖𰁦𰃊𰃄𰂅𰃍𰁦𰃄𰃊𰁲𰃀𰂜𰂓𰂜𰂖𰁦𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃊𰁓𰀺𰂖𰃊
𰀉𰀌𰀪𰂂𰂜𰃄𰁵𰂜𰃙𰃊𰂅𰃄𰃊𰀺𰂖𰃊𰂅𰂖𰂖𰂜𰃘𰀺𰃍𰂅𰃘𰁦𰃊𰁵𰂜𰃙𰃊𰁓𰃛𰃍𰂜𰂓𰁦𰃍𰃀𰃛𰁮𰁊𰀺𰃄𰁦𰁝𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰃍𰂂𰀺𰃍𰃊
𰁊𰁦𰃊𰂅𰁝𰁦𰂖𰃍𰂅𰁳𰃊𰁦𰁝𰃊𰂅𰂖𰃊𰀺𰃊𰃄𰂅𰂖𰁹𰂎𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰁚𰁝𰁦𰂨𰁦𰂖𰁝𰂅𰂖𰁹𰂜𰂖𰃊𰃍𰂂𰁦𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰂽𰃄𰃊
𰁦𰂖𰀺𰁊𰂎𰁦𰃄𰃊𰀺𰁓𰃍𰂅𰃘𰀺𰃍𰂅𰂜𰂖𰂄𰃄𰃍𰀺𰃍𰁦𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰃊𰂜𰁲𰃊𰂓𰃒𰂎𰃍𰂅𰂨𰂎𰁦𰃊𰂨𰃀𰂜𰃍𰁦𰂅𰂖𰃄𰃊𰀺𰃍𰃊𰃄𰂅𰂖𰁹𰂎𰁦𰂄𰁓𰁦𰂎𰂎𰂎𰁦𰃘𰁦𰂎𰃄𰂮
𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂎𰁦𰃘𰁦𰂎𰃊𰀺𰂖𰁝𰃊𰃍𰂂𰁦𰃊𰃄𰁦𰂖𰃄𰂅𰃍𰂅𰃘𰂅𰃍𰃛𰂜𰁲𰃊𰃍𰂂𰁦𰃊𰂓𰀺𰃄𰃄𰃊𰃄𰂨𰁦𰁓𰃍𰃀𰂜𰂓𰁦𰃍𰁦𰃀𰂮
𰀉𰀌𰀪𰂂𰂜𰃄𰁵𰂜𰃙𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃄𰃊𰃒𰃄𰁦𰁝𰃊𰂅𰂖𰃊𰁓𰂜𰂓𰁊𰂅𰂖𰀺𰃍𰂅𰂜𰂖𰃊𰃙𰂅𰃍𰂂𰃊𰁓𰁦𰂎𰂎𰃊𰃄𰃒𰃀𰁲𰀺𰁓𰁦𰃊𰂓𰀺𰃀𰂍𰁦𰃀𰃄𰃊
𰀬𰁦𰃄𰁦𰀺𰃀𰁓𰂂𰁦𰃀𰃄𰃊𰀺𰃍𰃊𰀊𰁦𰂎𰂎𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰂂𰀺𰃘𰁦𰃊𰃒𰃄𰁦𰁝𰀪𰂂𰂜𰃄𰂨𰂂𰂜𰀭𰁓𰀺𰂖𰃊𰃍𰂜
𰂓𰀺𰂍𰁦𰃊𰂅𰃍𰃊𰂨𰂜𰃄𰃄𰂅𰁊𰂎𰁦𰃊𰃍𰂜𰃊𰃄𰃍𰃒𰁝𰃛𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰁦𰃘𰁦𰂖𰃍𰃄𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰃊𰂅𰂖𰃊𰃄𰂓𰀺𰂎𰂎
𰁝𰁦𰃍𰁦𰃀𰂓𰂅𰂖𰁦𰃊𰁓𰁦𰂎𰂎𰃒𰂎𰀺𰃀𰃊𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰃊𰂨𰃀𰂜𰁳𰂎𰁦𰃄𰃊𰂅𰂖𰃊𰂂𰃒𰂖𰁝𰃀𰁦𰁝𰃄𰃊𰂜𰁲𰃊𰁓𰁦𰂎𰂎𰂎𰂅𰂖𰁦𰃄𰁚
𰃄𰃒𰁊𰂨𰂜𰂨𰃒𰂎𰀺𰃍𰂅𰂜𰂖𰃄𰃊𰂜𰁲𰃊𰁓𰂜𰂓𰂨𰂎𰁦𰃚𰂨𰃀𰂅𰂓𰀺𰃀𰃛𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃄𰂮𰀮𰂂𰁦𰃊𰃀𰁦𰀺𰁹𰁦𰂖𰃍𰃊𰂎𰂅𰂖𰁦𰃊𰂅𰂖𰁓𰂎𰃒𰁝𰁦𰃄𰃊
𰃚𰁦𰂖𰂜𰁹𰃀𰀺𰁲𰃍𰃄𰁚𰃊𰀺𰂖𰁝𰂨𰃀𰂅𰂓𰀺𰃀𰃛𰂂𰃒𰂓𰀺𰂖𰃊𰃍𰃒𰂓𰂜𰃀𰃄𰂮
𰁓𰂎𰂜𰃄𰁦𰃊𰃍𰂜𰂣𰃟𰃟𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰃊𰁓𰂜𰂖𰂌𰃒𰁹𰀺𰃍𰁦𰁝𰂨𰂂𰂜𰃄𰂨𰂂𰂜𰃀𰃛𰂎𰀺𰃍𰂅𰂜𰂖𰂄𰃄𰂅𰃍𰁦𰂄𰃄𰂨𰁦𰁓𰂅𰁳𰃊𰁓𰃊𰀺𰂖𰃍𰂅𰁊𰂜𰁝𰂅𰁦𰃄𰂮
𰀊𰁦𰂎𰂎𰃊𰀭𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰀮𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛
𰀁𰂨𰃀𰂜𰂨𰃀𰂅𰁦𰃍𰀺𰃀𰃛𰁊𰃒𰁲𰁲𰁦𰃀𰃊𰃄𰃛𰃄𰃍𰁦𰂓𰃊𰁦𰂖𰀺𰁊𰂎𰁦𰃄𰃊𰀪𰂂𰂜𰃄𰁵𰂜𰃙𰃊𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰃊𰁝𰂅𰃀𰁦𰁓𰃍𰂎𰃛𰃊𰂅𰂖𰃙𰂂𰂜𰂎𰁦𰃊
𰀓𰂜𰃀𰃊𰂅𰂖𰁲𰂜𰃀𰂓𰀺𰃍𰂅𰂜𰂖𰃊𰂘𰃇𰁫𰂄𰁫𰃈𰃇𰂄𰃑𰃎𰃟𰃟
𰁊𰂎𰂜𰂜𰁝𰃊𰃄𰀺𰂓𰂨𰂎𰁦𰃄𰂮𰀮𰂂𰂅𰃄𰃊𰃍𰁦𰁓𰂂𰂖𰂜𰂎𰂜𰁹𰃛𰃊𰂖𰂜𰃍𰃊𰂜𰂖𰂎𰃛𰁊𰂜𰂜𰃄𰃍𰃄𰃊𰁓𰁦𰂎𰂎𰂄𰃄𰂅𰁹𰂖𰀺𰂎𰂅𰂖𰁹𰃊𰂖𰁦𰃍𰃙𰂜𰃀𰂍
𰃙𰃙𰃙𰂮𰁓𰁦𰂎𰂎𰃄𰂅𰁹𰂖𰀺𰂎𰂮𰁓𰂜𰂓
𰀺𰂖𰀺𰂎𰃛𰃄𰂅𰃄𰃊𰀺𰂖𰁝𰂂𰂅𰁹𰂂𰃊𰁓𰂜𰂖𰃍𰁦𰂖𰃍𰃊𰃄𰁦𰁓𰂜𰂖𰁝𰀺𰃀𰃛𰃊𰃄𰁓𰃀𰁦𰁦𰂖𰂅𰂖𰁹𰃊𰁲𰂜𰃀𰃊𰁝𰃀𰃒𰁹𰁝𰂅𰃄𰁓𰂜𰃘𰁦𰃀𰃛𰃊𰀺𰂖𰁝
𰂰𰃥𰁳
ScienceCareers.org POSITIONS OPEN
DIRECTOR
POSITIONS OPEN
The University of Southern Mississippi

School of Ocean and Earth Sciences
Classified Advertising The College of Science and Technology at the Uni-
versity of Southern Mississippi seeks a DIRECTOR
for the School of Ocean and Earth Sciences (SOES). FACULTY POSITION
The University of Southern Mississippi (USM), a Northwestern Feinberg School of Medicine
Carnegie Doctoral/Research Extensive and a South- Department of Neurology and Neuroscience
ern Regional Education Board (SREB) Category 1 The Neuromuscular Disorders Program of the
institution, is the only comprehensive university in Department of Neurology announces a new search
the state that has dual-campus status (Hattiesburg to recruit outstanding individuals for full-time,
and Long Beach). USM has several other teaching tenure-track appointments at the level of ASSIST-
and research sites on the Mississippi Gulf Coast. The ANT PROFESSOR, depending upon prior experi-
SOES was established in 2005, and comprises the ence and research accomplishments.
academic Departments of Coastal Sciences (Ocean Applications will be considered in the areas of
Springs), Geography and Geology (Hattiesburg), and novel therapeutic applications to amyotrophic lateral
Marine Science (Stennis Space Center) as well as the sclerosis (ALS) or study of the biology of motor
Gulf Coast Research Laboratory (Ocean Springs). For neurons.
more information, see websites: http://www.usm. The Ph.D. or M.D. appointees are expected to
edu/colleges/cost/ and http://www.usm.edu/ have demonstrated exceptional potential in either
From life on Mars cost/soes.htm. therapeutic approaches to ALS research or study of
to life sciences The Director will guide development of a shared motor neurons. Responsibilities of the positions are
vision and a strategic plan for the future of ocean and to develop dynamic, independently funded research
earth sciences at USM that facilitates interactions programs and to participate in medical, graduate,
For full advertising details, go to among the diverse units of SOES. The Director will and postgraduate teaching. High-quality laboratory
www.sciencecareers.org and click on be an advocate for the School at local, regional, and space and excellent startup support will be provided.
For Advertisers, or call one of our representatives. national levels, including fundraising, and will pro- Salary will be negotiable depending upon experience.
mote diversity and interdisciplinary expertise. The The appointees will have access to new state-of-
Director will coordinate an integrated program of the-art animal facilities and to shared facilities for
United States & Canada research, teaching, outreach, and service in support tissue culture, cell imaging, transgenic and knockout
of the School_s mission. projects, monoclonal antibodies, gene and protein
The successful candidate will have a Ph.D. and micro-arrays, structural biology, and biotechnology.
Fax: 202-289-6742
will be eligible for tenure in one of the academic Additional information about the Neuromuscular
IAN KING Sales Manager/Industry disciplines within SOES and will demonstrate a Disorders Program can be found on our web pages
Phone: 202-326-6528 record of achievement in research, extramural fund- (website: http://www.neurogenetics.northwestern.
ing, and teaching, and will have strong administra- edu). Applicants must include the following ma-
NICHOLAS HINTIBIDZE West tive and supervisory skills. Qualified persons should terials: (1) current curriculum vitae and list of pub-
Phone: 202-326-6533 submit a letter of intent, current curriculum vitae, lications, (2) brief statement of research interests (three
and the names of three references who will be con- pages or less), and (3) three letters of reference sent on
DARYL ANDERSON Midwest/Canada tacted only with the explicit permission of the can- their behalf to either: John A. Kessler, M.D. (e-mail:
Phone: 202-326-6543 didate. Nominations are welcomed. Review of [email protected]) or Teepu Siddique,
ALLISON MILLAR Northeast/Southeast applications and nominations will begin May 1, M.D. (e-mail: [email protected]), Co-
Phone: 202-326-6572
2007, and will continue until the position is filled. Chairs, Amyotrophic Lateral Sclerosis Search
All correspondence will be held in confidence and Committee, Northwestern University, Feinberg
should be mailed or e-mailed to: Dr. Rex Gandy, School of Medicine, Ward 10-185, 303 E. Chica-
Dean, College of Science and Technology, The go Avenue, Chicago, IL 60611.
Europe & International University of Southern Mississippi, 118 College Please refer to academic search number P-128-06.
Drive, Number 5165, Hattiesburg, MS 39406- Completed applications must be received by July 1,
E-mail: [email protected] 0001. E-mail: [email protected], telephone: 601- 2007. Appointments will commence on or after
Fax: +44 (0) 1223 326532 266-4883. Visit the USM website: http://www. October 1, 2007.
TRACY HOLMES Sales Manager usm.edu. Northwestern University is an Equal Opportunity/Affirma-
Phone: +44 (0) 1223 326525
The University of Southern Mississippi is an Equal Op- tive Action Educator and Employer and invites applications from
portunity Employer; diversity is highly valued. Minorities and all qualified individuals. Applications from women and
CHRISTINA HARRISON women are encouraged to apply. Affirmative Action/Equal minorities are especially sought. Hiring is contingent upon
Phone: +44 (0) 1223 326510 Opportunity Employer/ADAI. eligibility to work in the United States.
LOUISE MOORE
FACULTY POSITIONS
Phone: +44 (0) 1223 326528 Pathology Research
The Department of Pathology at the University of
Japan Illinois at Chicago (UIC) College of Medicine is
searching for candidates to fill two mid-senior level
JASON HANNAFORD tenure-track/tenured positions at the ASSISTANT, CARDIOVASCULAR PHARMACOLOGIST
Phone: +81 (0) 52-757-5360 ASSOCIATE, or FULL PROFESSOR level in its The Department of Veterinary Physiology and
E-mail: [email protected] Research Division. Desirable candidates will have a Pharmacology, College of Veterinary Medicine and
Fax: +81 (0) 52-757-5361 Ph.D. in a biomedical field (or an M.D. with equiv- Biomedical Sciences, Texas A&M University an-
alent research experience), and a record of at least nounces the availability of a tenure-track position for
three years of productive research as an independent ASSISTANT/ASSOCIATE PROFESSOR in the
investigator. Hiring of two individuals who have area of pharmacology. A strong research program in
To subscribe to Science:
In U.S./Canada call 202-326-6417 or 1-800-731-4939
worked together or have linked programs in the same cardiovascular science is desirable. A proven record
In the rest of the world call +44 (0) 1223-326-515 area is possible. The Department has a research focus in teaching is required. The successful candidate will
Science makes every effort to screen its ads for offensive
in cancer prevention and molecular epidemiology; be expected to contribute to a team-taught pharma-
and/or discriminatory language in accordance with U.S. however, investigators with interests outside of can- cology course in the professional curriculum. Depart-
and non-U.S. law. Since we are an international journal, cer will be considered. Preference will be given to mental faculty and their interests can be identified at
you may see ads from non-U.S. countries that request
applications from specific demographic groups. Since U.S.
individuals conducting research that involves human website: http://www.cvm.tamu.edu/vtpp. Re-
law does not apply to other countries we try to accom- samples, or that has obvious near-term translational view of applications will begin immediately and con-
modate recruiting practices of other countries. However, potential. Excellent new laboratory facilities, tissue tinue until the position is filled. Candidates should
we encourage our readers to alert us to any ads that they
feel are discriminatory or offensive.
resources, clinical collaborations, and infrastructure send curriculum vitae, letter of application, and names
support are available. Rank and salary will be com- and addresses of three references to: Randolph H.
mensurate with qualifications. For fullest considera- Stewart, D.V.M., Ph.D., Department of Veteri-
tion, submit curriculum vitae and list of three nary Physiology and Pharmacology, TAMU 4466
references by May 1, 2007, to: Dr. Peter Gann (e-mail: Texas A&M University, College Station, TX
[email protected]), University of Illinois at Chicago, 77843-4466. Telephone: 979-862-7764, fax:
Department of Pathology, 840 S. Wood Street, 979-845-6544. E-mail: [email protected].
Room 113 CSN, MC847, Chicago, IL 60612. Texas A&M University is an Equal Opportunity
UIC is an Affirmative Action/Equal Opportunity Employer. Employer/Educator.
www.sciencecareers.org SCIENCE VOL 316 6 APRIL 2007 129

The Gerstner Sloan-Kettering Graduate
School of Biomedical Sciences offers the next
generation of basic scientists a program to
study the biological sciences through the lens
of cancer — while giving students the tools
they will need to put them in the vanguard of
research that can be applied in any area
of human disease.
PhD Program
An Internationally Recognized Research Faculty in: An Innovative, Integrated Curriculum Provides
• Cancer genetics a Fundamental Understanding of:
• Genomic integrity • The nature of genes and gene expression
• Cell signaling and regulation • Cellular organization
• Structural biology • Tissue and organ formation
• Immunology • Cell-cell interactions
• Chemical biology • Cellular response to the environment
• Developmental biology • Enzyme activity
• Computational biology
• Experimental therapeutics Student Support and Services:
• Experimental pathology Students receive a fellowship package that includes
• Imaging and radiation sciences a stipend, tuition, textbook allowance, and health
• Oncology insurance. Students also have access to affordable
• Genomics housing within easy reach of the school.
• Animal models of disease
Please visit our Web site to learn how to apply, for application
deadlines, and for more information about our PhD program.
www.sloankettering.edu
[email protected] | 646.888.6639
New York City

FOCUS ON CAREERS
Careers in Cancer Research AAAS/Science Business Office Feature
MANY PATHS TO CHOOSE

Ask a cross-section of scientists how they got into cancer research, and you’ll hear
©iStockphoto.com/Andresr
about a dizzying variety of routes from fields as diverse as biology, pharmacology,
mathematics, and medicine. And with certain attributes – an inquiring mind, self-disci-
pline, and a dash of ambition – it seems that there’s no limit to what can be achieved.
By Julie Clayton
C
ancer research has moved with the times, embracing new technologies enabling
scientists to pursue more varied research goals than ever. Cancer researchers
can find themselves in various settings linked to either academia or industry,
working in many areas, from tackling basic questions in the laboratory to testing new
drugs and vaccines in the clinic.
Many young scientists at the basic research end of the spectrum will admit that they
were initially attracted more by the desire to investigate fundamental questions in biol-
ogy than to work specifically on cancer. But they often then realize the importance and
applicability of their work to understanding the underlying biological processes lead-
ing to cancer. From such understanding, new treatments can arise.
Laura Buttitta, for example, is a postdoctoral fellow at the Fred Hutchinson Cancer
“ You look
down the microscope
at the diversity of cancer

Research Center in Seattle. Armed with a Ph.D. in mouse embryology she was drawn by
the center’s top-class reputation for basic research. She is investigating the molecular and it makes you want to
switches that govern cell division – using fruit flies as a model system.
“I have to confess that I was more interested in how our cells work at the molecular
level than in cancer research. But since coming to the Hutch, I’ve been able to attend
[clinical research] seminars and have become more interested in that.”
“The Hutch” is one of 39 comprehensive cancer centers across the United States
designated as such by the National Cancer Institute at the National Institutes of Health
understand more.
”
(NIH). It brings together, under one roof, basic research into cancer at the cellular level,
clinical research, and epidemiology – for studying the cause and prevention of can-
cer at the population level. It offers various research training and support initiatives,
including internal postdoctoral awards, interdisciplinary training programs, and pilot
startup initiatives that enable researchers to embark upon new projects that are too
speculative initially to win short-term awards.
Cancer Research Competition Increases

In fiscal year 2005, The Hutch received more than $10 million in funding from the
National Institutes of Health – the world’s largest cancer research funder. This is
typical of the favorable status of cancer centers; their portion of NIH funds has
risen by around 30 per cent in the past five years. On the surface, NIH spend-
ing appears to have increased significantly all-round over the past decade. The
rby
agency’s total spending on cancer research rose from just over $2 billion in fiscal
e
nd
year 1995 to almost $5 billion in FY 2005. But taking inflation into account, actual E
tt-
Wi
spending by NIH on research has flattened over the past five years, including on cancer Paula
research. “Obviously, this has negatively impacted cancer research,” comments The
Hutch’s chief financial officer Randy Main.
At the same time, competition has intensified: Since 1996, the number of grant ap-
plicants to the NIH has more than doubled, leading to a fall in the proportion of grants
awarded, from 27 percent in 1996 to 19 percent in 2005. Researchers are turning in- UPCOMING FEATURES
stead to smaller funders. The Susan G. Komen Breast Cancer Foundation, for example,
Careers for Postdoc Scientists: Transferable Careers — April 20
received more than four times its usual number of applications owing to cuts in NIH
spending on breast cancer research, according to Paula Witt-Enderby, a researcher at Biotech and Pharma — April 27
Duquesne University in Pittsburgh and a member of the charity’s grant review panel. Interdisciplinary Research — May 4
In Europe, spending on cancer research is highly variable from one continued »
www.sciencecareers.org/businessfeatures 131
Ludwig Institute for Cancer Research
the global cancer institute
Group Leaders in Cancer Biology

The Cancer Research Center of Hawai‘i, an NCI-designated The establishment of a new Branch of the Ludwig Institute for Cancer
cancer center, is embarking on a major expansion of its Research (LICR) in Oxford University will focus its research endeavours
research programs. With dedicated funding from the State on cancer biology including suppressing tumour growth and preventing
of Hawai‘i through a tax on cigarette sales, the center is cancer metastasis. We are actively recruiting Research Group Leaders at
the Assistant Member (Assistant Professor/Lecturer), Associate Member
about to construct a state of the art 320,000 sq. ft. research (Associate Professor/Senior Lecturer/Reader) and Member (Professor)
and clinical care facility on the Honolulu waterfront. We plan levels.
to recruit tenure track faculty at the Assistant, Associate and
The LICR Oxford Branch will be housed in a new state of the art building
Full Professor ranks in the following areas: in the Institute of Cancer Medicine and be affiliated to the Nuffield
• Associate Center Director for Clinical Affairs and Department of Clinical Medicine (NDM); part of the Medical Sciences
Director of the Clinical Research Program. This posi- Division, University of Oxford. The LICR is a global non-profit organization
tion requires an MD degree with board certification in with nine Branches and numerous Affiliates worldwide.
an area of oncology. The successful candidate will have As an LICR faculty member, you will enjoy the benefits of being a distinctive
an extensive track record of extramural research funding, part of a dynamic local environment at a world-class university, and also
scholarly and scientific accomplishments, administrative of belonging to an internationally recognized Institute that is actively
responsibilities, and experience in clinical trials. pursuing the translation of its research discoveries into applications for
human benefit.
• Clinical Sciences. Multiple positions are available for
clinical scientists at all ranks and in all areas of oncology. For further details about these positions and how to apply, please email
Sarah Barnsley, [email protected], quoting ref – oxf-sci. The
• Director of Cancer Prevention and Control. An indi-

closing date is Monday 30th April 2007.
vidual with national funding is sought to lead a program of
behavioral research related to cancer prevention, detection
and control. Candidates with an interest in tobacco control The Ludwig Institute for Cancer Research is
are particularly encouraged to apply but other areas will an Equal Opportunity Employer. All qualified
be favorably considered. applicants will receive consideration for
employment without regard to race, color,
• Cancer Prevention and Control. Multiple faculty posi-
religion, sex or national origin.
tions at all ranks are available for scientists with research www.licr.org
interests in any area of cancer prevention and control.
• Thoracic Oncology. We are seeking several basic research
or clinical scientists with nationally funded research in lung
cancer, asbestos, mesothelioma or other thoracic malignan-
cies. Individuals with research interests in environmental
carcinogenesis and viral oncology are also strongly encour-
aged to apply. Vice Chair for Clinical Research
• Cancer Epidemiology. We are seeking several individu-
als with a background in clinical or molecular aspects of
Substance Dependence
cancer epidemiology to complement ongoing population An open-rank research faculty position is now
research which takes advantage of the unique ethnic diver- available at Montefiore Medical Center, The University
sity of Hawai‘i. Individuals at all ranks are encouraged to Hospital for the Albert Einstein College of Medicine.
apply.
• Biostatistics. Multiple positions are available at all ranks We seek an M.D. with the ability to maintain an active,
for biostatisticians to support research projects and to extramurally funded program in medications development
conduct related methodological research. for substance dependence disorders; build a Research
• Natural Products and Cancer Biology. We are seeking Division; and, ultimately, establish a Center. Our
scientists with research interests in fundamental molecular hospital network oversees treatment of some 5,000
mechanisms of cancer cell biology and/or with an interest patients with substance dependence disorders.
in the identification and development of novel anti-cancer
agents. The successful candidate will have an appointment in
the Department of Psychiatry and Behavioral Sciences.
Applicants should hold an MD and/or PhD or other doctoral
degree. Strong preference will be given to applicants with A highly competitive salary and a generous start-up
nationally funded cancer research grants. We offer a highly package will be available. Send curriculum vitae and
statement of research interests to: T. Byram Karasu,
MONTEFIORE
interactive and multidisciplinary research environment,

excellent startup packages and competitive salaries. For M.D., Silverman Professor & the University Chairman,
more information please see our website at www.crch.org Department of Psychiatry & Behavioral Sciences;
or contact Alan McClelland PhD, Associate Director for e-mail: [email protected]. We are an equal
Scientific Administration, at [email protected]. opportunity employer.
Applications including a curriculum vitae and a statement
of current and future research interests should be sent to
[email protected] or Search Committee,
Cancer Research Center of Hawai‘i, 1236 Lauhala Street,
Better. Believe it.
Suite 510, Honolulu, HI 96813. www.montefiore.org/careers
An Affirmative Action/Equal Opportunity Employer.
AAAS/Science Business Office Feature
FOCUS ON CAREERS
Careers in Cancer Research AAAS/Science
Careers BusinessResearch
in Cancer Office Feature
country to another. The UK spends the most, and has seen around a
9 percent growth in cancer research funding over the past five years “It’s the only way forward – to have
– supported mainly by charities – with £343 million spent in 2005 by
tight collaboration and mutual
the partners of the UK’s National Cancer Research Institute. Accord-
ing to Michael Stratton, director of the Cancer Genome Project at respect between clinicians and
the Wellcome Trust Sanger Institute in Cambridge, UK, Europe has a scientists.”
lot to offer, and it is “to be expected” that postdoctoral researchers
—Fran Balkwill
will move from one country to another to gain experience. Stratton
arrived at his position originally from a clinical background. He made
such a leap after specializing in pathology at the Hammersmith we can’t just spend the next 25 years finding out more. We can and
Hospital in London. “It brought me closer to the scientific basis of should tackle the big divide between what we know and what we can
disease – you look down the microscope at the diversity of cancer do for those patients on the ward.”
and it makes you want to understand more. I always wanted to do Balkwill’s first clinical trial starts later this year, based on develop-
research, but it was a rude shock not to go back to medicine.” ments in her laboratory. She will be using therapeutic antibodies to
Top-level cancer research institutes around Europe are now thriv- dampen the inflammation around ovarian tumors. This, she hopes,
ing, including The Netherlands Cancer Institute (NKI), Amsterdam; will enable “the good guys” – cells of the immune system – to spe-
CNIO in Madrid; the Radium Institute, Oslo; The Karolinska Institute, cifically attack and destroy tumors.
Stockholm; the Marie Curie and Gustav Russie Institutes in Paris; the Her lab is embedded within a cancer center, which allows closer
World Health Organization-funded IARC in Lyon; and the German Na- contact between scientists and clinicians, and which offers several
tional Cancer Institute (DKFZ) in Heidelberg. Each of these institutes advantages, including an improved flow and quality of tissue sam-
has something unique to offer, drawing excellent local researchers ples from clinic to laboratory and greater commitment from doctors.
as well as exceptional international scientists. “It’s the only way forward – to have tight collaboration and mutual
respect between clinicians and scientists,” she says.
Translational Potential Back in the US, and further along the translational pipeline is
Rather than focus on basic cancer research, many investigators are Doug Lowy, of the NIH in Bethesda, Maryland, who has already ex-
moving into the burgeoning area of translational research, taking perienced the potential of harnessing basic research for clinical use.
basic developments toward the clinic. Witt-Enderby at Duquesne is He has devoted the past 20 years to studying human papilloma vi-
using her expertise in molecular pharmacology to study the effects ruses (HPV), including their ability to cause cancer of the cervix. This
of melatonin on a mouse breast cancer model. She is now in discus- includes the development of the HPV-based vaccines now licensed
sions with the University of Pittsburgh about starting a small clinical to GlaxoSmithKline and Merck for the protection of women against
trial. “It’s a very big therapeutic strategy, and I just love it that all this disease. Lowy’s next goal is to develop a second generation of
this research on signaling and how a cell works is finally paying off,” vaccines that can be produced more easily and cheaply, making them
she says. more available for use in developing countries, and active against a
Witt-Enderby enjoys the academic setting as well as her mentor- broader range of HPV strains than the currently approved versions.
ing role toward graduate students. She notices, too, that increas- Many of Lowy’s former graduate students, postdocs, and clinical
ing numbers of graduate students show a specific interest in cancer fellows have moved on to prominent cancer research positions in
research. “They read about it or they know someone who has had academia and industry. He attributes their success to a combination
cancer. They want to know how drugs work and how to apply that of skills and qualities, including initiative, curiosity, technical and
to cancer.” intellectual abilities, and last but not least,
Witt-Enderby is on the grant review panel self-discipline. “There are many brilliant peo-
Cancer Research UK
for the Susan G. Komen Breast Cancer charity, www.cancerresearchuk.org
ple who are not successful as scientists be-
which is placing greater emphasis on trans- cause they don’t focus, or are not innovative,
lational research, and a corresponding in- Duquesne University or do not have sufficient technical skills. And
www.duq.edu
crease in suitable bids. “We’re starting to see you need patience, because it takes a long
that bridging. I’ve read so many grants where Exelixis, Inc. time to achieve anything meaningful.”
you see M.D.s teamed up with Ph.D.s.” Other www.exelixis.com
fund-holders are also encouraging the trend, Fred Huchinson Cancer Research Center
Industry Beckons
including the biggest of them all, the U.S. Na- www.fhcrc.org Biotechnology companies are eagerly pro-
tional Cancer Institute. moting translational cancer research. Two
National Cancer Institute
In Europe, a similar boon is occurring, www.cancer.gov
years ago, Keith Luhrs moved to Peregrine
with increasing numbers of scientists be- Pharmaceuticals after completing his first
coming involved in running clinical trials. Peregrine Pharmaceuticals postdoc at nearby University of California,
www.peregrineinc.com
Fran Balkwill, head of the London-based Irvine. He is now developing antibody-based
Translational Oncology Laboratory of Cancer The Wellcome Trust Sanger Institute therapeutics for cancer and other diseases.
Research-UK, sums up the mood: “We’ve www.wellcome.ac.uk “I was looking for something a bit more ap-
learned so much in the past 25 years but plied. Having that goal of continued »
Cancer Research UK Career Development Fellowship and Senior Cancer Research Fellowship
Would you like to work with one of the world’s leading cancer research organisations? Can your research be relevant to preventing, diagnosing or
treating cancer? Are you aiming to become a leader in your field? Cancer Research UK is committed to developing the next generation of leaders
in all areas of cancer research. If this is the opportunity you are looking for then our fellowship schemes can help you achieve your goals.
Career Development Fellowship Senior Cancer Research Fellowship

We will provide: We will provide:
• 6 years support • 6 years support
• Salaries for you, a post-doctoral researcher and a technician • Salaries for you, 2 post-doctoral researchers, a technician and a student
• Consumables costs and equipment • Consumables costs and equipment
About you: About you:
• You are a promising post-doctoral scientist who wishes to achieve • You are an outstanding scientist who wishes to develop your independence
independence in your research for the first time • You have shown exceptional ability in your previous work
• You have developed a strong track-record of research • You have between 6 and 10 years of postdoctoral research experience
• You have between 3 and 6 years of postdoctoral research experience • You may already be running your own research group.You may even
• You will not have previously run your own research group. However, hold a tenured position. We would still be interested in hearing from
you are now ready to step up and direct a small programme of work you, as long as you have been doing this for no more than six years
Cancer Research UK is also keen to support the broader development of its fellows, through opportunities for training and regular fellows’ meetings.
You must check your eligibility for these awards on our website at: fellowships.cancerresearchuk.org You will also find the application forms
and information about recently appointed fellows here.
The deadline for preliminary applications is Friday 8 June 2007.
If you would like to discuss your application for any of these fellowships, or have any other questions, please contact Dr Matthew Wakelin at
[email protected] or call 020 7438 5333.
Charity no: 1089464
Harold M. Weintraub Graduate

Student Awards – 2007
Cell and Molecular
The Fred Hutchinson Cancer Research Center Biology
congratulates the following recipients of the
2007 Harold M. Weintraub Graduate Student The Leukemia & Lymphoma Society’s
Award in recognition of outstanding achieve- Research Grant Program is based on the Postdoctoral Fellowship Award from
ment during Graduate Studies in the Biological belief that scientifically sound research the Francis Goelet Charitable Lead
Sciences. toward the cure or control of leukemia, lym- Trust, at the Sidney Kimmel Cancer
Sung Hee Ahn-Upton phoma, and myeloma should be encouraged Center in San Diego
Rockefeller University on a worldwide basis. The Society supports
We are pleased to offer a 3-year Francis Goelet
Michael A. Crickmore basic laboratory research and its application
in clinical settings. The Society’s Research Postdoctoral Fellowship in the laboratories of
Columbia University
Drs. Robert Margolis and Rati Fotedar at the
Ellen Ezratty Grant Programs include:
Sidney Kimmel Cancer Center. SKCC is one of
Columbia University
Career Development Program: Stipend sup- the few centers nationwide to have been honored
Douglas Fowler
port for basic and clinical investigators at the to offer this prestigious award. The applicant
Scripps Research Institute
level of Scholar, Special Fellow and Fellow. will have the opportunity to study mechanisms
Goarav Gupta
Deadlines: Preliminary Application (submit of mitotic exit and cell cycle checkpoints in
Cornell University Medical College
Gianna Hammer via website) - September 15; Full Applica- mammalian cells. The Goelet Fellowship
University of California, Berkeley tion - October 1 is intended for outstanding candidates with
Seyun Kim exceptional profiles and leadership qualities. The
Johns Hopkins University School of Medicine Specialized Center of Research Program: candidates will receive generous remuneration
Carolyn Phillips Support for team-based projects that are inter- and will work in the newly constructed research
University of California, Berkeley disciplinary, cohesive, and sharply focused. facility on the 10-acre SKCC campus in the
Zachary Pincus Deadlines: Preliminary Application (submit- middle of the densest concentration of health
Stanford University ted via website and hard copy) - November science research institutes and companies in the
Scott Tomlins 1; Full Application (by invitation only) United States. The Francis Goelet Fellow will
University of Michigan - March 15 have available state-of-the-art techniques and
Omer Yilmaz equipment in cell and molecular biology and of
University of Michigan Translational Research Program: Funding the Drug Development Core at the SKCC.
Hui Zhu for projects that translate laboratory findings
to clinical application. Please send c.v. with recommendations to: Dr.
Case Western Reserve University Albert Deisseroth, Chair, Goelet Fellow Search
Deadlines: Preliminary Application (submit-
The recipients will participate in a Sympo- Committee, Sidney Kimmel Cancer Center-
ted via website) - March 1; Full Application
sium this spring honoring Hal Weintraub and PDGS, 10905 Road to the Cure, San Diego,
his commitment to innovative science. More - March 15
CA 92121; [email protected].
information on this award can be found at: http: Guidelines are available at: www.LLS.org
//www.fhcrc.org/science/basic/weintraub EOE
AAAS/Science Business Office Feature
FOCUS ON CAREERS
Careers in Cancer Research AAAS/Science
Careers BusinessResearch
in Cancer Office Feature
Keith Luhrs, Missag Parsaghian

ity of new anticancer compounds. These are aimed specifically at
“If you step into the pathways involved in either the initiation and progression of cancer
right small company you or its resistance to conventional drugs. One successful compound,
will end up having to sharp- named Exel 647, that targets nonsmall-cell lung cancer in previously
untreated patients is now in a phase 2 clinical trial.
en your skills in a wide vari-
ety of the life sciences.” Right Person for the Job?
—Missag Parsaghian As for personal qualities, it is perhaps no surprise that cancer re-
searchers have much in common with other scientists. Bradley em-
something that is useful for human disease is definitely a driving phasizes the importance of academic knowledge. “You need a deep
force. I wanted to get things done at a faster pace, and make some- and broad knowledge, very good molecular biology skills, and the
thing beneficial.” ability to generate ideas.” He also emphasizes the need for speed in
What also attracted Luhrs was the prospect of honing many skills. translating ideas into results by being technically competent so as
“In a given week I’ll do cell culture, protein chemistry, molecular bi- to be able to perform experiments “quickly and efficiently ahead of
ology, microscopy, and structural biology on the computer,” adds someone else who’s going to compete with you on that idea.”
Luhrs. Witt-Enderby prizes students and postdocs with an open mind and
“If you step into the right small company you will end up having a willingness to read about topics as diverse as molecular biology,
to sharpen your skills in a wide variety of the life sciences,” adds endocrinology, drug mechanisms, pharmacology, and biostatistics.
Missag Parsaghian, Peregrine’s director of research and develop- “Because mine is a very interdisciplinary lab you have to learn so
ment. much. You have to know a lot because out of that will come a very
New technologies have enabled a whole slew of biotech compa- good answer to a research question.”
nies to form in recent years. Lexicon Genetics of Houston, Texas, for Stratton often recruits graduate students and postdocs with an
example, grew from the use of mouse embryonic stem cell technol- inclination toward bioinformatics, who can organize and process
ogy to create mouse models of disease. Its founder, Allan Bradley, data from genomewide screens, and improve high throughput DNA
was at the time based at the M.D. Anderson Cancer Research Center sequencing and analysis. On top of this, he values the involvement
in Houston, Texas, and now heads the world’s largest mouse genetics of trained bioinformaticians to be “roaming wild and free through
program at the Wellcome Trust Sanger Institute, which has a strong all our data to find something interesting, not constrained by goals
cancer component. Many of his original team took up positions with and targets.”
Lexicon Genetics.
“There has been a significant trickle of people who’ve gone to in- The Future of Cancer Research
dustry from my lab,” Bradley said. “They had unique skills that in- Looking ahead, cancer research looks set to thrive – particularly in
dustry did not have and they could get very lucrative salaries. I’ve the translational research arena and the development of new thera-
had people who’ve gone to Merck, Pfizer, either into the laboratory peutics.
or into management and business.” “This is one of the most exciting times in cancer research. I believe
Bradley himself resisted the lure of industry, preferring instead the that the next 10 years is going to be a golden age,” says Gendreau.
academic freedom of his current position. This enables him to make This is partly due to what he describes as “a more sophisticated
his resources – embryonic stem cell lines and mouse models – freely view that these cancers are similar in their molecular footprint – in
available to researchers outside the institute. He also prefers the the pathways that have caused them to become tumorigenic.” New
idea of having greater control of his research. “In a company you’re drugs targeting these pathways may be useful against a variety of
not in charge of your own destiny. Projects come and go. If the com- different cancers.
pany decides it, a project ends,” he says. In turn, Parsaghian of Peregrine Pharmaceuticals predicts that an-
Such sudden changes of direction in industry are something tibody-based therapeutics – and diagnostics – will continue to be a
that Steve Gendreau, a cell biologist and project leader at Exelixis growth area in cancer research, together with other protein-based
in South San Francisco, has experienced five times in the past nine biological agents and stem cell technology.
years. But rather than give up, he supports the change as necessary The excitement over translational research is tempered, however,
for keeping his company’s R&D portfolio moving toward a successful by the need to maintain “a balanced portfolio” between blue-sky re-
product. “These projects can feel like your own children. It’s some- search and clinical studies, according to Balkwill. “You still need the
times difficult to say goodbye. But the pragmatic view is that the basic research, because there’s still a lot to learn, and you need clini-
ability to remain flexible is absolutely key – we have to move people cal trials. But you also need this not-so-well-developed in-between
from one project to another,” he says. world,” says Balkwill.
One of the trade-offs, he adds, is avoiding what some see as the “It’s just as important that people do basic science,” agrees Witt-
hang-ups of academia. “I am not required to teach classes, write Enderby. “If you have to show that everything has translational po-
grants, or oversee graduate committees. I am paid to do what I love: tential within a short space of time, then you would lose out.”
discover life-changing cancer therapeutics.” Julie Clayton, a freelance science writer and journalist, works out of
Part of this involves developing assays for assessing the activ- Bristol, UK.
CAREERS IN CANCER RESEARCH
Abraham A. Mitchell OSI scientists have turned early research opportunities into medical advances
Distinguished Clinical Cancer Investigator Awards that focus on the needs of patients. If you are a bench scientist with excellent
communication skills and strong knowledge of cell biology, join our Cancer
The USA Mitchell Cancer Institute (USAMCI) at the University Biology group and work in our fast-paced team oriented environment.
of South Alabama (USA) invites nominations and applications of
highly qualified, clinically active physician-scientists for senior Research Scientist
faculty positions in oncologic sciences and interdisciplinary
Scientists with excellent molecular biology skills are sought to
clinical oncology within the USAMCI. A successful candidate generate clones and stable cell lines for EMT models. Candidates
will bring to the USAMCI an established, well-funded research must possess a Ph.D. with 0-3 years post-doc experience, or an MS
program, and will receive from the USAMCI a research expan- with 10+ years of relevant industry experience in cell and molecular
sion funding award in the aggregate total of $1,000,000 over a 3-5 biology. An understanding of different methods of cell line
year time period, with the goal of further growing the individual’s generation, gateway cloning and lentiviral systems are required.
and the institution’s research grant funding base and enhanc- Must have working knowledge of Vector NTI. Ref # FA00129
ing translational research links both within and external to the
USAMCI. Funds from the award may be used for partial salary Research Scientist
support for the awardee (research time only) and/or member(s) Scientists with strong cell biology and microscopy skills are invited
of his/her research team, materials and supplies, other opera- to design, develop and implement cell-based 2D and 3D EMT
models used to study the impact of small molecule inhibitors on the
tional expenses and/or equipment. Awardees will be expected epithelial to mesenchymal transition. A Ph.D. with 0-3 years post-
to participate in the Institute’s interdisciplinary clinical services doc experience in EMT biology, confocal microscopy along with an
not to exceed 2 days/week, with the balance of time devoted to excellent publication record and proven ability to engage in
research. Preference may be given to candidates whose research collaborative research efforts are required. Knowledge of cell
and/or clinical specialty interests are most complementary to signaling pathways as it relates to oncology is a plus. Ref #ME00190
existing or planned future USAMCI programs. Appointees to OSI offers excellent salaries and benefits including 401K, vacation,
these positions will receive highly competitive salary and ben- stock and much more. Interested candidates, please apply online at
efits, and academic rank commensurate with training, experience http://careers.osip.com. Please include ref#. EOE M/F/D/V.
and accomplishments.
Faculty Positions in Cancer Research

The University of South Alabama Mitchell Cancer Institute
(USAMCI) is seeking highly qualified candidates for cancer
research faculty positions at all ranks. Preference may be given POSITIONS OPEN
to candidates whose research background and expertise comple-
ment current or future thematic areas of emphasis within the The Weizmann Institute of science
USAMCI including genomics and gene expression, proteomics,
developmental therapeutics, molecular pathology and diagnos- will award a limited number of
tics, metastasis research and molecular oncology. The positions Distinguished Postdoctoral Fellowships
will be filled with researchers capable of establishing cutting- Known as
edge, core technologies and independent research projects in the
respective fields, providing collaborative core research support Koshland Scholars
for other investigators within the USAMCI, and achieving/
maintaining self-sufficiency through peer-reviewed external The fellowships are available in all the fields of scientific research that are
funding. The USAMCI is being organized and developed to pursued at The Weizmann Institute in the following areas:
facilitate highly interactive research involving laboratory and Biology, Chemistry, Computer Science, Physics,
clinical investigators, and unique collaborative opportunities Mathematics, Biochemistry
between experts in different fields. Appointees to these posi- All courses and seminars at the Institute are conducted in English.
tions will have attractive start-up resources and compensation,
and academic rank commensurate with training, experience and Applications may be submitted at any time, but the awards will be made
accomplishments. Appointees will have dedicated space within a shortly after the usual deadlines for the submission of Fellowship applications:
new state-of-the-art interdisciplinary basic/translational research January 5 and May 15 of each year. Candidates for Koshland fellowships
and clinical facility scheduled for completion in 2007. must be sponsored by a Faculty Member of the Weizmann Institute. Interested
candidates are advised to contact prospective sponsors directly.
The USAMCI is ideally located in Mobile, AL, a progressive, For additional information and application forms, consult the Feinberg Home
mid-sized port city of rich cultural history, in the beautiful Page at http://www.weizmann.ac.il/feinberg or write to: Postdoctoral
upper Gulf coastal region. Moderate climate, abundant outdoor Fellowship Program, The Feinberg Graduate School, The Weizmann Institute
of Science, Rehovot 76100, Israel; Fax: 972-8-934-4114; e-mail:
recreational opportunities, low cost-of-living and a “college- [email protected]
town” atmosphere all contribute to a high “quality-of-life”
opportunity.
The Weizmann Institute of Science invites outstanding Ph.D. students at
Applicants please send letter of interest and curriculum vitae to: universities abroad who are nearing graduation (or who have recently completed
Office of the Director - USAMCI, 307 N. University Blvd., MSB their Ph.D. studies) to visit the Institute for a few days, meet its scientists,
explore postdoctoral research opportunities, and experience its warm and relaxed
2015, Mobile, AL 36688 or e-mail [email protected].
atmosphere. Alimited number of such visits are subsidized by the Institute on
a competitive basis. Those who are invited are under no obligation to the
The University of South Alabama is an Affirmative Action and Institute to continue their postdoctoral studies here. For more details, see
Equal Opportunity Employer. http://www.weizmann.ac.il/feinberg/student_visit
COME SEE US AT AACR BOOTH 845
HealthCare www.myBayerjob.com
Lucia Rosano wants to make the world a better place - for everyone. As a biosci-
entist at Bayer, Lucia knows she is doing just that. Searching for solutions and
never giving up. That is the passion that unites all of us at Bayer. We call it the
Bayer Spirit. If you feel it, too, then it is high time we had a chance to talk about a
career at Bayer.
Postdoctoral Research Scientist (m / f)

Responsibilities The Target Discovery group at Bayer HealthCare in Wuppertal, Ger-
many offers a three year Postdoctoral Research Scientist position funded by the Marie
Curie Research Training Network.
The project is to identify changes in chromatin plasticity during heart failure.
Qualification The candidate will have a PhD and be highly motivated with qualifications
in molecular/cellular biology and in vivo pharmacology. A good knowledge in the area of
expression profiling using chip technology, primary cell cultures, RNAi gene knock down
and cardiovascular pharmacology would be desirable Good communication and organi-
zational skills are also essential, along with the ability of writing reports and publications
for international journals, present and disseminate project results in national and inter-
national meetings.
Your application If you are interested in the above position, please apply online, quo-
ting box number 0000000480 and attaching the relevant documents. Please also indi-
cate your salary expectations and the earliest possible date on which you could take up
employment. Visit the website www.chromatin-plasticity.org for more information on the
Marie Curie Research Training Network and www.bayerhealthcare.com for information
on Bayer HealthCare.
www.myBayerjob.com Phone +49 214 30 99 777

Senior Investigator in Bioinformatics
Research Triangle Park, North Carolina
National Institutes of Health
Biostatistics Branch – The NIEHS in Research Triangle Park, North Carolina, publication record and proven history of research leadership.
is seeking a senior investigator in Bioinformatics/Computational Biology to Salary is commensurate with experience and level of accomplishments.
assume directorship of a Bioinformatics Section in the Biostatistics Branch,
Environmental Diseases and Medicine Program, Division of Intramural Research. Applications from women and members of minority groups are particularly
NIEHS is one of the National Institutes of Health. The incumbent will develop welcome. To apply, submit a curriculum vitae, bibliography, brief statement
and direct a strong research group to carry out independent and collaborative of research interests and arrange for three letters of recommendation to
research in the general area of bioinformatic and computational biology, be sent by June 15, 2007, to the below address. Applications received
particularly as related to biological networks, analysis of high-dimensional after that date will be considered as needed.
data, proteomics, comparative and functional genomics, gene expression, and Ms. Barbara Curtis (DIR07-03)
epigenetics. This work will provide a bioinformatic infrastructure and innovative National Institutes of Health
data mining approaches to advance intramural research aimed at understanding National Institute of Environmental Health Sciences
biological responses to environmental stressors, in the context of cell biology, P.O. Box 12233, Maildrop A2-06
animal experimentation, clinical research and epidemiology. 111 T.W. Alexander Drive, Room A235
A Ph.D. or equivalent degree is mandatory. The ideal candidate is a senior Research Triangle Park, NC 27709
investigator with an international reputation in a specific area within the broad E-mail: [email protected]
context of bioinformatics and a genuine passion for science. Possible research
backgrounds include but are not limited to mathematics, computational biology, http://www.niehs.nih.gov
physics, statistics, genetics, biochemistry, bioinformatics, bioengineering DHHS and NIH are
and molecular biology. The successful candidate will have an outstanding Equal Opportunity Employers
HEALTH SCIENTIST ADMINISTRATOR

National Institute of General Medical Sciences

Cell Biology and Biophysics Division

The National Institute of General Medical Sciences (NIGMS), a major research component
of the National Institutes of Health (NIH) and the Department of Health and Human Services
(DHHS), is seeking applications from exceptional scientists to serve as Chief of the Biophysics

Branch in the Division of Cell Biology and Biophysics Division. The Biophysics Branch
supports major research grant programs in such areas as physical and chemical studies of proteins

and nucleic acids, structural analyses of macromolecules, development of physical techniques
for the analysis of molecular structure and function, and theoretical approaches to molecular
biophysics. The Institute is seeking an individual with scientific, administrative, and leadership
credentials who can manage individual grant programs in biophysics as well as serve as Chief
of the Biophysics Branch. Information about the Division of Cell Biology and Biophysics can

be found at: http://www.nigms.nih.gov/About/Overview/CBB.htm
Qualifications: The successful individual will possess a Ph.D., M.D. or equivalent degree in

a field relevant to the position, have research experience in biophysics, biochemistry or related
fields, an in-depth knowledge of biological processes, leadership and managerial skills, and
strong oral and written communication skills. Applicants must be U.S. citizens.

Salary: The current salary range is $110,363 – 143,471, depending on experience and accom-
plishments; a full Civil Service package of benefits (including retirement, health, life and
long term care insurance, Thrift Savings Plan participation, etc.) is available. Recruitment or
relocation incentive may be awarded and moving expenses will be paid.
How to Apply: Position requirements and detailed application procedures are provided in
vacancy announcement NIGMS-07-172576, which can be obtained by accessing the NIGMS
!" website at http://www.nigms.nih.gov. All applications and supplemental information must be
#""$#% received no later than April 16, 2007. For additional information, contact Ms. Eric Bandak
at (301) 594-2035.
Help Us Help Millions
Are you ready for an exciting career that could help improve millions of lives around the world?
Then consider joining the scientific and medical forces at the National Institute of Allergy and Infectious Diseases (NIAID). NIAID supports and
conducts basic, applied and clinical research to better understand, treat, and prevent some of the world’s most deadly diseases. The Division
of Microbiology and Infectious Diseases (DMID), an extramural research division of NIAID, supports extramural research related to the control
and prevention of diseases caused by virtually all human infectious agents (over 250 pathogens) except HIV. DMID has the following scientific
opportunities available:
Chief, Office of Clinical Research Affairs (OCRA) Program Officer
As the Chief of OCRA, DMID, the selected candidate will provide As part of DMID, the Parasitology and International Programs
scientific/medical leadership and direction for the planning, Branch (PIPB) is responsible for planning and conducting
implementation, management, and evaluation of a broad, programs of extramural research aimed at understanding
coordinated national/international program that deals with critical the biology of protozoan and helminth parasites and their
biomedical research issues. In this capacity, has oversight, provides interaction with the human host as well as their vectors and
medical information and resolves critical medical issues related intermediate hosts. As a Program Officer for PIPB, the selected
to the study design, safety/efficacy, monitoring and data analysis candidate will provide leadership and scientific/medical expertise
of clinical trials. The Chief of OCRA determines the programmatic and guidance in the planning, development, implementation
structure of the Branch; establishes priorities for new clinical trials and evaluation of basic and clinical research concepts, projects
program initiatives; oversees medical/scientific liaison with the and initiatives to appropriate advisory groups; identify opportunities
FDA, other government agencies and pharmaceutical companies. and problem areas, research gaps and relevant program needs
Candidates must have a Doctor of Medicine or Doctor of Osteopathy. and make recommendations for and facilitate new research
The selected candidate will have experience in the methodology, efforts, clinical studies, clinical trials or other initiatives; and
design, implementation, monitoring and assessment of clinical communicate with grantees/contractors, cooperative group
research trials, providing scientific/medical leadership and direction members/representatives and others on policy interpretation,
for the planning and management of a national/international program merit review and evaluation processes and procedures, and on
and demonstrated expertise in oral and written communication. decisions, concerns or other issues/matters of a medical/scientific
Experience in Regulatory Affairs, Vaccine Development, and nature. In order to be considered for this position, applicants should
International Studies is desirable. have experience in basic and/or clinical research to examine the
causes, diagnosis, treatment and prevention of infectious diseases;
This vacancy is being advertised under the Title 5 and Title 42 research experience in bacteriology, mycology, virology, or parasitic
hiring authorities. Salary is commensurate with experience and other tropical diseases, is required. Candidates with a Ph.D.
and accomplishments. and relevant experience are highly desired.
Title 5 vacancy: Applicants must be a U.S. citizen. To apply for this To apply for this vacancy, please visit http://usajobs.opm.gov
vacancy, please visit http://usajobs.opm.gov . Vacancy number: Vacancy number: NIAID-07-170616-DE and NIAID-07-170616-MP
NIAID-07-170767-DH. Specific application procedures apply and Salary: $79,397-$121,967. Specific application procedures apply.
applications must be submitted to a Human Resource Specialist. Applications must be submitted to a Human Resource Specialist
Title 42 vacancy: Non-citizens may apply. Please submit curriculum by May 4, 2007.
vitae/bibliography and three letters of reference to Denise Blackwell, A full Civil Service package of benefits (including retirement, health,
6610 Rockledge Drive, Room 6015, Bethesda, MD 20892 or life and long-term care insurance, Thrift Savings Plan participation,
electronically to [email protected]. You may direct inquires etc.) is available for both positions.
to Denise Blackwell at [email protected] or 301-402-5598.
Deadline for receipt of all applications is April 27, 2007.
We invite you to explore our Institute and other available opportunities at http://healthresearch.niaid.nih.gov/dms
Department of Health and Human Services

National Institutes of Health
National Institute of Allergy and Infectious Diseases
Proud to be Equal Opportunity Employers
Agency for
Science,Technology
and Research
Chair, Department of Neurosciences
College of Medicine
The University of Toledo
Health Science Campus
The University of Toledo College of Medicine seeks an internationally-rec-
ognized neuroscientist as Professor and Chair of the Department of Neu-
rosciences. The recent merger of the University of Toledo with the former
Medical University of Ohio has created dramatic growth opportunities, and
neuroscience research has been targeted for significant development. Substan-
tial resources including new research space and faculty positions are avail-
able to create a nationally recognized department, complementing current
departmental research strengths (http://hsc.utoledo.edu/depts/neurosciences/
index.html) including sensory physiology, neurodevelopment, neuroendocrine
synaptic physiology, and related areas in structural anatomy and intravital
imaging. State-of-the-art imaging, proteomics/genomics and flow cytometry
cores as well as AAALAC-accredited animal facilities are available on the
modern Health Science Campus. The new Chair must have a distinguished
record of extramural funding, strong leadership and administrative skills, and
should promote alliances with clinical neuroscientists to stimulate research
with translational implications in such areas as drug abuse, developmental neu-
robiology, stroke, epilepsy, neurooncology, neuroimmunology, functional brain
imaging, Parkinson’s disease or neurodegeneration. The successful candidate
must also be committed to medical and graduate education, supporting the use
of innovative educational technologies in gross and microscopic anatomy, and
fostering development of the Neurosciences and Neurological Disorders Ph.D.
training program. The University of Toledo is a state supported institution in
the vibrant port city of Toledo (http://www.toledo.com).
Candidates should send a curriculum vitae and a cover letter sum-
marizing research, educational and administrative background to:
Chair, Neurosciences Search Committee, c/o Shirley Joseph, COM
Dean’s Office, 3045 Arlington Avenue, University of Toledo Health Sci-
ence Campus, Toledo, OH 43614; or e-mail: [email protected]
(PDF format).
The University of Toledo is committed to diversity and equal
opportunity. Applications from women and minority candidates
are strongly encouraged.
Dean of Multicultural Affairs

Tufts University School of Medicine
Tufts University School of Medicine is seeking nominations and
applications for the position of Dean of Multicultural Affairs. The School
www.tufts.edu/hr/jobs/
of Medicine embraces the philosophy of providing a workplace that

Director – Imaging Core Facility emphasizes a diverse faculty, staff and student body, and multicultural
University of Maryland Baltimore competence in its curriculum. This is a dynamic, highly visible, new
opportunity for someone who is seeking to make a demonstrable
School of Medicine difference and have a major impact on the Medical School.
Position #03-305-45
Responsibilities: Reporting to the Dean of the School, the successful
The University of Maryland School of Medicine is seeking a Director candidate will develop and manage programs to promote a diverse
faculty, staff and student body; develop strategies for enhancing the
of The University of Maryland, Baltimore Core Imaging Facility http:
school’s environment and curriculum with respect to cultural sensitivity
//medschool.umaryland.edu/confocal/. The Core Imaging Facility was and competence; and serve as the school’s principal representative to
established in 1994 and serves over 250 users from 28 Departments. We the University and external groups addressing diversity.
are now looking for a highly motivated individual at the Assistant Professor
Qualifications: Applicants should have at least five years of experience
level who desires to manage the facility as we enter a period of rapid related to increasing diversity and/or multicultural competence within an
expansion. The successful candidate will be committed to the continued academic institution. M.D. and/or a Ph.D. and a record of academic
development of a multi-disciplinary Core facility that contains cutting edge accomplishment consistent with a senior faculty level appointment are
imaging technologies not available elsewhere on campus. In collaboration required. Prior supervisory experience is desirable. The successful
with faculty and other Programs of the University, the Director will play a candidate will be an active faculty member in their particular specialty
key role in obtaining extramural funding for the support of the facility. Day- area in the Medical School. Interested candidates should apply online
to-day responsibilities will include business management of the facility, via www.tufts.edu.
training of users, and oversight of maintenance. Candidates should have a Nominations/Applications may be sent to:
PhD or equivalent degree, experience in fluorescence, confocal and multi- Jeff Glassroth, M.D.
photon microscopy, and/or other imaging modalities, and should possess Vice Dean
Tufts University School of Medicine
strong managerial and inter-personal skills. 136 Harrison Avenue
Please submit before April 30, 2007 your curriculum vitae, a brief Boston, MA 02111
or electronically to [email protected].
description of your career goals, and the names, addresses, telephone
numbers, and e-mail addresses of three potential references by e-mail to Applicants should provide a letter addressing their qualifications
W. Gil Wier Ph.D., Chair, Core Imaging Facility Search Committee and a copy of their curriculum vitae. Tufts University is an equal
opportunity/ affirmative action employer.
at [email protected].
The University of Maryland, Baltimore is an Equal Opportunity,
Affirmative Action Employer. Minorities, women, veterans and
individuals with disabilities are encouraged to apply.
Skirball Institute of
Biomolecular Medicine
Faculty Positions
The Skirball Institute and the Kimmel Center of
Biology and Medicine at New York University
School of Medicine invite applicants for tenure-track
positions at the assistant, associate or full professor
level. We seek applicants with an exceptional record
of achievement to join our existing programs in
Molecular Neurobiology, Developmental Genetics,
Structural Biology and Molecular Pathogenesis.
These programs are interdisciplinary and reflect
strengths at NYU’s School of Medicine and College
of Arts and Sciences. Special priority will be given to
applicants with broad interests working at the cutting
edge of mammalian genetics, stem cell research,
neurobiology or molecular cell biology.
NYU School of Medicine offers excellent resources

to support new faculty, including generous start-up
packages and core facilities for cell sorting, imaging,
proteomics, mouse molecular genetics, genomics
and structural biology.
Successful candidates are expected to initiate and

maintain vigorous independent research programs
that will enrich and be enriched by the highly
collaborative environment at the Skirball Institute
and throughout the NYU research community.
This is an electronic application process. No mail

applications will be accepted. Create your application
packet by formatting it as a single PDF document.
Use the following page order: (1) Cover Letter -
indicating Program preference, (2) Curriculum
Vitae, (3) Research Statement.
Email application packet to
[email protected].
Three letters of reference should be sent independently

to: [email protected]
New York University School of Medicine was

founded in 1841 and is an equal opportunity
affirmative action employer. Women and minority
candidates are encouraged to apply.
http://saturn.med.nyu.edu
From life on Mars
to life sciences
For careers in science,
turn to Science
If you want your career to skyrocket, visit of Science, the premier scientific journal, and
ScienceCareers.org. We know science. We are the long experience of AAAS in advancing
committed to helping you find the right job, science around the world. ScienceCareers.org is
and to delivering the useful advice you need. the natural selection.
Our knowledge is firmly founded on the expertise www.sciencecareers.org
Features include:
• Thousands of job postings • Resume/CV Database
• Career tools from Next Wave • Career Forum
• Grant information
ASSISTANT PROFESSOR
THE CHINESE UNIVERSITY OF HONG KONG NEUROSCIENCE AND CELL BIOLOGY
Developmental Neurobiology, Molecular and Cellular
Applications are invited for:- Neuroscience, and Structural Neurobiology
School of Chinese Medicine Assistant Professor Positions
Professor / Associate Professor / Assistant Professor The Department of Neuroscience and Cell Biology at the University of
(Ref. 07/052(665)/2) (Closing date: April 27, 2007) Texas Medical Branch at Galveston (UTMB) seeks applicants for full-
Applicants should have (i) a PhD degree in life science, preferably in Chinese time tenure-track positions at the Assistant Professor level. The candidates
medicine or related areas; (ii) established scholarship with a track record of should hold an M.D. and/or Ph.D. within the following preferred areas of
high-quality publications and award of competitive research grants; preferably specialty: developmental neurobiology, molecular and cellular neuroscience,
(iii) teaching and clinical experience in Chinese medicine; and (iv)
qualifications for practising Chinese medicine in Hong Kong. Applicants for neurogenomics and neuroinformatics, or neuroproteomics and structural
Professorship should also have extensive teaching experience and an neurobiology. Candidates should have an established level of expertise in
outstanding publication record in related fields. Duties include (a) teaching one of these areas as evidenced by postdoctoral research work, publications,
undergraduate and postgraduate courses; (b) supervising research projects; external funding, and relevant national and international reputation.
(c) conducting research in own field(s) of specialization; and (d) assisting in
administration of the School and curriculum development. Appointment will The successful applicants are expected to establish vigorous, externally
normally be made on contract basis for up to three years initially, leading to funded research programs at UTMB. Teaching and service contributions
longer-term appointment or substantiation later subject to mutual agreement. to the School of Medicine and the Graduate School for Biomedical Sci-
[Note: Those who have responded to the previous advertisement for Associate ences will be expected. Levels of appointment will be commensurate with
Professorship/Assistant Professorship (under Ref. no. 07/013/2) need not re-
apply on this occasion.] experience and accomplishments.
Salary and Fringe Benefits The positions offer a competitive salary and benefits package and generous
Salary will be highly competitive, commensurate with qualifications and start-up funds. Applications should include curriculum vitae, summary of
experience. The University offers a comprehensive fringe benefit package, research interests and goals, and the name and contact information (including
including medical care, plus a contract-end gratuity for an appointment of e-mail) of three references. Applications and references should be addressed
two years or longer; and housing benefits for eligible appointees.
to: Dr. Henry F. Epstein, Cecil H. and Ida M. Green Distinguished
Further information about the University and the general terms of service for University Chair in Neuroscience and Cell Biology, Department of
appointments is available at http://www.cuhk.edu.hk/personnel. The terms
mentioned herein are for reference only and are subject to revision by the Neuroscience and Cell Biology, The University of Texas Medical
University. Branch, 301 University Boulevard, Galveston, TX 77555-0625; E-
Application Procedure mail: [email protected].
Please send full resume, copies of academic credentials, a publication list UTMB is an Equal Opportunity, Affirmative Action Institution which
and/or abstracts of selected published papers, together with names, addresses
and fax numbers/e-mail addresses of three referees to whom the applicants’
proudly values diversity. Candidates of all backgrounds
consent has been given for their providing references (unless otherwise are encouraged to apply.
specified), to the Personnel Office, The Chinese University of Hong Kong,
Shatin, N.T., Hong Kong (Fax: (852) 2603 6852). The Personal Information
Collection Statement will be provided upon request. Please quote the reference
number and mark ‘Application - Confidential’ on cover.
From physics to nutrition
For careers in science,
turn to Science
If you want your career to bear fruit, don’t leave it to firmly founded on the expertise of Science, the premier
chance. At ScienceCareers.org we know science. We are scientific journal, and the long experience of AAAS in
committed to helping you find the right job, and to advancing science around the world. ScienceCareers.org
delivering the useful advice you need. Our knowledge is is the natural selection. www.sciencecareers.org
Features include:
• Thousands of job postings • Resume/CV Database
• Career tools from Next Wave • Career Forum
• Grant information
OTOLARYNGOLOGIST
The Section of Otolaryngology - Head and Neck Surgery at
Dartmouth-Hitchcock Medical Center seeks a board certified Director, Program in Emerging Infectious Diseases
or board eligible Otolaryngologist for a full-time faculty posi- The Duke-NUS Graduate Medical School Singapore (GMS) is unique in
tion.The candidate should possess an interest in an academic bringing post-baccalaureate, research-intensive medical education to Asia,
career and in the education of medical students and resi- and represents a truly global partnership between two leading universities:
dents.This position will combine a general otolaryngology National University of Singapore and Duke University. The GMS shares a
with a subspecialty practice in otology or pediatric otolaryn- modern campus with Singapores largest hospital and several national research
gology. Fellowship training in otology/ neurotology or pedi- centers. The GMS is creating a world-class, academically based Program in
Emerging Infectious Diseases that will both enhance health care in Singapore
atric otolaryngology is desirable. Research interests will be and serve as a national and international resource of excellence in emerging
encouraged. Academic rank will be commensurate with qual- infectious diseases. The mission of the Program faculty will be to conduct
ifications and experience. high-level basic and applied research, and to train graduate students, post-
doctoral fellows, and physician scientists in the disciplines relevant to
Interested applicants are encouraged to send letters emerging infectious diseases.
of inquiry and CV to:
We are seeking an individual with exceptional scientific credentials and
Daniel Morrison, MD, Chairman leadership skills to head the Program. The position of founding Director
will include full salary, a very generous start-up and five years of annual
Section of Otolaryngology - Head & Neck Surgery
research funding. The Director will be provided with the space and resources
Dartmouth-Hitchcock Medical Center necessary to recruit 6-8 outstanding faculty members at all academic ranks.
One Medical Center Drive The packages for these faculty recruits would include full salary, generous
Lebanon, NH 03756 start-up, and five years of annual research funding of up to S$500K/p.a.,
Telephone: 603-650-8123 assuring a stable base of support that can be supplemented by competitive
grant awards, which are expanding rapidly in Singapore. The director and
the faculty members he/she recruits will join the pioneering Duke and
Singapore investigators already affiliated with the GMS (see
www.gms.edu.sg).
Interested candidates should send a CV and the names of three references to:
Dartmouth-Hitchcock Medical Center is an affirmative action/equal opportunity Mariano A. Garcia-Blanco, M.D., Ph.D., Chair, Search Committee on
employer and is especially interested in identifying female and minority candidates. Emerging Infections, Duke-NUS Graduate Medical School, Singapore
by email to: [email protected]
www.DHMC.org The GMS is a collaboration of the Duke University School of Medicine and
the National University of Singapore.
FULL TIME ASSISTANT RESEARCH

ANATOMIST POSITION University of Cincinnati
UNIVERSITY OF CALIFORNIA
SAN FRANCISCO
DEPARTMENT OF ANATOMY Post Doc Fellow - Ophthalmology
The candidate will study the transmission and control of (27UC3488) The University of Cincinnati (UC) is
pain messages as well as the neuronal circuits that pleased to announce availability of endowed fund-
underlie the production of pain. The candidate will ing for research in macular degeneration or other vi-
oversee the work of students and technicians, and work sual disorders of the elderly and invites application for
Lancaster Environment Centre a Crawley Postdoctoral Scholar Award as part of
directly on bench research involving transgenic mice
(ZW mice) to investigate the CNS circuitry implicated in Department of Environmental Science the Edith J. Crawley Memorial Scholars Program.
pain transmission. In addition, he/she will work with the An award of this type will fund up to three years of
Principal Investigator and other scientists in the
laboratory to develop overall experimental strategies, to
Professor or Reader mentored vision neuroscience research at UC.
Qualified applicants should hold a doctoral degree
design specific experiments and controls, and to
interpret experimental results.
Atmospheric Science in life sciences (i.e., M.D., Ph.D. and/or equivalent
£41,544 - £46,758 p.a. degree) and be committed to a career in vision
Applicants must have a Ph.D. in biology and at least neuroscience research. Successful candidates may
four years postdoctoral research experience (foreign Professor min £50,565 (negotiable) either participate in ongoing projects in vision-neu-
training/experience acceptable). The candidate must
We invite applications for the post of Professor or roscience or new projects or avenues of research ap-
have demonstrated experience in use of viruses to
introduce new genes into the CNS, "in vitro" and "in Reader in Atmospheric Science in this research- proved by the mentor. The awardee will be expected
vivo" approaches, immunohistochemistry, western blot, led Department (which is an integral part of the to develop one or more independent research proj-
cell culture, DNA constructs for knock-ins, genetics Lancaster Environment Centre). ects worthy of extramural support upon completion
(including the generation of transgenic mice) and mouse of training.
The anticipated start date is on or before
behavior. This candidate will also have a track record of 1 September 2007. Interested individuals should submit a curriculum
publishing in highly regarded peer-reviewed journals.
Informal enquiries may be addressed vitae, statement of research interest, and three ref-
Send curriculum vitae and names of references prior to to [email protected] or erences to Winston W.-Y. Kao, Ph.D., Ben and Louise
May 4, 2007 to: [email protected] Tate Professor and Director, Ophthalmic Research,
Department of Ophthalmology, Health Professions
Yvonne Dea, Analyst Building, Suite 350, University of Cincinnati College
Department of Anatomy To apply or receive further information online,
of Medicine, 3233 Eden Avenue, Cincinnati, OH
513 Parnassus Ave, Box 0452 please visit http://www.personnel.lancs.ac.uk/ or,
45267-0527 (Phone: 513-558-2802, Fax: 513-558-
San Francisco, CA 94143 telephone Personnel Services, quoting reference
3108, [email protected]) For additional infor-
A763R, on answerphone +44 (0)1524 846549.
mation about the position #27UC0988, please see
UCSF seeks candidates whose expertise, teaching, research, Closing date: 9 May 2007. www.jobsatuc.com.
or community service has prepared them to contribute to our
commitment to diversity and excellence. UCSF is an Interviews: 5 June 2007. The University of Cincinnati is an affirmative
affirmative action/equal opportunity employer. The action/equal opportunity
University undertakes affirmative action to assure equal
employment opportunity for underutilized minorities and Aiming for Greater employer.
women, for persons with disabilities, and for covered Diversity
UC is a smoke-free
veterans, All qualified applicants are encouraged to apply, work environment.
including minorities and women.
FOCUS ON CAREERS
Looking for
Career Advice?
Find a wealth of information relevant to your
current career and future employment decisions in
the Science Career Features.
UPCOMING FEATURES:
April 20: Postdoctoral Careers: Transferable Skills
©2007 JupiterImages Corporation
April 27: Biotech and Pharma

May 4: Interdisciplinary Research
Also available online at:

www.sciencecareers.org/businessfeatures
Open Space.
TENURE TRACK FACULTY POSITIONS THE METHODIST HOSPITAL Open Minds.
DEPARTMENT OF BIOCHEMISTRY AND RESEARCH INSTITUTE
MOLECULAR BIOLOGY
VIRGINIA COMMONWEALTH UNIVERSITY The Methodist Hospital Research Institute of
SCHOOL OF MEDICINE The Methodist Hospital, Houston, Texas, seeks
As part of an ongoing major expansion in bio-
an exceptional physician scientist to lead its
effort in clinical research. The Methodist Hos-
ASSISTANT PROFESSOR,
medical research at Virginia Commonwealth
University Medical Center, we invite applications
pital System consists of 1,450 beds, including IMMUNOLOGY
1,000 located in the Texas Medical Center in
from outstanding individuals with expertise and
Houston. With our partners at Weill Cornell The University of Idaho is seeking a talented educator
interest in cellular and molecular signaling for
Medical College and New York-Presbyterian
tenure-track positions. We welcome candidates and researcher to join our faculties in the Department of
in all areas of biochemistry and biomolecular sci-
Hospital in New York City, there are 3,500 beds
available for clinical investigation and clinical Microbiology, Molecular Biology & Biochemistry in the
ences. Candidates should have an active research area of immunology.
program with a record of sustained research pro- trials. The successful candidate will be respon-
ductivity. More senior candidates should have sible for organizing and leading the Institute’s
clinical research in Houston and collaborating The individual selected will be expected to contribute
current extramural funding. Applicants should
have a M.D., Ph.D. or equivalent degree and with our Cornell and NYP colleagues. We to the overall research, teaching, and service mission
will be expected to contribute to the Department’s encourage applications from individuals who of the department, college and university by serving on
teaching mission as well as develop vigorous col- currently lead substantial funded programs committees, advising students, participating in semi-
laborative efforts with other VCU researchers. conducting clinical research. The hospital has nars and interacting with faculty and students.
The Department’s and VCU’s research expertise entered an unprecedented expansion phase that
is spread across several programs having national includes construction of a 420,000 SF state- Requires M.D., Ph.D. or equivalent in Molecular Life
prominence in terms of NIH-funded research of-the-art research building and a 750,000 SF
Sciences or a related field with at least two years of
ranking. More information about the School of ambulatory care building, both designed to
foster interdisciplinary collaboration. The suc-
postdoctoral research experience; strong record of ac-
Medicine and the Department, and this open posi-
cessful applicant will receive a generous salary, complishments in research as demonstrated by publi-
tion can be found at www.medschool.vcu.edu/
and www.pubinfo.vcu.edu/facjobs/. Applicants fringe benefits, and a relocation package, and cations in peer-reviewed journals; teaching experience
should submit by email a CV with names and e- may be eligible for an endowed chair. at the undergraduate and/or graduate level.
mail addresses of three references to: Dr. Robert For a full description and application materials, visit
F. Diegelmann ([email protected]), Depart- Individuals interested in this unique career
ment of Biochemistry & Molecular Biology, opportunity should send via e-mail a cur-
http://www.hr.uidaho.edu/
Virginia Commonwealth University School of riculum vitae, including grant funding infor-
Medicine, Richmond, VA 23298-0614. mation to: James M. Musser, M.D., Ph.D., Review of applications
c/o Ms. Ginny Gittemeier, Co-Director and begins 4/30/07.
Virginia Commonwealth University is an Equal Executive Vice President, The Methodist
Opportunity/Affirmative Action Employer. Hospital Research Institute; gittemeier@
Women, persons with disabilities, and minori- grantcooper.com; Phone: 636-240-2090;
ties are encouraged to apply. To enrich education through diversity, the University of Idaho is an
Fax: 314-726-5294. Equal Opportunity/Affirmative Action Employer.
IF YOU THINK OUR
PIPELINE IS IMPRESSIVE,
Dave Jensen
Industry YOU SHOULD SEE
Recruiter
THE SCIENCE
BEHIND IT!
Every day, your work has the ability to impact lives. Many of
GSK Consumer Healthcare’s well-known products are global
brands, and offer people a new lease on life. Whether smoking
cessation, weight loss, pain management or even restoring a
quiet night’s sleep, our products make life better for millions.
Recently named the 2006 Pharmaceutical Company of the

Year by Med Ad News, GSK is no stranger to the power of
exceptional science and global product marketing. As we
expand our portfolio of consumer products, which already
include such market leaders as: Nicoderm, Breathe Right,
Panadol, Abreva, Polident, and Alli, the first FDA approved OTC
weight loss product, GSK Consumer Healthcare is eager to
Science
attract the most passionate scientific talent in the marketplace.
Our New Product Development and New Product Research

departments, located in Parsippany, New Jersey, currently
Careers have the following opportunities available for exceptional

professionals:
Forum • Principal Development Scientists, NPD

(Req. ID: 38859 & 39590)
• Principal Scientists, NPR

• How can you write a resume that (Req. ID: 40776, 40204, & 41512)
stands out in a crowd?
• What do you need to transition from We also have a Principal Scientist, Oral
academia to industry? Healthcare (Req. ID: 41875) posi-
• Should you do a postdoc in academia tion available on our Aquafresh
or in industry? team at our London Innovation
Centre. Company provided
Let ScienceCareers.org help you answer relocation available.
these questions. ScienceCareers.org has
To learn more about these posi-
partnered with moderator Dave Jensen
tions and to apply online,
and four well-respected advisers who,
please visit www.gsk.com/careers.
along with your peers, will field career-
Indicating Req. ID is essential to search.
related questions.
Visit ScienceCareers.org and

start an online dialogue. gsk.com/careers
GlaxoSmithKline is dedicated to supporting you with career-long opportunities and
learning. We offer a competitive benefits and compensation package designed to
attract and retain the very best. We are proud to be an equal opportunity employer.
“
As a child I got very interested in space travel.
Who’s helping bring
When I was six my father gave me some
books on rockets and stars. And my universe
the gift of science suddenly exploded in size because I
realized those lights in the sky I was
to everyone? looking at were actually places.
I wanted to go there. And I discovered

that science and technology was a gift
that made this possible. The thrill of most
Christmas presents can quickly wear off. But I’ve
found that physics is a gift that is ALWAYS exciting.
I’ve been a member of AAAS for a number of years.

I think it’s important to join because AAAS represents
scientists in government, to the corporate sector, and
to the public. This is very vital because so much of
today’s science is not widely understood.
I also appreciate getting Science because of the

breadth of topics it covers. It gives me a great
grounding for many activities in my professional life,
such as advising government agencies and
private corporations.
”
Jim Gates is a theoretical physicist and professor at
the University of Maryland. He’s also a member
of AAAS.
See video clips of this story and others at

www.aaas.org/stories
S. James Gates Jr., Ph.D.

Theoretical physicist
and AAAS member
Who’s working
to increase support
for science?
Top quality research depends on comprehensive support. governments around the world. And only AAAS Funding
AAAS is present at every stage of the process – from Updates – sent out monthly – provide continual coverage
advising on funding policy initiatives to tracking the US of R&D appropriations. By actively working to increase
Federal R&D budgeting process. As the experts, we support for research, AAAS advances science.
brief Congressional staffers and representatives from To see how, go to www.aaas.org/support
POSITIONS OPEN POSITIONS OPEN
The University of California, San Diego (UCSD)
Center for Human Genetics and Genomics (website:
http://chgg.ucsd.edu/) in conjunction with the
Departments of Pediatrics and Medicine at the
ASSISTANT/ASSOCIATE PROFESSOR
The University of Minnesota Cancer Center, the
UCSD School of Medicine invites applications for
several TENURE-TRACK/TENURED POSI-
TIONS from outstanding individuals to develop a
vigorous research program in any area of contempo-
What’s
your
Department of Laboratoroy Medicine and Patholo- rary human genetics and genomics. The appoint-
gy, and the Department of Urologic Surgery are ments will be at any academic level, and will involve
expanding the Cancer Center Research Program in teaching at both the graduate and medical school
Prostate Cancer Research and invite applications for levels. There are openings for both junior and senior
a Tenure-Track Faculty Position. We are interested
next
faculty, including DIVISION CHIEF positions in
in candidates with specific research interests, experi- medical genetics in both the Departments of Pediat-
ence, publication, and external funding in prostate rics and Medicine. Candidates must possess a
biology, prostate cancer, and the cellular and mo- doctoral degree and be well trained in any area of
lecular mechanisms associated with prostate cancer contemporary human genetics and genomics with a
career
genesis, invasion and metastasis, and experimental demonstrated track record of outstanding peer-
therapeutics. The goal of this search is to attract reviewed research. For the Division Chief positions,
faculty members that will enhance ongoing research Board certification in any area of medical genetics is
programs in the following areas: (1) Genesis and desirable. Space will be provided in the Center for
move?
development of neoplastic changes in the prostate. Human Genetics and Genomics on the fourth floor
Tumor microenvironment with a specific focus on of the new Skaggs School of Pharmacy and Pharma-
musculoskeletal tumor growth, the bone/tumor mi- ceutical Sciences building or other sites within the
croenvironment, tumor metastasis to bone, animal UCSD Health Sciences campus. In addition, an
models of bone metastases and/or emerging ther- attractive and competitive startup package will be
apies for bone cancer; (2) Progression associated provided. The goal of the Center is to galvanize
changes in signal transduction pathways associated
with alterations in the structure/function of plasma
membrane microdomains, signalosome assembly/
already existing genetics and genomics efforts
throughout the School of Medicine and UCSD
Get help
campus, and these recruitments will play a major
function, or other tumor associated changes in cell
growth/survival signal transduction pathways; (3)
Proteomic, genomic, and tumor/stromal changes
role in this exciting effort.
Applicants should e-mail their curriculum vitae and
from the
names and addresses of three references to e-mail:
associated with primary tumor growth/progression
of prostate tumors. Preference will be given to in-
[email protected] or mail to: Christine Coffey, As-
sistant to Anthony Wynshaw-Boris, M.D., Ph.D.,
experts.
dividuals who use novel three dimensional and/or Director, Center for Human Genetics and Ge-
co-culture systems to model progression in vitro and nomics University of California, San Diego School
in vivo. of Medicine, 9500 Gilman Drive, 0627 La Jolla,
Successful candidates will be expected to develop
projects that have translational potential in pre-
clinical models, with a long-term goal of developing
CA 92093-0627.
Review of applications will begin on April 1, 2007, www.sciencecareers.org
and will continue until positions are filled. UCSD is
novel clinical approaches for the diagnosis/treat- an Equal Opportunity/Affirmative Action Employer committed
ment of prostate cancer in patients. The candidate to excellence through diversity.
must hold a Ph.D. or M.D./Ph.D. degree and pref-
erence will be given to candidates with external
funding, established research programs, and training
in prostate biology. Academic rank and salary are • Job Postings
commensurate with training and experience.
Submit curriculum vitae, a brief research state- TENURE-TRACK POSITIONS in ANATOMY,
ment, and contact information of three references
online to requisition #145924 at website: http://
PHYSIOLOGY, and GENETICS
F. Edward Hebert School of Medicine
• Job Alerts
employment.umn.edu. Information about the Uniformed Services University of the
Health Sciences
Cancer Center is available at website: http://
www.cancer.umn.edu. The University of Minnesota is The Department of Anatomy, Physiology and • Resume/CV
an Equal Opportunity Educator and Employer. Genetics, Uniformed Services University School of
Medicine, Bethesda, Maryland, chaired by Harvey B. Database
POSTDOCTORAL ASSOCIATE Pollard, M.D., Ph.D., invites applications for tenure-
A Postdoctoral Associate position for a Ph.D., track positions at the ASSISTANT/ASSOCIATE
M.D., or M.D./Ph.D. is available immediately in
the Division of Hematology, Oncology, and Trans-
PROFESSOR level. The Department has strong
commitments to medical education and biomedical
• Career Advice
plantation, Department of Medicine. The duties and
responsibilities of this position will be to study the
research and is located in an interactive scientific
environment adjacent to the National Institutes of
from Next Wave
effects of cytochrome P450 enzymes on breast Health and National Library of Medicine. Outstand-
cancer cell proliferation in vitro and in vivo. Studies
will involve analysis of eicosanoids and other lipids
ing core facilities are available to support medical
education and biomedical research in cell biology, • Career Forum
by mass spectrometry, using tumor xenograft mod- genomics, and proteomics. The successful applicant
els. The job duties will include, but not be restricted will have a Ph.D. and/or M.D. degree and will be
to, performing the isolation of cell lines expressing
recombinant signaling proteins, performing cell growth
expected to: (i) contribute to the Department_s in- • Graduate
terdisciplinary anatomy and physiology curriculum
inhibition assays and soft agar colony formation as-
says, performing tumor xenograft assays and the use
for first-year medical students and (ii) establish an
independent and extramurally funded research pro-
Programs
of mass spectrometry assays for eicosanoids. The suc- gram. Teaching experience in physiology is desir-
cessful applicant must have the ability to work indepen-
dently, analyze and report research results. Writing
able. Research interest in regenerative biology and
stem cell research is an asset. Candidates should submit
• Meetings and
and figure production skills necessary for drafting
manuscripts is essential, as well as familiarity with
curriculum vitae, an outline of their proposed research
program, and have three letters of reference sent to: Announcements
statistical analysis of biochemical and pharmacological Gregory P. Mueller, Ph.D., Search Committee
data. Working experience with eicosanoid biochem- Chair, Department of Anatomy, Physiology, and
istry, experimental pharmacology, mass spectrometry, Genetics, Uniformed Services University of the
enzymology, and breast cancer xenograft models will Health Sciences, 4301 Jones Bridge Road, Bethes-
be helpful. da, MD 20814-4799 (e-mail: gmueller@usuhs.
Apply online at website: http: //employment. edu). Positions open until filled. Foreign nationals
umn.edu. Applications should consist of curriculum will be considered if qualified United States citizens are
vitae and names of three references. not available. USUHS is an Equal Opportunity Employer
The University of Minnesota is an Equal Opportunity with a strong commitment to racial, cultural, and ethnic
Employer and Educator. diversity.
152 6 APRIL 2007 VOL 316 SCIENCE www.sciencecareers.org

CONFERENCE
Conferences organised at the Institut Pasteur, Paris, France in 2007
FROM MOLECULES TO COGNITION: A TRIBUTE TO JEAN-PIERRE CHANGEUX

September 17-19, 2007
Website: http://www.pasteur.fr/infosci/conf/sb/neuroscience_pasteur
Information: [email protected] - Fax: +33 1 40 61 37 21
EARLY STEPS OF THE VIRUS LIFE CYCLE: MOLECULAR AND CELLULAR INSIGHTS
October 4-5, 2007
Website: http://www.pasteur.fr/infosci/conf/sb/virus_cell/
GENETICS AND MECHANISMS OF SUSCEPTIBILITY TO INFECTIOUS DISEASES
meetings and announcements

November 21-24, 2007
Website: http://www.pasteur.fr/infosci/conf/sb/host_genetics
VIBRIO 2007
November 28-December 1, 2007
Website: http://www.pasteur.fr/infosci/conf/sb/vibrio2007
Science Alerts in Your Inbox

Get daily and weekly E-alerts on the latest breaking news and research!
k Get the latest news and research

his Weejournal’s
News T from Science as soon as it is
Science mmaries of the
y Alert
Brief su ntent OW Weekl published. Sign up for our e-alert
news co ScienceNeadline summary
Weekly h n services and you can know when
ing Notificatio
Notification Science Post ekly issue is posted the latest issue of Science or
ess
Science Expr ed in advance of print Alert when we
Articles publish Science Express has been posted,
Alert* peruse the latest table of contents
ScienceNOW Daily mary
Daily headline sum for Science or Science’s Signal
Transduction Knowledge Environ-
Science Magazine TOC ment, and read summaries of
Weekly table of contents the journal’s research, news
content, or Editors’ Choice column,
STKE TOC all from your e-mail inbox. To start
Weekly table
of contents receiving e-mail updates, go to:
http://www.sciencemag.org/ema
Editors’ Choice
Highlights of the
recent literature
This Week in Science

Summaries of research
content
POSITIONS OPEN POSITIONS OPEN POSITIONS OPEN
FACULTY POSITIONS in PHYSIOLOGY ECOLOGIST and ENVIRONMENTAL/
The Department of Physiology at the Medical RESOURCE ECONOMIST
College of Wisconsin (MCW) invites applications for Woods Hole Research Center
two tenure-track faculty positions at the ASSIST- The Woods Hole Research Center (WHRC) seeks:
ANT or ASSOCIATE level with research interests an Ecologist who will work on the relation between
FACULTY POSITIONS in the physiological function of the cardiovascular the condition of forest ecosystems and the Becosystem
Academia Sinica, Taiwan system, the kidney, or pulmonary system. Favored ECOLOGIST
services[ they deliver;andand:ENVIRONMENTAL/
an Environmental/Resource
Applications are invited for faculty positions to par- candidates will be those that: (a) complement the Economist RESOURCE
who will focus ECONOMIST
on the economic valuation
Department_s strengths in connecting genes to com- Woods Hole Research
of the products and services derivable Centerfrom forest
ticipate in the Summit Project at Academia Sinica.
Through this project, we plan to make several stra- plex functional pathways; (b) are highly focused on The Woods
ecosystems Hole Research
and mechanisms Center (WHRC)
for reflecting the value of
tegic appointments in research programs of (1) stem endothelial or epithelial ion channels and cell signal- seeks:
these an Ecologist
products who will work on the
and services.
cell, (2) infectious disease, and (3) drug discovery. ing; (c) are capable of extending their research to relation
For morebetween the condition
information, of foresthttp://
go to website: eco-
We are seeking for outstanding scholars at ALL more integrated level; (d) are interested in develop- systems and the Becosystem services[ they
whrc.org/about_us/jobs.htm
FACULTY LEVELS in the following areas. ing translational collaborative projects with clinical deliver; and:
WHRC is an an Environmental/Resource
Equal Opportunity Employer and Econ-
actively
Stem cell (code: SC): Functional genomics, cel- scientists. The overall goal of the Department is to seeksomist whopool
a diverse will
of focus on in
candidates thethiseconomic
search. valua-
lular and molecular biology of stem cells, including sustain a breadth of scientific expertise and research tion of the products and services derivable from
cancer stem cells. spanning from genome and cell to the whole or- forest ecosystems and mechanisms for reflect-
Infectious disease (code: ID): Development of ganism. Superb opportunities exist for collaborative ing the value of these products and services.
antiviral and antibacterial strategy, drug resistance, research and our faculty are closely affiliated with the For more information, go to website:
vaccine and disease models. MCW Human and Molecular Genetic Center (hous- http://whrc.org/about_us/jobs.htm
Drug discovery (code: DD): Synthetic organic ing the National Heart, Lung, and Blood Insititute WHRC is an Equal Opportunity Employer and
chemistry and medicinal chemistry, drug design and Program of Genomic Applications), the Cardiovascular actively seeks a diverse pool of candidates in this search.
synthesis. Center (housing the National Institute of Neurolog-
The funding for the Summit Project is substantial ical Disorders and Stroke-Program Project Grant in
and long-term. In addition, Academia Sinica has es- stroke), the Biotechnology and Bioengineering Cen- ASSISTANT PROFESSOR of PHYSIOLOGY
tablished several national core facilities, including ter (housing the NHLBI Center of Proteomics), and Department of Pharmacology and Physiology
mouse mutagenesis and disease models, genotyping, the Center of Kidney Research. Candidates will be The Department of Pharmacology and Physiology
microarrays, RNAi, mass spectrometry and proteomics, expected to participate in both the graduate and med- at Oklahoma State University-Center for Health Sci-
X-ray, nuclear magnetic resonance, magnetic reso- ical curriculums. The candidate must hold a Ph.D. ences invites applications for one tenure-track phys-
nance imaging, nanotechnology and bioinformatics, and/or M.D. degree, and demonstrate clear evidence iology faculty position at the level of ASSISTANT
and recently has established two P3-level laborato- of research independence such as current or immi- PROFESSOR. For all applicants, a Ph.D. is required
ries and an ultra-high throughput screening (uHTS) nent grant support. The positions will remain open with at least two years of postdoctoral experience.
system. Familiarity with the operation of such envi- until filled and applicants should send their curricu- The successful applicant is expected to develop an
ronment and equipment would be an advantage. lum vitae, statement of interest, and three letters of extramurally funded research program and contrib-
Qualification: Candidates with doctoral degrees recommendation to: Allen W. Cowley, Jr., Ph.D., ute to medical and graduate teaching. Individuals
and strong track records in the above areas are en- Chairman, Department of Physiology. E-mail: with interests in neurobiology of social behavior are
couraged to apply. Please indicate the code on your [email protected]. Website: http://www.phys. invited to apply. Applicants must apply online at
cover letter describing your research experience and mcw.edu/index.htm. website: https://jobs.okstate.edu, job # 02963.
interest, and send a copy of curriculum vitae, includ- Affirmative Action/Equal Opportunity Employer.
ing a list of publications along with name and contact
information of three references to: Vice President, MEETINGS
Dr. Andrew H.J. Wang, Academia Sinica, 128 MEETING ANNOUNCEMENT
Academia Road, Section 2, Nankang, Taipei 115, PENN STATE UNIVERSITY The University of Georgia will host an interna-
Taiwan. Telephone: þ886-2-2789-9407; fax:
Penn State York invites applications for an IN- tional conference on the BImmunobiology of Influ-
þ886-2-2788-2043. E-mail: [email protected].
STRUCTOR in biology (multi-year appointment, enza Virus Infection: Approaches for an Emerging
edu.tw. Website: http://www.sinica.edu.tw/.
36 weeks) starting August 2007, or as negotiated. Zoonotic Disease[ from July 29 through 31, 2007.
Teach freshman biology with laboratory sections and This meeting will assemble influenza experts from
ASSISTANT PROFESSOR advanced courses in area of expertise; prefer interest academia, government, and industry to foster effec-
Histology/Cell Biology in operating a small greenhouse. Participate in course, tive translation of new findings in basic influenza
Mercer University, Macon, Georgia curriculum, and program development and advise research into effective vaccines and therapies. It is
students. Qualifications: Ph.D. in biology (or related intended for students, scientists, and others who
Mercer University School of Medicine invites ap- discipline). Prefer background in botany, plant pathol- need to be informed about the latest scientific
plications for a 12-month salaried, tenure-track ogy, plant physiology, or entomology. developments in the influenza field. A keynote ad-
position in histology/cell biology at the rank of To learn more about the campus and Penn State, dress by 1996 Nobel Laureate Peter C. Doherty
Assistant Professor. The successful candidate must visit website: http://www.psu.edu/ur/cmpcoll. will precede presentations by eminent leaders in the
have a strong commitment to medical education html. field of viral immunity, and poster sessions will pro-
excellence in a multidisciplinary, case-based curricu- To learn more about the position and how to apply, vide cutting-edge research on virus biology, vac-
lum, and will be expected to develop an indepen- visit website: http://www.psu.jobs/Opportunities/ cines, therapeutics, detection, diagnostic procedures,
dent, externally funded research program. Applicants Opportunities.html and select location: Penn State and other topics of interest. For further information
should have a doctoral degree with an expertise in York. refer to the following website: http://www.virus-
human histology and cell biology, and three years Affirmative Action/Equal Opportunity. EID.org.
of postdoctoral training. Review of applications
will continue until the position is filled. For online
submission of applications, visit website: https:// MARKETPLACE
www.mercerjobs.com. Affirmative Action/Equal Op-
portunity Employer/Americans with Disabilities Act. Widely 8¢/u
POSTDOCTORAL POSITIONS
POSTDOCTORAL FELLOWSHIPS
in the Epigenetics of Disease
Johns Hopkins University School of Medicine
Recognized
Original &
Guaranteed
KlenTaq1 Truncated
Taq DNA
Polymerase
Available at Sloan-Kettering Institute Withstand 99oC
Postdoctoral Fellowships are available for the US Pat #5,436,149 e-mail: [email protected]
Seeking RESEARCH SCIENTISTS, with or study of the epigenetic basis of human cancer and Call: Ab Peptides 1•800•383•3362
without postdoctoral experience, who wish to join other human disease. Studies include the epigenetic Fax: 314•968•8988 www.abpeps.com
a laboratory focused on defining the molecular events progenitor origin of cancer (Nat. Rev. Genet. 7:21-
underlying normal or leukemic hematopoiesis. Appli- 33, 2006), and development of high throughput
cants should have considerable interest and expertise epigenomic technology, through a Center of Excel-
in the use of mouse models to study hematopoietic lence in Genome Sciences, extending epigenetics to
stem cell biology, mechanisms of transformation, or
therapeutic approaches to hematologic malignancies.
common disease, such as bipolar disorder and autism.
The candidate should be a prospective or recent
Oligo Synthesis Reagents
Send a description of your research interests, curric- recipient of a Ph.D. or M.D./Ph.D. with good Specialty CPG Supports
ulum vitae, and the names and telephone numbers publication(s) in genetics or genomics. Please e-mail
of three references to: Dr. Stephen D. Nimer, Me- Dr. Andrew P. Feinberg curriculum vitae and
Linkers, Spacers, & Modifiers
morial Sloan-Kettering Cancer Center, 1275 York names and e-mail addresses of three references, but Bulk Reagent Pricing Available
Avenue, P.O. Box 575, New York, NY 10021. Me- no other attachments, to e-mail: [email protected]. +1.800.GENOME.1
morial Sloan-Kettering is an Affirmative Action/Equal Opportu- Johns Hopkins University is an Equal Opportunity/Affirmative www.btisynthesis.com
nity Employer. Action Employer.
154 6 APRIL 2007 VOL 316 SCIENCE www.sciencecareers.org

The power of small
1μl analysis — increased throughput

The NanoDrop® ND-8000
8-Sample Spectrophotometer
x 8
1μl samples. No cuvettes. No dilutions.
Revolutionary technology. 8 readings in under 30 seconds. dynamic range

The NEW NanoDrop® ND-8000 8-Sample Spectrophotometer (e.g., 2-3700 ng/μl dsDNA),
is powerful — eight 1μl samples at once. virtually eliminating the need for dilutions. Then just a quick
wipe clean and you’re ready for your next samples. What
Full spectrum UV/Vis analysis of 1μl samples for quantitation,
could be easier — or more powerful?
purity assessments and more: nucleic acids, microarrays,
proteins and general spectrophotometry. And for the power of small in single-sample absorbance
or fluorescent measurements, check out the NanoDrop®
Measurement is quick and easy — pipette up to eight samples
ND-1000 Spectrophotometer or the NanoDrop® ND-3300
and measure. Each sample is read using two path lengths
Fluorospectrometer (ultra low fluorescent detection limit of
to achieve an extensive
sample mass — e.g., 2 pg dsDNA).
Ready to experience the power of small x 8? Test a
NanoDrop® ND-8000 8-Sample Spectrophotometer
in your own lab.
FREE one-week evaluation www.nanodrop.com

© 2007 NanoDrop Technologies, Inc.
302.479.7707

Science Apr 06 07

Uploaded by

Copyright:

Available Formats

Science Apr 06 07

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Science Apr 06 07

Uploaded by

Copyright:

Available Formats

Tools for Cell Biology Research

R&D Systems Surveyor™ IC Immunoassays

15 Akt MCF-7 cells were incubated with or

Selection expanding weekly—visit www.RnDSystems.com

The Benchmark System Just Got Better

Products for your research needs

Tet-On Advanced Tet-Off Advanced

Clontech Laboratories, Inc.

Bringing protein analysis

GE Healthcare Bio-Sciences AB, a General Electric Company.

NEWS OF THE WEEK LETTERS

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 5

Prepare for results

I Protein, RNA, and DNA purification

Contact QIAGEN today or visit www.qiagen.com/goto/SampleTech .

Sample & Assay Technologies

CLIMATE CHANGE CELL BIOLOGY

TECHNICAL COMMENT ABSTRACTS BREVIA

CONTENTS continued >>

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 7

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 9

Bio-Rad and RNAi.Come have a look.

With a broad range of proven delivery technologies, award-winning

Practice of the polymerase chain reaction (PCR) may require a license.

Visit us on the Web at discover.bio-rad.com

GLOBAL: Special Feature—Getting Published in

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 11

T4 DNA Ligase from New England Biolabs

I T4 DNA Ligase* r Advantages:

For large or difficult constructs reaction conditions

Ocean Conditions known about the structure of dislocations, which

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 13

The More You Know, the More You Learn

Understanding Inositol Pyrophosphates

Sizing Up Man’s Best Friend

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 15

Announced in early 2007, the Open to New Investigators Who

A Two-Pronged Climate Strategy

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 17

Introducing Our Second Generation Genome Sequencing System

Genome Sequencer FLX

...more Flexibility and more Applications.

Roche Diagnostics GmbH

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007 19

<< Better Bones Without Bax

www.sciencemag.org SCIENCE VOL 316 6 APRIL 2007

For a career in science,

You know, ScienceCareers.org

That means they’re putting

With thousands of job postings,

• Thousands of job postings • Funding information

24 6 APRIL 2007 VOL 316 SCIENCE www.sciencemag.org

25 years of delivering next-generation systems

Accelerate your time to publish

To ﬁnd out more, visit: info.appliedbiosystems.com/hdr

More Flexible LCM

Unique IR-enabled LCM—never lose contact with the sample!

Expect more. We’ll do our very best to exceed your expectations.

LIFE SCIENCE TECHNOLOGIES

Also available at www.sciencemag.org/products/articles.dtl