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Dear Harvard Community,

Let me begin by congratulating the editors of the Harvard Undergraduate Research Journal on the wonderful

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have a critical place in science education, but a deep understanding of science and engineering requires hands-

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University Planning Committee on Science and Engineering and the Harvard University Science and Engineering

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The Harvard Undergraduate Research Journal demonstrates the fruits of such “hands on learning” and the ability

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Sincerely,

Steven E. Hyman Provost

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
i
 L e t t e r s

THE HARVARD UNDERGRADUATE RESEARCH JOURNAL

Dear Harvard Community,

It is our great pleasure to present to you the inaugural issue of The Harvard Undergraduate Research Journal

(THURJ), a biannual publication that seeks to showcase the extraordinary research performed by Harvard

undergraduates. The journal has been in the works since the past summer, and we are very excited to see it come to

fruition. The idea behind THURJ, however, originated even earlier when we noticed that many other universities—

including Stanford, Columbia, and MIT—had established journals that showcased undergraduate science research

at their respective universities. Surely, we thought, Harvard and its students deserved an undergraduate research

journal dedicated to the profound work being done here in Cambridge and Boston, MA.

Though the seed of THURJ grew from the good-natured rivalry we felt with the other universities, we

encountered many important reasons for having such a journal while putting it together. It truly is the case that

many undergraduates are passionate about the sciences and perform advanced research; we believe that such

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undergraduate research has not been lost on Harvard, which has developed several programs to foster research.

THURJ also gives both student manuscript submitters and evaluators insight into peer reviewing, which is central

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As with any large endeavor, we and THURJ owe thanks to many people and institutions. The peer review process

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our Faculty Advisory Board, consisting of Harvard faculty, who volunteered their time and energy to support us.

THURJ’s Content, Business, and Design boards all made similarly tremendous efforts to bring you the publication

that you now hold. Certainly this inaugural issue would not have been possible without the gracious sponsorship of

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who submitted their research to THURJ, who made our lives easier by doing such excellent work. Finally, thank you

for opening the pages and experiencing the fantastic research going on at Harvard. Research is meant to be shared

and we are glad that you can join us.

Sincerely,

John Zhou, Co-Editor-in-Chief Shoshana Tell, Co-Editor-in-Chief

ii w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

About us
EXECUTIVE BOARD
CO-EDITOR-IN-CHIEF
John Zhou ‘10 (johnzhou@fas.harvard.edu)
The Harvard Undergraduate Research
CO-EDITOR-IN-CHIEF
Journal (THURJ) showcases peer-reviewed
undergraduate student research from Shoshana Tell ‘10 (stell@fas.harvard.edu)
DOO VFLHQWLÀF GLVFLSOLQHV $V D ELDQQXDO
BUSINESS MANAGER
publication, THURJ familiarizes students
Xin Pan ‘10 (xinpan@fas.harvard.edu)
with the process of manuscript submission
and evaluation. Moreover, it provides a MANAGING EDITOR OF CONTENT
FRPSUHKHQVLYHIRUXPIRUVFLHQWLÀFGLVFRXUVH Aditi Balakrishna ‘10
on the cutting-edge research that impacts our (balakris@fas.harvard.edu)
world today.
At its core, THURJ allows students to gain MANAGING EDITOR OF
insight into the peer review process, which PEER REVIEW AND SUBMISSIONS
LV FHQWUDO WR PRGHUQ VFLHQWLÀF LQTXLU\ $OO Lisa Rotenstein ‘11 (lrotenst@fas.harvard.edu)
THURJ manuscripts are rigorously reviewed
DESIGN CHAIR
by the Peer Review Board (consisting of Justine Chow ‘10 (jschow@fas.harvard.edu)
Harvard undergraduates), and the top
manuscripts that they select are further BUSINESS BOARD
reviewed by Harvard professors. This
Yi Cai ‘11
process not only stimulates faculty-student Aubrey Huynh ‘11
collaboration and provides students with Jessie Jiang ‘11
valuable feedback on their research, but also Kevin Liu ‘11
promotes collaboration between the College Alec Pinero ‘11
and Harvard’s many graduate and professional
CONTENT BOARD
schools. In addition to publishing original
student research papers, THURJ is also an Yi Cai ‘11
important medium for keeping the Harvard Ada Lio ‘11
Alterrell Mills ‘10
community updated on science research- Fernando Racimo ‘11
related news and developments. Peyton Shieh ‘10
Sophie Warton ‘11
Contact Farhan Murshed ‘11
Please email: contact@thurj.org DESIGN BOARD
Advertising Ada Lio ‘11
John Liu ‘11
Please email: advertising@thurj.org Kevin Liu ‘11
Yan Yan Mao ‘10
Subscriptions Kate Xie ‘10
Please email: subscriptions@thurj.org
PEER REVIEW BOARD
Submissions John Liu ‘11 - Head Copy Editor
Please email: submissions@thurj.org Meng Xiao He ‘11 - Copy Editor
Charlotte Seid ‘10 - Copy Editor
Ana Garcia ‘11
Website Daniel Handlin ‘11
http://www.thurj.org Iddosshe Hirpa ‘11
Kevin Liu ‘11
Tim Markman ‘11
Copyright 2008 The Harvard
Yan Yan Mao ‘10
Undergraduate Research Journal. Alterrell Mills ‘10
No material appearing in this publication Joe Pollard ‘11
may be reproduced without written Abby Schiff ‘11
Sheng Si ‘11
permission of the publisher. The opinions
Helen Yang ‘11
expressed in this magazine are those of the
contributors and are not necessarily shared by PRODUCTION ADVISOR
the editors. All editorial rights are reserved.
Roque Strew, Harvard employee
w w w . T H U R J . o r g T H U R J V o l I I s s u e
C o n t e n t s
FACULTY ADVISORY BOARD
Paul Bamberg, Ph.D
SENIOR LECTURER IN MATHEMATICS
Michael Brenner, Ph.D
GLOVER PROFESSOR OF APPLIED MATHEMATICS AND APPLIED PHYSICS
Myron Essex, D.V.M., Ph.D
MARY WOODARD LASKER PROFESSOR OF HEALTH SCIENCES IN THE FACULTY
OF PUBLIC HEALTH
Jeffrey Flier, M.D.
DEAN, HARVARD MEDICAL SCHOOL, AND GEORGE C. REISMAN PROFESSOR
OF MEDICINE
Steven Freedman, M.D., Ph.D
ASSOCIATE PROFESSOR OF MEDICINE
David Haig, Ph.D
GEORGE PUTNAM PROFESSOR OF ORGANISMIC AND EVOLUTIONARY BIOLOGY
Dudley Herschbach , Ph.D
FRANK B. BAIRD JR. RESEARCH PROFESSOR OF SCIENCE
Richard Losick, Ph.D
MARIA MOORS CABOT PROFESSOR OF BIOLOGY
Hongkun Park, Ph.D
PROFESSOR OF CHEMISTRY AND OF PHYSICS
Steven Pinker, Ph.D
JOHNSTONE FAMILY PROFESSOR OF PSYCHOLOGY
Pardis Sabeti, M.D., Ph.D
POSTDOCTORAL FELLOW, THE BROAD INSTITUTE OF MIT AND HARVARD
Zhigang Suo, Ph.D
ALLEN E. AND MARILYN M. PUCKETT PROFESSOR OF MECHANICS AND
MATERIALS
In the creation of this publication,
we have received guidance,
funding, and even written and
visual contributions from the
following, without whom this
publication would not exist:
Provost Hyman, Dr. Steven Freedman,
Dean Jeffrey Flier, Felice Frankel, Dean
Venkatesh Narayanamurti, Dr. Caren
Solomon [deputy editor of the New
England Journal of Medicine], Dr. Katrina
Kelner [deputy editor of Science],
Dr. Edward Benz and Dana-Farber,
Dr. Steven Gygi, Dean Judith Kidd,
Dean Paul McLoughlin, Dean Jeremy
Bloxham, Wei Hong, Janice Ahn,
Jae Hur, Sirinya Matchacheep, Frances
Chu, Ethan Karp, Jessica Tibbits,
Radcliffe Institute, The Undergraduate
Council, Harvard College
Thank you!
1 S p r i n g 2 0 0 8
iii
 C o n t e n t s
Features
6 $IRRWEDOODÀFLRDGRFDOOLQJ
the plays in cancer research

9 Unraveling the neural

circuitry behind moral
judgments

17 Studying abroad

22 Using small tools to tackle a 14 7KHHPHUJLQJÀHOGRI 

big problem neuroeconomics

1 Manuscript Summaries
Book Review Opinion/Editorial
28 The ABC’s of learning
your ABC
Proust and the Squid: the story
and science of the reading brain

24 Engineering: linked in
to almost everything

ON THE COVER: “NANOTUBES” BY FELICE FRANKEL

Felice Frankel is a Senior Research Fellow in FAS and heads the
Envisioning Science Program at Harvard’s Initiative in Innovating
Computing. For her next book, No Small Matter, (Harvard Uni-
versity Press 2009), with Harvard University Professor George M.
Whitesides, she constructed an 8-inch long acetate cylinder printed
with a hexagonal pattern to represent the structure of a nanotube.
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WKHGLJLWDOÀOHZLWK3KRWRVKRS:HWKDQN)HOLFHIRUJLYLQJXVRXU
ÀUVWFRYHULPDJH

iv w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 C o n t e n t s
Manuscripts
CELL BIOLOGY

CELL BIOLOGY
37 Thrombospondin-1 switches nitric oxide function
from inhibitory to stimulatory for gene expression
of human endothelial tumor markers
CELL
CHEMISTRY BIOLOGY

44 Bright, luminescent silicon nanoparticles for bio-

logical applications
31 Targeting TRAF proteins as an anti-
ENGINEERING
tumor strategy CHEMISTERY
50 0LFURÁXLGLFFHOOXODUGRXEOHHQFDSVXODWLRQLQD
polymer shell
MEDICAL SCIENCE
NEUROSCIENCE
62 NADPH oxidase-derived free radicals are required ENGINEERING
for cortex-controlled motor activity

PHYSICS
69 Probing non-linear rheology with free oscillations
MEDICAL
PHYSIOLOGY SCIENCE

73 3HOYLFÀQPRWLRQGXULQJVWHDG\VZLPPLQJDQG
maneuvers
54 Synthesis of a bioresorbable elec-
trospun guide tube for spinal cord NEURO-
regeneration SCIENCE

Visit http://www.thurj.org for news,

SUSTAINABILITY SCIENCE
details about the organization, and other
information on science at Harvard. PHYSICS

PHYSIOLOGY

80 Biological, socioeconomic, and po- SUSTAINABILITY

SCIENCE
litical aspects of the Nassau grouper
(SLQHSKHOXVVWULDWXVÀVKHU\LQWKH
Turks and Caicos Islands

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F e a t u r e s

Unraveling the Neural Circuitry

Behind

Moral Judgments
BY SOPHIE WHARTON, THURJ STAFF

C an moral reasoning be mapped in our neurons?

Josh Greene, an assistant professor of psychology
at Harvard, believes that studying the brain can indeed
reveal the mechanisms that underlie our moral decision-
making. Greene has begun to combine moral philosophy
with advanced neuroimaging technology, pioneering an
H[FLWLQJQHZÀHOGNQRZQDVQHXURSKLORVRSK\

9 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

A Psychological Philosopher
A s an undergraduate at Harvard, Greene pursued
a philosophy concentration and became inter-
ested in the questions of where our moral intu-
itions come from and whether we can trust them.
His senior thesis was a philosophical exploration
of psychological biases in moral judgment. Af-
WHU JUDGXDWLRQ WKH VHOIVW\OHG ´VFLHQWLÀFDOO\PLQGHG
skeptic” went on to Princeton to pursue a graduate
degree in philosophy.
Green soon pitched the idea of coupling neuroimag-
ing with philosophical dilemmas. Teaming up with Jona-
than Cohen of Princeton’s Center for the Study of Brain,
0LQGDQG%HKDYLRU*UHHQHFRQGXFWHGRQHRI WKHÀUVW
neuroimaging studies of moral judgment in 1999 and is
now continuing his research here at Harvard.
What was Greene looking to discover about moral
judgment? The question of how humans should make
decisions about moral dilemmas has been the central con-
cern of moral philosophy. The German philosopher Im-
manuel Kant believed that decisions ought to be rational
and reasoned with the intellect, while Scottish philoso-
pher David Hume argued that moral judgment is actually
driven by emotion. Greene hypothesized that both reason
and emotion are critical to judgment and that different
brain processes might be involved when we use different
w w w . T H U R J . o r g T H U R J V o l
F e a t u r e s
by Justine Chow, THURJ Staff
modes of judgment.
Moral Dilemmas Meet Modern Technology
G reene tested his hypotheses by presenting volun-
teers with classic moral dilemmas and monitoring
their brain activity using a functional magnetic resonance
imaging scanner (fMRI). One such classic moral question
is the trolley problem that had captivated Greene early in
his career:
“A trolley is running down a track out of control. If it
NHHSVJRLQJLWZLOOUXQRYHUWKHÀYHXQVXVSHFWLQJSHRSOH
hanging out on the track. You can prevent this disaster
by throwing a switch, redirecting the trolley onto a siding
where it will kill one person. Do you hit the switch?”
Most people answer yes, making the utilitarian judgment
WKDWLWLVZRUWKVDFULÀFLQJRQHOLIHWRVDYHÀYH+RZHYHU
consider this slight variation on the same dilemma:
I I s s u e 1 S p r i n g 2 0 0 8
10
F e a t u r e s

“You’re watching the same impending situation from a in the personal moral situation, justifying the act as be-
IRRWEULGJHDQGWKHRQO\ZD\WRVDYHWKHÀYHSHRSOHIURP ing “for the greater good,” they took a longer time to
certain death is to push a large person next to you off respond, because, Greene hypothesizes, they had to over-
the bridge and onto the tracks. He’ll be crushed by the come their initial emotional reaction to the situation.
WUROOH\DQGZLOOGLHEXWWKHÀYH
people will live, as he will have “One important difference Further Investigation
stopped the train.”
Although logically this situ-
ation is identical to the previ-
is intention—people feel it
is different harming another
G
moral
reene’s current research
LQYROYHV DQRWKHU GLIÀFXOW
dilemma:
RXVRQHDQRWKHUÀYHRYHURQH person as a means to end, versus “It’s wartime and you’re hiding
trade-off, people are much less in the basement with some fellow
ZLOOLQJWRVDFULÀFHWKHRQHSHU- as a side effect, as collateral villagers and your baby. The en-
VRQLQWKLVFDVH:KLOHWKHÀUVW damage.” emy soldiers are outside and they
variation is what Greene labels have orders to kill all remaining
an “impersonal moral dilemma,” the second is a “per- FLYLOLDQV DQG LI  WKH\ ÀQG \RX WKH\·UH JRLQJ WR NLOO \RX
sonal moral dilemma.” People seem to have an emotional your baby, and everybody else. Your baby starts to cry
aversion to the idea of pushing the large person off the and if you don’t cover your baby’s mouth, the soldiers
bridge. ZLOOÀQG\RXDQGNLOOHYHU\ERG\%XWLI \RXGRWKHQ\RXU
“One important difference is intention—people feel it is baby will smother to death. Is it morally acceptable for
different harming another person as a means to end, ver- you to do this?”
sus as a side effect, as collateral damage,” Greene says. Greene hypothesizes that when presented with this ter-
The other vital distinction between the two scenarios rible choice, emotional and utilitarian impulses will com-
is the close body contact in the latter case, what Greene pete and people may take a long time to come to a moral
calls “personal force.” Responses to other versions of decision. He predicts that the anterior cingulated cortex,
the trolley problem have shown that intention or personal a region associated with competing behavioral responses
force alone are not enough to elicit the emotional reac- of all types (not just moral), will be activated during the
WLRQEXWWKHFRQMXQFWLRQRI WKHWZRIDFWRUVZLOOLQÁXHQFH decision-making process.
response. He is also currently conducting more research involving
In his neuroimaging study, Greene tested subjects with the trolley problem, but is varying the
these two scenarios and other similar personal and imper- parameters of the scenario
sonal moral dilemmas, and found that the medial fron- (the number of peo-
tal gyrus, the posterior cingulate gyrus, and the angular ple you are saving,
gyrus—all regions of the brain involved in emotional the probability that
DQGRU VRFLDO FRJQLWLRQ³ZHUH VLJQLÀFDQWO\ PRUH DFWLYH they will die, etc)
when the variation to the original dilemma was brought and studying—using
up. economic-type calcu-
Areas associated with working memory --the right mid- lations—how the ex-
GOHIURQWDOJ\UXVDQGWKHSDULHWDOOREHZHUHVLJQLÀFDQWO\ pected moral value
less active in the second situation. Greene’s research changes with the
appears to suggest that the integration of an emotional variations. Greene
component in personal moral judgments seems to inter- is also planning on
fere with the functioning of other cognitive faculties and testing psychopaths
affect a person’s ability to reason with pure utilitarian- in order to study their
ism. responses to the same
The reaction times of subjects in Greene’s study were moral dilemmas.
also revealing. When the occasional person answered,
“Yes, you should push the fat man off the footbridge”
by Justine Chow, THURJ Staff

11 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

The Purpose of Emotion
I f, as Greene’s research suggests, emotion sometimes in-
terferes with our rational, utilitarian system of judgment,
what then is the evolutionary purpose of incorporating the
emotional system?
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FRQÁLFWEHWZHHQUHDVRQDQGHPRWLRQH[SODLQLQJ´ZKDWZH
UHDOO\PHDQLVDFRQÁLFWEHWZHHQ¶DXWRPDWLF·V\VWHPVZKHUH
the emotion leads to a plan for action quickly and without
PXFKUHÁHFWLRQDQG¶FRQWUROOHG·V\VWHPVZKHUHHPRWLRQV
VXSSRUWDVORZHIIRUWIXOGHOLEHUDWHSURFHVVRI UHÁHFWLRQµ
Often, rapid, automatic and emotional impulses lead us to
make good moral decisions.
“We expect people to have rapid, immediate moral be-
liefs,” Cushman says. “Imagine somebody who had to think
carefully, deliberately
and rationally about
whether it was best
to save his younger
brother from drown-
ing in a pool. Even
w w w . T H U R J . o r g T H U R J V o l
F e a t u r e s
if he made the right decision in the end, we would tend
to think there’s something wrong about even having to
reason about it. Instead, we expect people to have an
LPPHGLDWHHPRWLRQDOUHVSRQVHWKDWVD\V¶VDYHKLP·µ
Cushman also points out that people with brain dam-
age often lack those emotional impulses and thus end
up acting in ways the majority consider inappropriate
or antisocial. Thus, emotion may have evolved to play
a role in the decision-making of a normally functioning
individual because it leads people to value the lives of
others and make rapid decisions in times of peril.
Relevance to Philosophy?
F rom the trolley car study, Greene has found that
moral judgments are arrived at through complex,
often competing neurological processes, involving both
rational and emotional systems.
Will this research change our philosophical concep-
tions of morality? Many philosophers, including Andy
Reath, visiting professor in moral philosophy and expert
on Kantian ethics, advise that we take caution before
PDNLQJ DQ\ SKLORVRSKLFDO FODLPV IURP QHXURVFLHQWLÀF
research.
While psychology reveals how we
actually think, Reath explains, “phi-
losophy is concerned with how we
ought to think, which is something
brain scanning cannot tell us.”
But is psychology research entirely
LUUHOHYDQW WR WKH ÀHOG RI  SKLORVR-
phy?
Psychology professor Steven
Pinker raises the Kantian argument
He’ll be crushed by
that
the trolley and will
“ought GLHEXWWKHÀYHSHR-
implies can,” and
thus psychologi- ple will live.
cal research into
our intuitive morals can help delineate what is possi-
ble, what we can morally aspire to given our biological
makeup. Pinker also believes we can act more morally
by learning to disregard certain visceral emotional re-
actions, such as disgust towards homosexual practices,
which may have arisen thousands of years ago as a lega-
I I s s u e 1 S p r i n g 2 0 0 8 12
F e a t u r e s

cy of evolution, but are no longer appropriate emotional

responses.
Cushman too believes that psychological research is
connected to the philosophical “ought”.
“I’m inclined to think that morality is something hu-
mans create, not something they discover,” Cushman
says. “And for that reason, a psychological understanding
of morality is critically important to the work that moral
philosophers aspire to accomplish.”

No Role Left for the Soul?

F indings like Greene’s may disturb people who prefer

to attribute morality to a soul that is apart from the
physical entity. Some believe that dualism—a mind/body
distinction—is increasingly unfeasible, and that research
like Greene’s shows that moral reasoning is the interplay
of physical brain processes and does not necessitate a
separate, immaterial soul.
While the purpose of Greene’s experiments is not to
disprove ideas about the soul, discussions of these ideas
invariably arise as a side-effect of his research.
Although he is wary of completely dismissing the pos-
sibility of the soul, Greene says his research “challenges
people’s intuitive assumptions of what a person is.”
“If there’s anything your soul does, it’s make moral de-
cisions,” Greene says. “When we are able not only to see
brain regions activate, but to go further with each new
study in understanding what exact regions are used for
ZKDW VSHFLÀF VFHQDULRV WKLV SXWV WKH VRXO RXW RI  EXVL-
ness.”
Cushman does not think dualism is dead quite yet, but
agrees with Greene that the knowledge gained from ad-
vances in neuroscience may edge the concept out. He
cautions, though, that neuroimaging cannot truly dis-
prove dualism.
“Neuroimaging allows us to look at physical, chemi-
cal reactions in the brain,” Cushman says. “Dualism pos-
its that there are non-physical causes of behavior. Since
those causes are non-physical, they’re not going to show
up in a brain scan—so the fact that we can’t see them
does not provide direct evidence that they don’t exist.”
Whatever the impact of neurophilosophy research on
our beliefs about dualism, it is evident that work like
Greene’s is expanding our understanding of the physi-
ological processes involved in moral reasoning. Perhaps
as we gain more knowledge about our intuitive responses
and instincts, we will be better able change and improve
our moral behavior.
13 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 F e a t u r e s

7KHHPHUJLQJíHOGRI

Neuroeconomics

BYALTERRELL MILLS,
THURJ STAFF

W
hat makes us havioral economics,
decide what which considers the
we purchase? psychological ef-
A classical econo- fects on economic
mist might say that decisions, may be
we choose based on limited in that
calculating expected emotions and
utility and maximiz- motivation are
by Justine Chow, THURJ Staff often qualitative
ing our personal
gains, essentially by characteristics that cannot be ex-
predicting value plained but by the individual ex-
based on an economic theory. A periencing them. As a result, eco-
psychologist might claim that we nomic predictions are divided into
choose products that evoke pleas- normative and positive branches.
ant memories or conjure thoughts Normative economics describes
feelings of happiness. A neurosci- which economic decisions maxi-
entist might posit that goods that is able to integrate the theories of these mize personal gain, while positive
activate our dopamine centers (an- three scientists in order to make his pre- economics describes which eco-
atomical centers associated with diction. nomic decisions humans actually

reward and motivation) would in-
ÁXHQFHRXUVSHQGLQJKDELWV
All of these scientists are correct,
in part. But a new individual to
the table—the neuroeconomist—
N
choose.
euroeconomics is a relatively new Neuroeconomics furthers the
ÀHOGRI VWXG\WKDWWDNHVLQWRFRQ- understanding of human cogni-
sideration several of the social tion within the framework of eco-
sciences that are involved in choice. Be- nomic and behavioral decision-

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
14
 F e a t u r e s

K
ahneman and Tverysky’s Prospect Theory is a de-
making with brain science. Neuroanatomical measures of scriptive model for positive economic decisions, as
preference, belief, and cognition are found using Positron it models the observed, not expected, behavior of
Emission Topography (PET) scans and Functional Mag- individual choice. The main tenet of the theory is that
netic Resonance Imaging (fMRI) to shed light on which consumers are averse to losses, instead preferring gains.
areas of the brain are activated as an individual makes an More importantly, consumers are averse to perceived
economic decision. losses when compared to perceived gains.
In the 1979 study, participants were presented with the
Û2XU HPRWLRQV RYHUULGH UDWLRQDO following scenario: There is a viral epidemic with an ex-
pected death toll of 600. The participants are given two
GHFLVLRQPDNLQJÜ program proposals. In Program A, 200 people will be
saved, and in Program B, there is a one-third chance that
For example, Harvard Professor of Economics, David 600 people will be saved, and a two-thirds chance that no
I. Laibson ’88, found in a 2004 study that brain activity in one will be saved. Participants were then presented with
the frontoparietal system and dopaminergic reward sys- an alternate scenario. They were asked to choose between
tem predicted economic behavior. The experiment asked Program A, in which 400 people will die, and Program
participants to choose a smaller or larger award; the con- B, in which there is a one-third chance that no one will
dition for the larger award was receiving it in the future, die and a two-thirds
compared to the present. Participants exercised the im- chance that 600
mediacy effect, which dictates that human choice is heav- will die.
LO\LQÁXHQFHGE\ZDQWLQJLPPHGLDWHJUDWLÀFDWLRQ The research-
Why does emotion underlie such a deviation in consum- es found that
er’s actual economic choices, diverging from economic 67% of par-
theories and models? According to Laibson, “Emotional ticipants
cognition is fundamentally unlike analytical cognition, choose pro-
both in terms of mechanism and goals. Hence it is natural gram A in the
that economics—a theory of long-run, rational decision- ÀUVWVFHQDULR
making (optimization)—is very different from emotional where 200
cognition. In essence, our emotions override rational de- people lived
cision making. The impact of this obvious fact is that it and 400
FDQEHXVHGWRLQÁXHQFHHFRQRPLFFKRLFHVE\DOWHULQJWKH died, and
emotional state of the consumer.” only 33% of

15 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

participants choose program A in the second scenario,
where 400 died and 200 people lived. In both variations,
program A kills 400 and saves 200, and program B kills
200 and saves 400. The “economic” behavior performed
by the participants demonstrated that humans are adverse
to perceived loss, not simply actual loss.
The Prospect Theory is representative of an error in
judgment, where human perception precludes the most
´SURÀWDEOHµ HFRQRPLF GHFLVLRQ 7KH KDUPRQ\ RI  HFR-
nomics and psychology is key in understanding this ap-
parent contradiction.
In another classic experiment, Read and van Leeuwen
found that 74% of subjects are more likely to choose fruit
over chocolate as a snack when the food will be delivered
“next week,” but that 70% of subjects were more likely to
choose chocolate over fruit for that same day.
Findings such as these—which demonstrate consum-
er impatience—are the basis behind mail-in
rebates and credit cards. With an under-
VWDQGLQJ RI  KRZ WLPH DQG LQÁXHQFH
can affect our decision, there comes
a need to discuss what is fair to
consumers. Is it fair to prime
consumers, using their
emotional state, to in-
ÁXHQFH HFRQRPLF
decisions?
Laibson
says,
“peo-
ple use
by Justine Chow, THURJ Staff
w w w . T H U R J . o r g T H U R J V o l
F e a t u r e s
HPRWLRQDOSULPHVWRLQÁXHQFHRXUFKRLFHVµVXFKDVZHDU-
ing makeup to attract attention or using celebrity endorse-
ments. It is a fair assertion that an advertiser’s priming
is no different than trying to attract desired praise with
rouge, , or other beauty-enhancing techniques, he says,
:LWK DQ XQGHUVWDQGLQJ RI KRZ
WLPHDQGLQîXHQFHFDQDIIHFWRXU
GHFLVLRQWKHUHFRPHVDQHHGWR
GLVFXVV ZKDW LV IDLU WR FRQVXP-
HUV
adding that consumers are aware of advertisers’ manipu-
lations. He further states that it would be “nice to know
when we are being emotionally manipulated,” but that in
practice, our cognitive systems do not need censors or
“paragraphs of disclosures that we would never read.”
N
euroeconomics works to predict economic de-
cision making, not to regulate how it should be
used, by looking one step deeper than behavior
economics—at the physiological machinery that governs
our actions. The use of brain imaging and emotional cog-
nition is a groundbreaking way to approach modeling
economic theory. If Plato is correct in that “Necessity
is the mother Invention,” then the problem of making
better models of economic choices may lie within neu-
roeconomics.
I I s s u e 1 S p r i n g 2 0 0 8
16
 F e a t u r e s

Studying
Abroad
BY YI CAI, THURJ STAFF

W
hile Jamie Greenwald ’08 conducted research Gaining New Perspective on Science
and took classes at a Botswana university dur-

ing the 2007 spring term, many of her fellow
Harvard College science concentrators trekked through
the relentless Cambridge winter snow to their laborato-
ries and classes to receive their academic credit.
Students like Greenwald—science concentrators who
venture hundreds of miles outside the gates of the Yard
to study or conduct research—are a minority in the Har-
YDUGFRPPXQLW\$FFRUGLQJWRGDWDIURPWKH2IÀFHRI 
International Programs (OIP), science concentrators are
vastly outnumbered by non-science concentrators when
participating in conventional study abroad programs (tak-
ing courses at a Harvard-approved institution overseas).
Though science concentrators have the additional option
RI  SXUVXLQJ VFLHQWLÀF UHVHDUFK DEURDG HVWDEOLVKHG SUR-
grams limit enrollment to only a handful of students and
many do not provide credit.
<HWHYHQWKHVHOLPLWHGRIIHULQJVUHSUHVHQWDVLJQLÀFDQW
step up from the status quo only a few years ago. Many
of these research and study abroad opportunities just
sprouted up in the past two years, and plans are in the
works to expand these programs.
S
tudy abroad is increasingly viewed as a vital part of a
student’s education. Giorgio DiMauro, the associate
director of the OIP, asserts that the purpose of study
abroad is to “broaden the mind, challenge assumptions,
WKLQNFULWLFDOO\DERXWKRZFXOWXUHLQÁXHQFHVDQGVKDSHV
our perspective, and become a responsible global citi-
zen.”
Studying or researching abroad has become more and
more relevant to an undergraduate science concentrator’s
Scientific advances are
increasingly achieved through
collaborative efforts between
multinational teams of scientists

17 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Photo courtesy of Batsirai Chidzodzo and Catherine Rosseel
Students currently participating in the CIEE “HIV in Africa” program climb Kgale Hill, overlooking Gaborone. In-
cluded are Sandy Bolm (‘09) in the yellow shirt, and Sarah Ashburn (‘09) in the background, with a blue bandana.
education in our shrinking world. Dr. Marie Dahleh,
assistant dean for academic programs at the School of
(QJLQHHULQJDQG$SSOLHG6FLHQFHVVD\VWKDWDVVFLHQWLÀF
advances are increasingly achieved through collaborative
efforts between multinational teams of scientists, “being
exposed to a different culture … and learning how people
[of those cultures] think is important so that students can
learn how to work with other societies.”
Research or study abroad may also provide a rich edu-
FDWLRQDOVXSSOHPHQWWRZRUNLQDVFLHQWLÀFFRPPXQLW\LQ
a different country. Professor Robert Lue, director of
life sciences education and senior lecturer in molecular
and cellular biology, says he sees study or research abroad
as “an opportunity to see how a different culture or so-
ciety approaches sciences.” Study abroad may be able to
provide students with the perspective to combine differ-
ent approaches to science, leading to the development of
QHZVFLHQWLÀFPHWKRGV
)XUWKHUPRUHWKHFXOWXUDOLPPHUVLRQEHQHÀWVRI VWXG\
or research abroad are undeniable to those who have ex-
w w w . T H U R J . o r g T H U R J
F e a t u r e s
perienced time abroad. Greenwald, who participated in
the Botswana “HIV in Africa” research and study abroad
program, recalled several visits to nearby villages exposed
her to much of the culture in Botswana. The experi-
ence also provided Greenwald with a “world view and
perspective” that allowed her to see the privilege students
at Harvard enjoy. Greenwald also studied and used Set-
swana, the national language of Botswana, during her
time there.
Simply being abroad may play an important, if not
crucial role, in the program. For example, the Harvard-
sponsored study abroad program, “Fauna and Flora of
Hispaniola,” is dependent on being set in the Dominican
Republic. The Botswana location of the “HIV in Africa”
DOORZHG*UHHQZDOGWRVHHWKHVLJQLÀFDQFHRI KHUUHVHDUFK
in person.
“The fact that we were doing [HIV research] in Botswa-
QD RQH RI  WKH PRVW DIÁLFWHG FRXQWULHV DGGHG D ZKROH
other level,” says Greenwald.
V o l I I s s u e 1 S p r i n g 2 0 0 8
18
F e a t u r e s

Diverse Options

A t present, there are a number of science-focused

programs for study abroad available through the
OIP as well as several research-abroad programs through
the Harvard Summer School and Harvard departments.
The programs take students to a diversity of locales, such
as South Africa, the Dominican Republic, India, Germa-
ny, and Malaysia.
Harvard-sponsored study abroad programs include
“Darwin and the Origins of Evolutionary Biology” in
Oxford, England and “The Biodiversity of Borneo” in
semester. Students participating in the RIKEN program
for immunology conduct immunology research in Yoko-
hama while receiving Japanese language training. How-
ever, no credit is awarded to the three students accepted
to the latter program.
Nevertheless, the number of science concentrators
study abroad remains quite low. Of the students who
studied abroad through OIP programs last academic year
(2006-07), only 13% of the declared students were sci-
ence concentrators.

Photo courtesy of Batsirai Chidzodzo and Catherine Rosseel

International Students attend a University of Botswana orientation meeting, January 2008.
Malaysia. The former program credits students with two
half-year courses while the latter program credits students
with a single full-year course. However, course enroll- The programs take students to a
ment is limited.
Harvard-sponsored research abroad programs include diversity of locales, such as South
“HIV in Africa” in Gaborone, Botswana and “RIKEN
Center for Allergy and Immunology” in Yokohama, Ja- Africa, the Dominican Republic,
pan. The HIV in Africa’s pilot program, held last spring,
allowed three students to spend a semester conducting India, Germany, and Malaysia.
HIV research and take elective courses at the University
of Botswana. Thus, the students receive credit for a full

19 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 F e a t u r e s

Limitations and Difficulties

S o, why aren’t there more science concentrators going

abroad to study or research? One of the most ob-
vious reasons is cost. Although the OIP provides some
In some cases, there are not
IXQGLQJWRRI DSSOLFDQWVVWXGHQWVLQDOOÀHOGVPD\ enough funding support
still be discouraged from going abroad because they may
still need to cover much of the expenses, according to Di-
Mauro. In some cases, there are not enough funding sup-
programs for them. It is
SRUWSURJUDPVIRUWKHP,WLVHVSHFLDOO\GLIÀFXOWIRUVWX-
dents to receive adequate funding if they pursue research
especially difficult for students
abroad through individually-crafted programs (those that
are not already established through Harvard). AJ Garcia, to receive adequate funding if
’09, who was a Herchel Smith Fellow, researched abroad
LQ%UD]LOODVWVXPPHU+HVD\VÀQDQFLQJKLVSODQV´UH- they pursue research abroad
quired a tremendous amount of independent research
DQGDQDPD]LQJDPRXQWRI SURELQJ>WKH2IÀFHRI &DUHHU
Services], [the David Rockefeller Center for Latin Ameri-
through individually-crafted
can Studies], and other Harvard organizations for infor-
mation, funding, and support.”
programs.
Also, many of the established research and study abroad
programs available can only accept a handful of students.

Photo courtesy of Batsirai Chidzodzo and Catherine Rosseel

International students meet the Kgosi, or chief, at the Tlokweng Kgotla, a traditional tribal meeting place, early this
January in Bostwana.
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
20
F e a t u r e s

The HIV in Africa program and the RIKEN program

each only take three students.
Though language barriers may sometimes be a chal-
lenge for students who wish to go abroad, many of the
host organizations abroad are no strangers to the English
language and many programs do not have language pre-
requisites.
Dr. Dahleh also suggests that some students avoid going
abroad because they would not receive enough credit.
“For example, for the Bachelor of Science degree in
Engineering Sciences, there are 20 half-courses worth of
requirements and, in that case, the student may not have
enough time,” Dahleh says.
Indeed, some research abroad programs sponsored Photo courtesy of Batsirai Chidzodzo and Catherine Rosseel
by Harvard departments do not award credit (Research Students enjoy the view from the top of Kgale Hill in Bo-
tswana. From left to right, Nathan Leiby (‘10), Elizabeth
Internships in Science and Engineering in Germany,
Loy (UPenn), Sarah Ashburn(‘09), Sandy Blom (‘09), and
RIKEN program in immunology, Cambridge University
Harvard Projects, among others). However, the study/
research abroad programs (HIV in Africa, Darwin and
the Origins of Evolutionary Biology, The Biodiversity
of Borneo, and more) through Harvard College Summer
School and the OIP do award credit.
Though none of these challenges are necessarily insur-
mountable, Dahleh says that “only a handful of students”
DWWKHHQJLQHHULQJVFKRROGURSE\KHURIÀFHWRWDONDERXW
going abroad. She suggests that undergraduates may not
be fully aware of the available opportunities, as many ex-
isting programs are relatively new. Indeed, study abroad
itself has increased in popularity of late. According to
the OIP, the number of students going abroad during
2006-07 was more than a 300 percent increase from the
number in 2001.
“It makes a great deal of sense to go abroad if you’re
studying international politics or economy, but may not
be so clear a trajectory for an inorganic chemist or a phys-
icist,” DiMauro suggesting another possible explanation.
Certainly, many science concentrators may think it un-
necessary to go abroad to study or research when there is
a wealth of resources here at Harvard. Greenwald recalls
that in Botswana, she had to “wait weeks for reagents to
arrive that [she] could have had shipped overnight in the
US.” Garcia shared similar lab setbacks in Brazil, and thus
recommends study and research abroad (especially in less
21 w w w . T H U R J . o r g T H U R J V o l
%RQQLH'HPXWK)DLUÀHOG
technologically-advanced countries) to students who are
´VHOIVXIÀFLHQWLQGHSHQGHQWDQGDUHULVNWDNHUVµ
Looking Forward
H owever, there are indications that more science con-
centrators may take advantage of study or research
abroad in the future.
“Traditionally, science concentrators have seen [study
or research abroad] as orthogonal or contradictory to
what they need,” Lue says. “But students are realizing that
[they] can combine the two [studying or researching with
going abroad].”
And more programs are being formulated for science
concentrators. The RIKEN program in immunology was
just crafted in the past year, and Lue says that a program
in Shanghai is in the works.
DiMauro says that the OIP also hopes to make study
or research abroad a more prominent option for science
concentrators. The OIP has been working with Harvard
science departments to do so, and there are plans to ini-
tiate more programs for both physical and life sciences
concentrators, he says.
I I s s u e 1 S p r i n g 2 0 0 8

using small tools
to tackle a
BIG PROBLEM
One lab’s application of nanotechnology in cancer research
BY FARHAN MURSHED, THURJ STAFF
C
ancer is the second most common cause of death
in the United States, according to the American
Cancer Society, second only to heart disease.
While traditional therapies—like chemotherapy and
radiotherapy—work on a large scale, by attacking both
cancer and normal cells, a group of researchers at Har-
vard Medical School have advanced a small but powerful
new paradigm in cancer drug delivery.
The lab of Dr. Omid Farokhzad, an assistant profes-
sor at Harvard Medical School, develops functionalized
nanoparticles that target chemotherapeutic drugs and
diagnostic agents to diseased cells and tissues. When
WKHVHVXUIDFHPRGLÀHGQDQRSDUWLFOHVUHDFKLQIHFWHGFHOOV
ligands synthetically conjugated to these particles bind to
antigens naturally found on membranes of targeted cell,
delivering drugs selectively to diseased cells. The nano-
particles come, they see, they conquer.
While these nano-Caesars are typically smaller than a
virus, they are providing therapy for a disease of diamet-
rically opposed magnitude. Nanotechnology utilizes ma-
terials on a scale of 1 to 100nm in size—the head of a pin
w w w . T H U R J . o r g T H U R J V o l
F e a t u r e s
is about the size of a millimeter, meaning it would have
to be divided a million times to bring it to the dimensions
of work being done in a nanomedicine lab. The size,
according to Dr. Carolina Salvador-Morales, a member
of the Farokhzad lab, is just one factor in characterizing
nanoparticles.
“The size of the particle, the charge, the density—all
WKHSK\VLFDODQGFKHPLFDOSURSHUWLHV³LQÁXHQFHKRZDS-
plicable nanoparticles are to biological systems,” Salva-
dor-Morales said.
<HWODERUDWRULHVÀQGLWYHU\GLIÀFXOWWRFKDUDFWHUL]HGLI-
IHUHQW W\SHV RI  QDQRSDUWLFOHV ZKLOH DWWHPSWLQJ WR ÀQG
useful applications for the technology.
The characteristics that endow nanoparticles with such
enormous potential—miniature size, large surface area to
volume ratio, solubility, and large drug-carrying capaci-
ty—simultaneously present advantages and challenges in
ÀQGLQJWKHUDSLHV
“The question is how to control size or charge,” Salva-
dor-Morales said. “The main aspect of our work is con-
trolling these chemical and physical characteristics.”
I I s s u e 1 S p r i n g 2 0 0 8
22
 F e a t u r e s

Harnessing nanoparticles’ poten- phobic), for triggering natural im-

tial has been a trying task. In a paper mune responses, and for diagnostic
on cancer nanomedicine published imaging (nanoparticles are conjugated
in the October 2007 issue of Clini- with quantom dots—a type of semi-
cal Pharmacology & Therapeutics, conductor nanocrystal with unique
Dr. Farokhzad reports that research- optical properties). With such rapid
ers have been attempting to use the GHYHORSPHQWV LQ WKH ÀHOG LW LV QRW
nanoparticle liposome for targeted- surprising that the NIH has deemed
drug delivery for over 20 years, but nanomedicine one of the branches
only a few particles have made it to of its Roadmap to “re-engineer the
clinical trials, and none have been ap- clinical research enterprise.”
proved for use. “There has been a sharp increase
The Farokhzad lab, however, discov- in funding regarding patients and it
ered one promising bioconjugate— has provided for research as a formal
known as the nanoparticle-aptam- mechanism,” replied Dr. Farokhzad
er—that was able to successfully when asked about NIH’s Roadmap
target pancreatic cancer cells and de- commitment, “There has also been
liver chemotherapy in vivo. a tremendous increase from the in-
In a landmark paper in the April vestment community and private
2006 issue of Proceedings of the sources- including those based on
National Academy of Sciences USA QDQRWHFKQRORJ\ SDWHQW ÀOLQJV RYHU
(PNAS), the Farokhzad lab demon- WKHSDVWÀYH\HDUVµ
VWUDWHG WKDW D QDQRSDUWLFOH PRGLÀHG An ultimate goal of the lab,
with an A10 RNA aptamer—a nucle- Farokhzad says, is to develop drugs
ic acid ligand—could target and bind that would be available for oral de-
WRSURVWUDWHVSHFLÀFPHPEUDQHDQWL- livery. The translational potential for
gen (PSMA) on the surface of pros- nanomedicine promises to make a
tate cancer cells. VLJQLÀFDQWLPSDFWRQPHGLFLQH$WD
These nanoparticles, which encap- FULWLFDOMXQFWXUHLQWKHÀHOG6DOYDGRU
sulated the chemotherapeutic drug Morales believes this is a critical time
docetaxel (Dtxl), would promote the for the lab.
uptake of Dtxl into the cancer cells “The nanotech boom has been de-
and increase the drug’s cellular toxic- veloping for some years now,” Mo-
ity. After just one injection of Dtxl- rales says. “In the last seven or eight
Nanoparticle-Apt bioconjugate, there years, many phenomena have been
ZDVFRPSOHWHWXPRUUHGXFWLRQLQÀYH illustrated, and with ideas derived
of seven prostate cancer-infected from these phenomena, we are com-
mice, and all seven mice survived the ing closer to translate research into
109-day study. medicine.”
In other studies, the Farokhzad lab Even if Dr. Farokhzad and his army
has shown the nanoparticles’ poten- of nanoparticles may not have con-
by Farhan Murshed, THURJ Staff quered Gaul, they represent a formi-
tial for carrying multiple types of
Scenes from the Farokhzad lab at
drugs (both hydrophilic and hydro- Harvard Medical School. dable threat to the disease of cancer.

23 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Engineering:
BY VENKATESH “VENKY” NARAYANAMURTI, DEAN, HARVARD SCHOOL OF ENGINEERING AND APPLIED
SCIENCES; JOHN A. AND ELIZABETH S. ARMSTRONG PROFESSOR OF ENGINEERING AND APPLIED SCIENCES;
PROFESSOR OF PHYSICS
O
ne cold December morning, I walked into Pierce
Hall, stepping around a piece of stray drywall
from the ongoing construction of our sizable new engi-
neering teaching lab. If things stay on schedule, sometime
later this spring, undergraduates will be busy delving into
CAD/CAM, mechanics, and photonics research.
7KHFUHDWLRQRI WKHODE³DEHDXWLIXOJODVVDQGVWHHOÀVK-
bowl I hope will become a haven for hands-on experi-
mentation and research—highlights the “renaissance”
You cannot do great
science...without
doing great engi-
neering.
w w w . T H U R J . o r g T H U R J V o l
O p / E d
L!"#$% I"
& '() *&
E+$,-&.!"/
engineering and applied sciences has experienced at Har-
vard, particularly during the past decade. Of course, one
future facility only tells part of the story.
With the launch of the School of Engineering and Ap-
plied Sciences in September, we are turning our attention
to implementing our ambitious plans—enhancing educa-
tion, advancing research, bettering society—and kicking
up a bit of dust in the process. Our launch did more than
simply mark a moment in time; the event celebrated our
past successes and highlighted the increasingly important
role of engineering at Harvard and in the world.
My mantra remains: You cannot do great science,
whether it is physics, biology, or chemistry, without doing
great engineering. Likewise, you cannot solve the “big”
problems, from global warming to clean water to better
health for all, without making advances in technology and
tools. Granted, you might expect to hear this pitch from a
dean of engineering. I think, however, with a look to his-
tory, I can readily convince you that everyone is linked to
some aspect of engineering research.
I I s s u e 1 S p r i n g 2 0 0 8
24
 O p / E d
E
ngineering has come a long way since the time
ZKHQ +DUYDUG VHW XS LWV ÀUVW IRUPDO SURJUDPV
in the applied sciences at the famed Lawrence School in
WKHODWHV7KHÀHOGLVQRORQJHUDGLVFLSOLQHRI ORQH
“tinkerers” but is as thoroughly systematic, analytical, and
rigorous as the other sciences. In short, engineering re-
25 w w w . T H U R J . o r g T H U R J V o l
by Justine Chow, THURJ Staff
search leads directly to new knowledge, not simply to new
gadgets.
For example, understanding the behavior (the funda-
mental physics and chemistry) of nanostructured ma-
terials advances basic science through the creation of
new tools and processes (such as laser “tweezers” for
manipulating individual atoms) and leads to innovative
I I s s u e 1 S p r i n g 2 0 0 8
 O p / E d

technologies such as solar photovoltaics. Taking this one
step further, such knowledge may then be channeled into
solutions for improving global health (new drug delivery
systems) or for sustaining a cleaner environment (alterna-
tive and green energy systems).
Today’s technologies, the ultimate products of engineer-
ing and applied sciences, shape how we understand our
biological and physical world (from cells to CAT Scans to
- developing novel delivery methods for common
vaccines; and
- devising solutions to controlling contaminated
water supplies in the Dominican Republic.
Moreover, to reinforce engineering’s fundamental role,
we are working hard to create a more engaging, hands-on
curriculum by:
- considering broad based seminars on the world

we have the of technology aimed at addressing questions like—“How

does a hydrogen car work?”, “What might the future of

potential to be a electronic currency look like?”, and “Can we create de-

vices to meld nanomotors with biological ones?”

‘connector and an - designing portal courses as multi-tiered entryways

into our concentrations as a way to guide those who want

integrator’ to build new technologies as well as those who want to

manage them; and
- integrating design-focused projects (like how to
ÀQGWKHEHVW´EULGJHµWROLQNWKH&DPEULGJHDQG$OOVWRQ
quantum computing.) Thus, the timing of SEAS, a truly campuses) throughout our courses and weaving in more
21st century institution, could not be better for Harvard research experiences (both term-time and summer), entre-
VWXGHQWVLQWHUHVWHGLQDQ\DVSHFWRI VFLHQWLÀFUHVHDUFK preneurship opportunities (from business competitions
to on-campus incubators), and international exposure.
In fact, I am most excited about the resulting educa-
tional and experiential opportunities that will come from

B
our transformation to a School. Already, we have quite a
few projects where undergraduates have worked along-
side graduate students, post-docs, and faculty from across
the sciences and engineering by:
- conducting research in nanophotonics and mi- y
FURÁXLGLFV DV SDUW RI  RXU WZR 16)IXQGHG FHQWHUV WKH doing so, our goal
1DQRVFDOH6FLHQFHDQG(QJLQHHULQJ&HQWHU16(&DQG LV WR ERWK UHÁHFW
the Materials Research Science and Engineering Center the changing na-
(MRSEC); ture of science and
- working at the frontiers of quantum science to society and to expose
implement a spin quantum computer; more students from all
- proposing clever ways to improve Harvard’s ath- areas to the interplay of
letic facilities and make Maxwell Dworkin more energy technology and society.
HIÀFLHQW Further, because of our smaller scale, we have the op-
- using E. coli to manufacture a biologically-based portunity to make engineering more personal, more rel-
“wire”; evant, and more directly related to the particular interests
 EXLOGLQJÁ\LQJPLFURUREWLFLQVHFWVDQGDÁHHWRI  and concerns of students. We believe that we have the
robotic soccer players that shoot and score thanks to el- potential to be a “connector and an integrator,” a School
egant AI programming; that links to the most exciting activities across the whole

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
26
 O p / E d
About the author:
campus.
With our increased visibility, new courses, and expand-
ed research opportunities I anticipate that we will attract
more Harvard students to concentrate in engineering
and applied sciences, or at the very least, to check out
a few classes. By proving varied avenues to engineering
research, I think we can be one of the “coolest” kids on
the block.
Moreover, current students and alumni have told me
stories about how one great class, research project, or a
RQHRQRQH FRQQHFWLRQ ZLWK D IDFXOW\ PHPEHU GHÀQHG
their entire experience at Harvard. As we go forward, all
of us at SEAS are keeping that in mind. After all, there’s
a reason why our new teaching labs have glass walls: to
remind those looking out to always keep the bigger links
and connections in view; and to remind those looking in 9HQNDWHVK´9HQN\µ1DUD\DQDPXUWLLVWKH-RKQ$DQG
that engineering can be a welcome surprise. Elizabeth S. Armstrong Professor of Engineering and
Applied Sciences and Dean of the School Engineering
For more information on future plans for the School of Engi- and Applied Sciences at Harvard University. He is also a
neering and Applied Sciences visit: http://www.seas.harvard.edu/ professor in Harvard’s Physics Department. He served as
highlights/celebration.html Dean of Physical Sciences at Harvard from 2003-2006.

The Dana-Farber Cancer Institute

provides expert, compassionate care
to children and adults with cancer.

Learn more at:

DANA-FARBER www.dana-farber.org
CANCER INSTITUTE

44 Binney Street
Boston, MA 02115

27 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 B O O K R E V I E W

The ABCs
of processing
Proust
and your ABCs
the Squid: A s we begin to look at the words
comprising this paragraph,

the
we may ask ourselves whether
reading the whole article is worth the
trouble, or if moving along to another

story
page may prove more worthy of our
time. Seldom will any of us actually
ponder the very act of decoding
the individual words, of processing

and the physiological effort involved in

the act of reading. Maryanne Wolf,
Director of the Center for Reading

science and Language Research at Tufts

University, succeeds in doing so in her
book Proust and the Squid: The Story

of and Science of the Reading Brain.

D rawing a parallel between one of

the the greatest writers of the 20th

century and a marine cephalopod
may seem improbable at best, yet

reading Wolf effectively uses them both in

explaining the complexity of the
reading brain. To Proust, reading

brain was a “sanctuary,” a “divine pleasure”

BY FERNANDO RACIMO, THURJ STAFF
w w w . T H U R J . o r g
that we learn to appreciate over
time. Squids that couldn’t swim were
used during the 1950s to understand
by Justine Chow, THURJ Staff neuronal activity in motor functions.
T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
28
 B o o k R e v i e w

Similarly, the study of individuals that can’t read may

provide important insights into the neurobiology of
reading, that special sanctuary of the mind. These two
literary images are Wolf ’s starting point, from which she
elegantly presents the latest insights into the cognitive
functions of the reading brain.

P URXVW DQG WKH 6TXLG KDV WKUHH SDUWV 7KH ÀUVW

HQFRPSDVVHVWKHKLVWRULFDOGHYHORSPHQWRI WKHÀUVW
writing systems, the second provides an account of the
acquisition of reading skills from childhood to adulthood,
and the third addresses dyslexic children and the causes
and implications of their differently-wired reading
machinery. Wolf takes the reader on a journey through
the cultural history of reading in human civilization and
the “natural” history of reading in the developing brain.
This journey is easy to follow, even for the layperson,
but the book stops short of describing many cognitive
processes and events that could have further enriched the
otherwise comprehensive picture Wolf paints.

T KHERRN·VÀUVWFKDSWHUVDUHOLYHO\DQGDSSHDOLQJ%\
tracing the development of written communication
forms—beginning with early symbolic systems, moving
on to Summerian cuneiform, and concluding with the
birth of the Greek alphabet—the author shows how the
...the transition from
an oral to a written
culture constitutes a
landmark achievement
of human societies, as
Courtesy of brainsciencepodcast.libsyn.com
human brain learned to rearrange itself as this process
occurred through a series of “cognitive breakthroughs.”
7KHVH´EUHDNWKURXJKVµVLPSOLÀHGWKHDFWRI LQIRUPDWLRQ
processing and freed up cortical space in order to maximize
the brain’s ability to process written information. Wolf ’s
views on this progression are eye-opening, though they
WHQGWRERUGHURQRYHUVLPSOLÀFDWLRQDVKHUDQDO\VLVIDLOVWR
SURSHUO\DFFRXQWIRUVRFLRFXOWXUDOLQÁXHQFHVRQOLWHUDF\
throughout history. Reading did not always follow a linear
evolution and was sometimes subject to regressions due

it opened up space and to shifts in power or military domination.

time for the brain to
have deeper and more
insightful thoughts.
29 w w w . T H U R J . o r g
F or Wolf, the transition from an oral to a written
culture constitutes a landmark achievement of human
societies, as it opened up space and time for the brain to
have deeper and more insightful thoughts. She remarks
that Socrates was fearful that this transition would mark
the end of creative rhetorical thinking. Socrates’s fears
are paralleled by Wolf ’s own fears regarding the present
T H U R J V o l
transition to a digital culture: “How would Socrates
I I s s u e 1 S p r i n g 2 0 0 8
 B o o k R e v i e w

UHVSRQGWRDÀOPHGYHUVLRQRI D6RFUDWLFGLDORJXHWRKLV one of her motivations for uncovering the secrets behind
entry in Wikipedia or to a screen clip on YouTube?” the reading machinery. This allows for a deep, personal
connection between Wolf and her reader, enriching the

A fter this section, Proust and the Squid reaches it’s message she conveys: the dyslexic brain’s alternate wiring
most compelling stage, as it makes the reader dive can prove more dexterous at activities other than reading,
into the intricacies of a child’s brain learning to read. like pattern recognition and artistic endeavors.
&KLOGUHQIDOOLQWRHVVHQWLDOO\ÀYHVWDJHVZKLOHOHDUQLQJWR
read: the “pre-reader,” the “novice reader,” the “decoding
UHDGHUµWKH´ÁXHQWUHDGHUµDQGWKH´H[SHUWUHDGHUµ$V A ll in all, Proust and the Squid constitutes a rich
and complete account of a key feature of human
civilization and development—people’s ability to decode
“How would Socrates ZRUGV VHQWHQFHV DQG VWRULHV 7KH ERRN·V JUHDWHVW ÁDZ
may in fact lie in its initial appeal: the themes covered

respond to a filmed are made overly easy to understand, sometimes bordering

RQ XQZDQWHG VLPSOLÀFDWLRQ  :ROI ·V RZQ UHDGHU PD\
perhaps be left wanting more than he or she expected
version of a Socratic from the book, but the author’s theories and conclusions
are sound, strong and informative. Her ability to expertly
dialogue, to his entry draw relevant facts and evidence from history, psychology,
developmental science and cognitive neuroscience makes
in Wikipedia or her book particularly fascinating. If you’re done reading
this article and wonder how it is that you did it, Proust
to a screen clip on and the Squid will surely provide the answer.

YouTube?”
they pass through these stages, ther brains adjust their
own processing areas to make reading faster, easier, and
PRUHHIIHFWLYH7KHVHFKDSWHUVDUHÀOOHGZLWKLQWHUHVWLQJ
UHVHDUFKÀQGLQJVIURPODEVDURXQGWKHZRUOGDQGSHUVRQDO
memories from the author herself, which may evoke
recollections of each reader’s own struggles with learning
to read at an early age. By this point, however, Wolf ’s
style of writing tends to get slow and repetitive. After a
while, the reader might get fed up of hearing words like
automaticity, logosyllabary, and morphophonemic get
H[SODLQHGDQGUHGHÀQHGUHSHWLWLRXVO\

T KHÀQDOFKDSWHUVSXWWKHVWXG\RI WKHUHDGLQJEUDLQ
into perspective by introducing us to an exception
to the rule: the dyslexic brain. To Wolf, there is no single
“dyslexic brain,” but instead a range of differently-wired
brains that, for a variety of reasons, cannot learn to
read the way most people do. The theories attempting
to account for this are numerous and complex, but
they are presented in a manner that is concrete and
easy to understand. Wolf also draws from her personal
by Justine Chow, THURJ Staff
experience with her dyslexic son, whom she reveals as
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
30
 Manuscripts

Targeting TRAF Proteins as an Anti-tumor

Strategy
YI CAI AND JILL SUTTLES
CELL BIOLOGY

University of Louisville, Department of Microbiology and Immunology, 319 Abraham Flexner Way,
Louisville, KY 40292
Secondary tumors resulting from metastasis are major causes of death associated with human can-
cer. To proliferate and metastasize, cancer cells express many secreted and membrane-bound pro-
teins. Better understanding of mechanisms involved in the production of these proteins will be useful
for developing treatment strategies in cancer therapy. Many studies have shown that interaction of
CD40, a member of the tumor necrosis factor (TNF) receptor superfamily, and its ligand, CD154,
plays an important role in tumor proliferation and metastasis and this signaling interaction is medi-
ated by TNF receptor associated factors (TRAFs). This study examined the roles of TRAFs in pro-
liferation and metastasis of Lewis lung carcinoma (3LLC) cells, as well as 3LLC production of the
monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases-9 (MMP-9), and vascular
endothelial growth factor (VEGF), which plays important roles in tumor proliferation and metastasis.
Flow cytometric analysis revealed that 3LLC express both CD40 and CD154, which makes 3LLC
a good model to study CD40-CD154 interaction in cancer cells. With cell-permeable TRAF bind-
ing peptides (TRAFBPs) that inhibit the interaction between CD40 and TRAF, we demonstrate that
TRAFBPs can act as effective inhibitors of 3LLC proliferation as well as MCP-1, MMP-9, and VEGF
production. Thus, the interaction of CD40 and CD154 is involved in the proliferation of 3LLC and
in the production of MCP-1, MMP-9, and VEGF. Therefore, TRAFs can affect multiple pathways
that contribute to cancer proliferation and metastasis and may be good targets for cancer therapy.
____________________________________________________

INTRODUCTION is associated with more blood vessels to the tumor, metastasis,

chemoresistance, and poorer prognoses. VEGF binds to
Cancer is a class of diseases characterized by uncontrolled VEGF receptor (VEGFR) on the endothelium of blood vessels,
proliferation and spreading of abnormal cells. Normal cells are stimulating endothelial cell proliferation and migration into the
under strict control of proliferation and are eliminated through tumor. It can also recruit endothelial progenitor cells from the
WKH SURFHVV RI  DSRSWRVLV LI  GDPDJHG RU DV D UHVXOW RI  VSHFLÀF bone marrow for endothelial vessel formation. The new blood
growth regulatory signals. However, cancer cells can avoid vessels also provide the tumor with a route by which tumor cells
apoptosis and continue to proliferate without control. Severity can enter into the body’s circulatory system.1-3
of cancer symptoms depends on the affected site and the ability Matrix metalloproteinases (MMPs) are a group of extracellular
of cancers to invade other tissues, either by direct growth into proteinases involved in degrading basal membrane and extracellular
adjacent tissue through invasion or by implantation into distant PDWUL[(&0SURWHLQVVXFKDVJHODWLQFROODJHQÀEURQHFWLQDQG
sites through metastasis. The secondary tumors resulting from laminin. They play an important role in invasion and metastasis
metastasis are major causes of mortality associated with human by degrading ECM in tissue surrounding primary tumors and in
cancer. To grow, migrate, and avoid the attack of the immune the new organ into which they migrate. In addition, MMPs are
system, cancer cells express a wide variety of both secreted and also involved in regulating cell behavior by cleaving non-matrix
membrane-bound proteins such as growth factors, proteases, proteins, thus playing a role in tumor angiogenesis, immune
immunosuppressive proteins, cytokines, chemokines, and evasion, growth, and progression.4 Among the human MMPs,
cytokine/chemokine receptors. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are expressed
As primary tumors (original tumors) and secondary tumors abundantly in various malignant tumors. They are considered key
grow beyond 2-3 mm3, they need to go through angiogenesis, a enzymes for tumor invasion and metastasis.
process to develop a blood supply, to support their nutrient and The chemokines represent about 50 small chemotactic proteins.
oxygen needs.1 They achieve this by secreting angiogenic growth They interact with their receptors, which are G protein-coupled
factors such as vascular endothelial growth factor (VEGF), which receptors with seven transmembrane domains.5 Chemokines
31 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

FIGURE 1. TRAF binding sites on
CD40. Binding of CD154 triggers
trimerization of CD40, which in-
FUHDVHVWKHELQGLQJDI¾QLW\WR75$)V
Sequences of the binding sites on
murine CD40 and the corresponding
TRAFs are shown
can recruit nearly all classes of
leukocytes, which would be expected
to be detrimental to the tumor and
Manuscripts
nodal or systemic metastasis at the time of diagnosis, while the
four CD40 negative cancer cell lines were stage I tumors, without
any evidence of regional or distant metastasis. These results
suggest that CD40 expression on lung cancer may play a role in
metastasis.
In some cases, both CD40 and CD154 are expressed on cancer
cells. The autocrine interaction between this receptor:ligand pair
has been shown to decrease drug-induced apoptosis on CD40-
expressing non-Hodgkin’s lymphoma and breast carcinoma cell

CELL BIOLOGY
EHQHÀFLDO WR WKH KRVW  +RZHYHU
lines.14 This effect was abrogated by anti-CD154 antibody, indicating
surprisingly, most invasive cancers
CD154 was involved in the protective effect. Coexpression of CD40
produce the broadest spectrum and
and CD154 was found in breast tumor biopsies.15 Coexpression of
the highest levels of chemokines.
CD40 and CD154 in the immobilized epithelial cell line increased
Therefore, the up-regulation of
proliferation, mobility, and invasion in vitro. Interestingly, transient
chemokines and their receptors may
stimulation of the parent cell line with recombinant soluble
EHEHQHÀFLDOIRUFDQFHUFHOOVWKURXJK
CD154 did not produce these changes. Therefore, CD40 needs
directing autocrine growth factors for
constitutive engagement to mediate these changes.15 Accumulating
cancer cells, providing paracrine growth advantages via angiogenesis, evidence suggests that high-dose transient stimulation of CD40
enhancing invasion, enhancing metastasis by vessel entry, determining may be favorable for apoptosis while constitutive engagement
the location of secondary tumors, and inducing immune evasion for might favor proliferation.8
VSHFLÀFFDQFHUV6 Selected chemokine receptors, including CXCR4, The mechanisms of CD40-CD154 signaling have not been fully
CCR7, and CCR10, are often up-regulated in a large number of characterized. CD40 does not encode a catalytic domain in its
common human cancers. They may potentially facilitate tumor cytoplasmic sequence. Upon ligation with CD154, CD40 forms
dissemination at several key steps of metastasis, including adherence a trimer and binds to TNF receptor-associated factors (TRAFs).
of tumor cells to the vessel endothelium, the passing of cancer TRAFs are a family of genetically conserved adaptor proteins and
cells through blood vessels, metastatic colonization, angiogenesis, VL[ 75$)V KDYH EHHQ LGHQWLÀHG LQ PDPPDOV18 Different TRAFs
proliferation, and activation of key survival pathways.7 have different biological functions17, but all share a common
Cluster of differentiation 40 (CD40), a member of the tumor stretch of amino acids at the C terminal TRAF domain. The TRAF
necrosis factor receptor superfamily (TNFRSF), is expressed at domain has been divided into TRAF-C and TRAF-N subregions.
uniformly high levels in carcinomas of the nasopharynx, bladder, The C terminal TRAF-C region mediates both homo- and hetero-
cervix, and ovary and at more variable levels in carcinomas of the dimerization of TRAF proteins and interacts with receptors that
breast, skin, and lung.8 The natural ligand of CD40 is CD154. It recruit them. At the N terminus TRAFs, with the exception of
is a member of the TNF superfamily (TNFSF), which exists 75$)FRQWDLQ5,1*ÀQJHUDQG]LQFÀQJHUGRPDLQVZKLFKDUH
both in a transmembrane-bound form and a soluble form. High- important for downstream signaling events. Among the six TRAFs,
levels of transient CD40 activation has been shown to inhibit cell TRAF4 is a predominantly nuclear protein and does not bind to
proliferation and/or increase apoptosis on human bladder, ovarian, cell surface receptors. The other TRAFs bind with the cytoplasmic
cervical, squamous cell, lung, and breast carcinomas.9 Ligation of domain of CD40 at two sites, a distal site for TRAF1/2/3/5 and a
CD40 on tumor cells can enhance immune responses and result in proximal site for TRAF6 (Figure 1). TRAF6 is not only involved
anti-tumor activity independent of host CD40 expression.10 CD40 in TNFR signaling, but also serves as an adaptor protein for the
ligation has also been shown to stimulate host anti-tumor immune IL-1/Toll-like receptor (TLR) family.17,18 Many of the biological
response by activating dendritic cells to enhance the antigen- effects of TRAF (also IL-1/TLR by TRAF6) signaling appear to
presenting capability and by increasing immunostimulatory cytokine EHPHGLDWHGE\WKHDFWLYDWLRQRI QXFOHDUIDFWRUNDSSD%1)ƪ%
production.8,9 Treatments based on the growth- and immune- extracellular signal-regulated kinase (ERK)1/2, and the activator
regulatory features of CD40-CD154 interaction in normal cells have protein-1 (AP-1) family that leads to induction of cytokines,
been explored to promote corresponding direct and indirect tumor chemokines, proliferation, and rescue from apoptosis. However,
growth-inhibitory outcomes. These treatments used agonistic anti- GHWDLOVRI WKHVLJQDOLQJSDWKZD\VKDYHQRWEHHQZHOOGHÀQHG
CD40 monoclonal antibodies, recombinant CD154, or CD154 gene Activation and blockade of CD40 have both been tested as a
WUDQVIHUVWRVWLPXODWH&'3RVVLEOHULVNRI V\VWHPLFLQÁDPPDWLRQ means of modulating tumor behavior. Inhibition of TRAF signaling
and autoimmune consequences of these treatments is a concern and might be an effective way to inhibit cancer cell proliferation,
the success of clinical trials is limited.9,11 because it may be able to block CD40 signaling as well as other
On the other hand, CD40 ligation may also increase proliferation pathways, such as interleukin-1 (IL-1)/TLR, that might favor cancer
in many tumors, such as acute myelomonocytic leukemia, low-grade proliferation. One way to inhibit CD40 signaling is to use TRAF
B cell malignancies, Burkitt’s lymphoma, HIV-related lymphoma, binding peptides (TRAFBPs) that have similar sequences to the
and Hodgkin’s diseases.9,12 In one study, 18 human lung cancer cell TRAF binding sites on CD40. The presence of such peptides will
OLQHVZHUHHYDOXDWHGXVLQJÁRZF\WRPHWU\13 Thirteen of fourteen compete with CD40 to bind with TRAF, and thus disrupt CD40
cancer cell lines that expressed CD40 on their surface had either
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
32
 Manuscripts
signaling by dramatically decreasing the interaction between CD40
and TRAF. To facilitate the permeability of the peptides to cells, a
signal sequence is added to the peptides. It has been shown that the
TRAF6 binding peptide was an effective inhibitor of CD40 induced
LQÁDPPDWRU\ F\WRNLQH P51$ V\QWKHVLV DQG SURWHLQ SURGXFWLRQ
in human monocytes and macrophages.19 This project studied the
effects of TRAFBPs on proliferation of the Lewis lung carcinoma
(3LLC) cell line, which expresses both CD40 and CD154.
CELL BIOLOGY
MATERIALS AND METHODS
Reagents and antibodies. Phycoerythrin conjugated anti- activated cell sorter (FACS) (Becton Dickinson, San Jose, CA).
%LRVFLHQFHV$IWHUWKHÀOWHUVZHUHGULHGRYHUQLJKW—/RI /6&
cocktail was added to each well and the amount of radioactivity in
each well was then counted on a TopCount scintillation counter
(Packard Biosciences).
Flow cytometric analysis. 3LLC cells were scraped and
treated with Fc Block (anti-mouse CD16/CD32 monoclonal
antibody) for 15 minutes at room temperature and then labeled with
both PE-conjugated anti-mouse CD40 and FITC-conjugated anti-
mouse CD154 antibody in Dulbecco’s phosphate buffered saline
with 5% FBS and 0.01% azide for an hour at 4 ºC. Afterwards,
WKHFHOOVZHUHZDVKHGDQGDQDO\]HGE\D9DQWDJH6(ÁXRUHVFHQFH
PRXVH &' DQWLERG\ DQG ÁXRUHVFHLQ LVRWKLRF\DQDWH ),7& Production of MCP-1, MMP-9 and VEGF by 3LLC cells.
conjugated anti-mouse CD154 antibody were obtained from The production of these proteins was measured using enzyme-
Beckman and Coulter (Fullerton, CA). Anti-mouse CD16/CD32 linked immunosorbent assay (ELISA). Cells were plated in 96-
monoclonal antibody was purchased from BD Pharmingen (San well microtiter plates in triplicates at 1"104 cells/well with none,
'LHJR &$ DQG ORZ HQGRWR[LQ D]LGHIUHH SXULÀHG DQWL&'   RU  Ƭ0 RI  75$)%3 RU 75$)%3  $IWHU 
antibody was purchased from BioLegend (San Diego, CA). Methyl- hour’s incubation, supernatants were harvested. Murine MMP-9
tritiated-thymidine (3H-thymidine, 37 MBq/ml, 1 mCi/ml) used and VEGF were assayed with ELISA kits from R&D Systems
for proliferation assays was obtained from Amersham Biosciences (Minneapolis, MN) and murine MCP-1 was assayed with an
(Piscataway, NJ). The liquid scintillation counting (LSC) cocktail ELISA kit from BD Pharmingen. Absorption of the samples
was acquired from Packard Biosciences (Billerica, MA). Cell was measured using an E-max Precision microplate reader from
culture medium RPMI 1640 and fetal bovine serum (FBS) were Molecular Devices (Sunnyvale, CA).
obtained from HyClone (Logan, Utah) and Atlanta Biologicals Statistical analysis. Data is presented as mean ± standard
(Lawrenceville, GA), respectively. GHYLDWLRQ6'HUURUEDUVLQÀJXUHVIRUDOODQDO\VLV$RQHZD\WZR
TRAF2,3,5BP and TRAF6BP: TRAF2,3,5BP and TRAF6BP sample t-test was used to compare treatments with the controls and
were purchased from Invitrogen (Carlsbad, CA). The sequence of SYDOXHVOHVVWKDQZHUHFRQVLGHUHGVWDWLVWLFDOO\VLJQLÀFDQW
murine and human TRAF2,3,5BP is AAVALLPAVLLALLAPN-
TAAPVQETLHGCQPV and the sequence of murine TRAF6BP RESULTS
is AAVALLPAVLLALLAPPAARRQDPQEMEDYPG. A signal Coexpression of CD40 and CD154 on 3LLC cells. To
VHTXHQFH LQ EOXH RI  .DSRVL·V ÀEUREODVW JURZWK IDFWRU .)*) ÀQGFHOOOLQHVVXLWDEOHIRUWKLVVWXG\ZHH[DPLQHGH[SUHVVLRQRI 
was added to the TRAFBPs to allow for cell permeability. The CD40 and CD154 on both murine and human lung carcinoma cell
additional amino acids represent a portion of the murine CD40 OLQHV  %RWK &' DQG &' ZHUH LGHQWLÀHG RQ WKH //& FHOO
protein that includes the TRAF binding sequences (shown as bold OLQH9LVLEOHVKLIWVLQÁXRUHVFHQFHZHUHGHWHFWHGDIWHUWKH//&
portions in red). cells were dual-stained for CD40 and CD154 (Figure 2). The
Cell culture. 3LLC cells were obtained from American Type 3LLC line has served as an important tumor model for metastatic
Culture Collection (ATCC) (Manassas, VA). They were cultured in and angiogenic studies.20 Our study focused on the 3LLC cell line
100 mm tissue culture dishes from BD Labware (Franklin Lakes, because the coexpression of CD40 and CD154 provided a good
NJ) in RPMI 1640 with 5% FBS, 0.01 M HEPES, and 50 !g/ml model for studying the autocrine interaction of CD40-CD154.
JHQWDPLFLQUHIHUUHGWRDV57KH//&FHOOVJUHZLQDKXPLGLÀHG
ž&LQFXEDWRUZLWK  &2 XQWLO FHOO FRQÁXHQF\ UHDFKHG 
Afterwards, they were removed using cell scrapers. The 3LLC cells
were plated in 96-well plates at either 5"103 cells/well for proliferation
assays or 1"104 cells/well for monocyte chemoattractant protein-1
(MCP-1), MMP-9, and VEGF experiments.
Proliferation assays. The 3LLC cells were cultured in R5
and plated in a 96-well plate in triplicates at 5"103 cells/well and
allowed 0.5 hours to adhere. The 3LLC cells were either untreated
RUWUHDWHGZLWKDQWL&'DQWLERG\DWƬJPODQGƬJPORU
with TRAF6BP or TRAF2,3,5BP at 100, 200, or 300 !M. The
plates were incubated at 37 ºC, 5% CO2 for either 1, 5, or 18 FIGURE 2. Surface coexpression of CD40 and CD154 on 3LLC
hours for the anti-CD154 antibody experiments or for 18 hours cells. The 3LLC cells were dual-stained for surface expression of
for the TRAFBP experiments. After incubation, 3H-thymidine &'OHIWSDQHODQG&'ULJKWSDQHODQGDQDO\]HGE\)$&6
was added at 1 !Ci/well. The plates were pulsed for 5 hours, and /LJKW OLQHV UHSUHVHQW EDFNJURXQG XQODEHOHG ¿XRUHVFHQFH ZKLOH
WKHQKDUYHVWHGRQWRÀOWHUVZLWKDÀOWHUPDWHKDUYHVWHUIURP3DFNDUG WKH GDUN OLQHV UHSUHVHQW WKH ¿XRUHVFHQFH LQWHQVLW\ RI WKH ODEHOHG
33 w w w . T H U R J . o r g T H U R J
cells.
V o l I I s s u e 1 S p r i n g 2 0 0 8

CD40-CD154 interaction contributes to 3LLC
proliferation. The CD40-CD154 interaction may lead to
proliferation or growth inhibition depending on cancer cell
types. In order to study the role of this interaction, 3LLC cells
were incubated with 0, 5, or 10 !g/mL of anti-CD154 antibody
for 1, 5, or 18 hours. The cell growth was then measured by
proliferation assays. The anti-CD154 antibody inhibited 3LLC
proliferation in a concentration-dependent manner (results from
1 hour incubation are shown in Figure 3), suggesting that the
autocrine interaction of CD154 and CD40 contributes to the
proliferation of 3LLC cells. At the 10 !g/mL level, anti-CD154
antibody decreased proliferation of the 3LLC cells by 36%, 24%,
and 9% respectively for 1, 5, and 18 hour incubation (5 and 18
hour data not shown). This weakening in inhibition over time may
EHFDXVHGE\WKHHQJXOÀQJRI WKHDQWLERG\E\WKH//&FHOOV7R
test this possibility, 3LLC cells were incubated with an anti-CD154
antibody for 3 hours and analyzed for CD154 surface expression,
DVFRPSDUHGZLWKIUHVKO\VWDLQHGFHOOV$FOHDUVKLIWLQÁXRUHVFHQFH
was detected from the freshly stained 3LLC cells, but there was no
Manuscripts
MCP-1 is one such chemokine that can attract macrophages which
facilitate tumor cell migration, invasion, and metastasis.21 Thus,
we examined the effect of TRAFBPs on MCP-1 production by
3LLC cells. The experimental design was similar to those of the
experiments examining the effect of TRAFBPs on cell proliferation
except that ELISA was used to measure MCP-1 production. High
levels of MCP-1 were detected from the untreated 3LLC cells,
EXW WKHVH OHYHOV ZHUH UHGXFHG VLJQLÀFDQWO\ LQ WKH SUHVHQFH RI  WKH
TRAFBPs in a dose-dependent manner (Figure 5). These results
CELL BIOLOGY
indicate that MCP-1 production by 3LLC cells can be inhibited by
the TRAFBPs.
TRAF6BP decreases production of MMP-9 by 3LLC
cells. Since MMP-9 is produced by many carcinomas and plays
an important role in cancer invasion, metastasis, and angiogenesis,
the role of TRAF6BP in MMP-9 production was studied. The
same approach as for the MCP-1 experiment was used, except
that MMP-9 was measured. Substantial levels of MMP-9 were
produced by 3LLC cells. Production decreased drastically by the
DGGLWLRQRI Ƭ0RI 75$)%3DQG003ZDVQRWGHWHFWDEOH
VLJQLÀFDQWVKLIWLQÁXRUHVFHQFHIRUWKH//&FHOOVVWDLQHGIRU ZKHQRUƬ075$)%3ZDVXVHG)LJXUH7KHUHIRUH
hours (data not shown). This indicates that there was not much TRAF6BP effectively inhibited production of MMP-9 by 3LLC.
anti-CD154 antibody left outside the cells, most likely because the TRAFBPs reduce VEGF production by 3LLC cells. VEGF
antibody was engulfed and destroyed by the 3LLC cells. is a potent pro-angiogenesis protein. Its importance in tumor
Proliferation of 3LLC is blocked by TRAFBPs. The growth has been well documented.1 The effect of TRAFBPs on
results of the previous experiment suggested a role of the CD40- VEGF production by 3LLC cells was examined. 3LLC cells were
CD154 interaction in 3LLC proliferation. However, treatment of incubated with or without
WKHFHOOVZLWKDQWL&'DQWLERG\ZDVRQO\PRGHUDWHO\HIÀFDFLRXV TRAFBPs and the VEGF in the cell supernatants was measured
in reducing proliferation. Hence, we considered the possibility that by ELISA (Figure 7).
use of TRAFBPs may be a better alternative to the anti-CD154 A similar reduction of the level of VEGF production was
antibody because they are taken up directly by cells and may be generated regardless of the concentration of TRAF2,3,5BP used,
less easily destroyed. Thus, we examined the effect of TRAF6BP suggesting that TRAFs2,3,5 may play a partial role in induction of
and TRAF2,3,5BP on 3LLC proliferation. The TRAFBPs contain VEGF production. However, production of VEGF was reduced
a KFGF signal sequence (to allow for cell permeability) and by TRAF6BP in a concentration-dependent manner and was more
sequences corresponding to those of the TRAF binding sites on effective than the TRAF2,3,5BP. This may be due to the ability of
CD40. Therefore, they can enter cancer cells and bind to TRAFs, TRAF6BP to inhibit multiple pathways or to inhibit a pathway(s)
preventing TRAFs from binding to CD40. The 3LLC cells were most critical to VEGF synthesis.
either untreated or treated with 100 !M, 200 !M, or 300 !M of
the TRAFBPs overnight and their proliferation was measured by DISCUSSION
proliferation assay. As shown in Figure 4, 3LLC cell proliferation
was inhibited by both TRAF2,3,5BP and TRAF6BP in a dose- Lung cancer is one of the most common and malignant cancers
dependent manner. The proliferation of the cells decreased by 90% notorious for its poor prognosis and high mortality, even with
and 99% with 300 !M of TRAF6BP and TRAF2,3,5, respectively. early detection. The deaths related to lung cancer are the most
TRAFBPs decrease MCP-1 production of 3LLC cells. common among cancer-related deaths for both men and women in
Cancer cells produce various cytokines and chemokines to recruit the United States.22 Better understanding of the causes of the high
leukocytes to help them to grow and invade surrounding tissues. proliferation and metastatic features of lung cancer will be useful
FIGURE 3. CD40-CD154 LQÀQGLQJWUHDWPHQWVIRUWKLVGLVHDVH
interaction contributes to The 3LLC tumor model is important for studies concerning
3LLC proliferation. 3LLC metastasis and angiogenesis because 3LLC is highly metastatic and
Control
cells were pretreated with can metastasize locally and distantly.20 Our experiments indicated
or without anti-CD154 Ab for that both CD40 and CD154 are expressed on 3LLC cells. Since
5 !g/mL
**  KRXU SXOVHG ZLWK +WK\-
Ab
midine for 5 hours, and as-
autocrine interaction of CD40 and CD154 has been reported to
VD\HGIRUUDGLRDFWLYHFRXQWV contribute to proliferation and metastasis, we examined its role
15
10 !g/mL
Ab ** Values and error bars are on proliferation and metastasis of 3LLC cells. The anti-CD154
0 0.5 1 1.5 2 means and SD of triplicate DQWLERG\ SURGXFHG VLJQLÀFDQW LQKLELWLRQ RI  //& SUROLIHUDWLRQ
CPM/1000 determinations (**: p < 0.01 but this effect decreased with the increase of initial incubation
E\WWHVW WLPH  5HVXOWV IURP WKH ÁXRUHVFHQW ODEHOHG DQWL&' DQWLERG\
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
34
 Manuscripts

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TRAF2BP *** TRAF6BP ** TRAF2BP * TRAF6BP **
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TRAF2BP *** TRAF6BP *** TRAF2BP ** TRAF6BP ***
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TRAF2BP *** TRAF6BP *** TRAF2BP *** TRAF6BP ***
0 5 10 15 20 0 5 10 15 20 0 1500 3000 4500 6000 0 400 800 1200 1600
CPM/1000 CPM/1000 MCP-1 (pg/ml) MCP-1 (pg/ml)
CELL BIOLOGY

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3LLC cells were pretreated with or without TRAF2,3,5BP or cells. 3LLC cells were pretreated with or without TRAF2,3,5BP or
75$)%3IRUKRXUVSXOVHGZLWK+WK\PLGLQHIRUKRXUVDQG TRAF6BP for 18 hours and MCP-1 in supernatant was measured
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t-test).
experiment indicated that most of the antibody was engulfed and
destroyed within 3 hours by 3LLC cells. The anti-proliferative
effects observed from 5 and 18 hour incubation experiments were
probably caused by the slow recovery from the inhibitory effects
originating from the antibody at the early stage of incubation.
The involvement of CD40-CD154 interaction in 3LLC
SUROLIHUDWLRQZDVIXUWKHUFRQÀUPHGE\WKH75$)%3H[SHULPHQWV
TRAFs are essential in the CD40-CD154 signal transduction
pathway. Their activity can be blocked by TRAFBPs, which have
the same sequences of TRAF binding sites on CD40, through
competitive binding with TRAFs. Both TRAFBPs were effective in
inhibiting 3LLC proliferation, especially the TRAF2,3,5BP, which
almost completely blocked proliferation. Thus, the TRAFBPs
are more effective than the anti-CD154 antibody in impeding
proliferation. Reasons for different effects of TRAF6BP and
TRAF2,3,5BP on 3LLC proliferation still need to be explored.
Cancer cells express a large variety of proteins to avoid attack by
the immune system and facilitate their proliferation and metastasis.
MCP-1, a strong attractor of macrophages, is one such protein
secreted by many cancer cells. Macrophages play an important role
membrane and ECM proteins, MMP-9 is also involved in cleavage
and activation of other proteins, such as VEGF. It was shown that
VEGF was expressed in non-angiogenic, angiogenic, and control
islets as well as tumor cells. MMP-9 doubled the release of VEGF
and induced angiogenesis, possibly by cleaving the ECM bound
164-amino-acid VEGF isoform to the freely soluble 120-amino-
acid VEGF isoform.28
Our results show that production of secreted VEGF was
inhibited by both TRAF2,3,5BP and TRAF6BP. The effect of
75$)%3OHYHOHGDWƬ0ZKLOHWKH75$)%3VHHPHGWR
be more effective at higher concentrations. The different effects
of TRAF2,3,5BP and TRAF6BP on VEGF secretion suggest that
different mechanisms might be involved in VEGF regulation. How
TRAF2,3,5BP and TRAF6BP produce different effects remains to
be investigated.
The effect of TRAFBPs was not due to cellular toxicity as
evidenced by the results of a trypan blue exclusion test and the
ÀQGLQJWKDWWKHV\QWKHVLVRI RWKHUSURWHLQVE\//&FHOOVLQWKH
presence of TRAFBPs was not affected (data not shown). For
H[DPSOH QR VLJQLÀFDQW GLIIHUHQFH LQ V\QWKHVLV RI  WXPRU JURZWK
in immuno-responses. They are known to have diverse activities IDFWRU7*)ƢZDVIRXQGEHWZHHQFRQWURODQG//&FHOOVWUHDWHG
depending on their microenvironment.23 A tumor’s ability to recruit with 300 !M TRAFBPs (which yielded 500 to 600 pg/mL of
macrophages and to create a microenvironment that promotes
PDFURSKDJHSURLQÁDPPDWRU\UHVSRQVHVFDQIDFLOLWDWHSUROLIHUDWLRQ
angiogenesis, and metastasis.21,24 CD40 and TRAF6 were shown to
be involved in MCP-1 production in human renal proximal tubule
epithelial cells.25 Our results indicated that both TRAF2,3,5 and
TRAF6 were involved in MCP-1 production in 3LLC cells. In
addition, the CD40-CD154 interaction also plays an important role
LQWKHSURLQÁDPPDWRU\UHVSRQVHIURPPDFURSKDJHV7KHUHIRUH
TRAFs may be good targets for cancer therapy.
Two additional important proteins to consider are MMP-9 and
VEGF, which facilitate tumor invasion and metastasis. MMP-9
is known to be up-regulated by the CD40 pathway in cervical
carcinoma cells.26 Also, TRAF2 and TRAF3 were reported to be
involved in induction of MMP-9 by latent membrane protein 1
(LMP-1) in nasopharyngeal carcinoma cells .27 In both cases, the
DFWLYDWLRQRI ERWKWKH1)ƪ%DQG$3SDWKZD\VZHUHQHFHVVDU\
Our results show that production of MMP-9 was inhibited by
TRAF6BP. A role of TRAF2,3,5 in MMP-9 production by 3LLC
35 is currently under investigation. In addition to digestion of basal
w w w . T H U R J . o r g T H U R J V
7*)ƢGDWDQRWVKRZQ
+RZHYHUWKH&'&'LQWHUDFWLRQLVQRWVSHFLÀFWRWXPRU
FHOOV7KXVLI 75$)%3VZHUHWRDFWLQDQRQVSHFLÀFZD\GDPDJH
to other tissues in a treated organism may result. We therefore
propose to study the effects of TRAFBPs in vivo with mice by
injecting TRAFBPs directly into the tumors so that the peptides
only interact with tumor cells. Also, the in vivo study may also reveal
WKH FRQFHQWUDWLRQ RI  75$)%3V QHFHVVDU\ WR DFKLHYH VLJQLÀFDQW
inhibition of cancer cell proliferation and metastasis. Finally, future
in vivo studies can also show whether TRAFBP treatment would
result in negative side effects.
In summary, both CD40 and CD154 are expressed on 3LLC
cells. Our data suggest that the autocrine interaction of CD40-
CD154 is involved in the proliferation of 3LLC and secretion
of MCP-1, MMP-9, and VEGF (Figure 8, left panel) that play
important roles in tumor proliferation, invasion, angiogenesis,
and metastasis. TRAFBPs can act as decoys to competitively bind
to TRAFs (Figure 8, right panel), thus preventing the binding of
TRAFs with CD40 and inhibiting proliferation and metastasis of
o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

FIGURE 6. TRAF6BP de-

Control
creases MMP-9 production Control Control
ȝ0 E\ //& FHOOV  //& FHOOV
TRAF6BP *** were pretreated with or with- ȝ0 ȝ0
TRAF2BP ** TRAF6BP ***
ȝ0 out TRAF6BP for 18 hours
TRAF6BP *** ȝ0 ȝ0
and MMP-9 in supernatant TRAF2BP *** TRAF6BP ***
ȝ0
TRAF6BP *** ZDV PHDVXUHG E\ (/,6$ ȝ0 ȝ0
Values and error bars are TRAF2BP *** TRAF6BP ***
0 150 300 450 600 750
means and SD of triplicate 0 50 100 150 0 40 80 120 160
MMP-9 (pg/ml)

CELL BIOLOGY
determinations (***: p < VEGF (pg/ml) VEGF (pg/ml)
E\WWHVW
3LLC cells. In addition, TRAF6BP may also block other pathways,
such as the IL-1/TLR pathway, that contribute to proliferation
and metastasis. Therefore, TRAFs can affect multiple pathways
that contribute to cancer growth and metastasis and may be good
SDQG
FIGURE 7. 75$)%3V UHGXFH 9(*) SURGXFWLRQ E\ //& FHOOV
3LLC cells were pretreated with or without TRAF2,3,5BP or
75$)%3IRUKRXUVDQG9(*)LQVXSHUQDWDQWVZDVPHDVXUHG
E\(/,6$9DOXHVDQGHUURUEDUVDUHPHDQVDQG6'RIWULSOLFDWH
GHWHUPLQDWLRQV
SE\WWHVW
targets for cancer therapy. Our data indicate that use of TRAFBPs FIGURE 8 (IIHFWV
may be an effective method for the inhibition of TRAF activity of TRAFBPs on
in tumor cells. Future in vivo studies will further reveal whether autocrine interaction
TRAFBP treatment can be used in anti-tumor therapy. of CD40-CD154.
Left panel: CD40-
REFERENCES CD154 interaction
f a c i l i t a t e s
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vival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med metastasis and
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TRAFs are required
2. McColl BK, Stacker SA, Achen MG. 2004. Molecular regulation of the VEGF
family – inducers of angiogenesis and lymphangiogenesis. APMIS 112(7-8):463- LQWKHSDWKZD\5LJKW
80. panel: TRAFBPs
3. Shibuya M, Claesson-Welsh L. 2006. Signal transduction by VEGF receptors in (in green) block
regulation of angiogenesis and lymphangiogenesis. Exp Cell Res 312(5):549-60. &' SDWKZD\ E\
4. Yoon SO, Park SJ, Yun CH, Chung AS. 2003. Roles of matrix metalloproteinases SUHYHQWLQJ 75$) WR
in tumor metastasis and angiogenesis. J Biochem Mol Biol 36(1):128-37. bind with CD40.
5. =ORWQLN$<RVKLH2&KHPRNLQHVDQHZFODVVLÀFDWLRQV\VWHPDQGWKHLU J Cell Sci 115(Pt 4):679-88.
role in immunity. Immunity 12(2):121-7. 18. Kobayashi T, Walsh MC, Choi Y. 2004. The role of TRAF6 in signal transduction
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genesis and treatment of cancer. Curr Opin Pharmacol 4(4):360-7. cent protein model. Clin Exp Metastasis 18(1):57-60.
9. Tong AW, Stone MJ.2003. Prospects for CD40-directed experimental therapy of 21. Condeelis J, Pollard JW. 2006. Macrophages: obligate partners for tumor cell mi-
human cancer. Cancer Gene Ther 10(1):1-13. gration, invasion, and metastasis. Cell 124(2):263-6.
10. Shorts L, Weiss JM, Lee JK, Welniak LA, Subleski J, Back T, Murphy WJ, Wil- 22. American Cancer Society. Cancer Facts and Figures 2006. Atlanta: American Can-
trout RH. 2006. Stimulation through CD40 on mouse and human renal cell FHU6RFLHW\KWWSZZZFDQFHURUJGRFURRW677VWWBDVS
carcinomas triggers cytokine production, leukocyte recruitment, and antitu- 23. Stout RD, Jiang C, Matta B, Tietzel I, Watkins SK, Suttles J. 2005. Macrophages
mor responses that can be independent of host CD40 expression. J Immunol sequentially change their functional phenotype in response to changes in mi-
176(11):6543-52. FURHQYLURQPHQWDOLQÁXHQFHV-,PPXQRO
11. Geldart T, Illidge T. 2005. Anti-CD 40 monoclonal antibody. Leuk Lymphoma 24. Pollard JW. 2004. Tumour-educated macrophages promote tumour progression
46(8):1105-13. and metastasis. Nat Rev Cancer 4(1):71-8.
12. Costello RT, Gastaut JA, Olive D. 1999. What is the real role of CD40 in cancer 25. Li H, Nord EP. 2002. CD40 ligation stimulates MCP-1 and IL-8 production,
immunotherapy? Immunol Today 20(11):488-93. TRAF6 recruitment, and MAPK activation in proximal tubule cells. Am J Physiol
13. Sabel MS, Yamada M, Kawaguchi Y, Chen FA, Takita H, Bankert RB. 2000. Renal Physiol 282(6):F1020-33.
CD40 expression on human lung cancer correlates with metastatic spread. Can- 26. 6PROD+HVV66FKQLW]OHU5+DGDVFKLN'6PROD+0DXFK&.ULHJ73ÀVWHU
cer Immunol Immunother 49(2):101-8. H. 2001. CD40L induces matrix-metalloproteinase-9 but not tissue inhibitor of
14. Voorzanger-Rousselot N, Blay JY. 2004. Coexpression of CD40 and CD40L on metalloproteinases-1 in cervical carcinoma cells: imbalance between NF-kappaB
B lymphoma and carcinoma cells: an autocrine anti-apoptotic role. Leuk Lym- and STAT3 activation. Exp Cell Res 267(2):205-15.
phoma 45(6):1239-45. 27. Takeshita H, Yoshizaki T, Miller WE, Sato H, Furukawa M, Pagano JS, Raab-
15. Baxendale AJ, Dawson CW, Stewart SE, Mudaliar V, Reynolds G, Gordon J, Traub N. 1999. Matrix metalloproteinase 9 expression is induced by Epstein-Barr
Murray PG, Young LS, Eliopoulos AG. 2005. Constitutive activation of the virus latent membrane protein 1 C-terminal activation regions 1 and 2. J Virol
CD40 pathway promotes cell transformation and neoplastic growth. Oncogene 73(7):5548-55.
24(53):7913-23. 28. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K,
16. Arch RH, Gedrich RW, Thompson CB. 1998. Tumor necrosis factor receptor- Thorpe P, Itohara S, Werb Z, Hanahan D. 2000. Matrix metalloproteinase-9 trig-
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death. Genes Dev 12(18):2821-30.
17. Chung JY, Park YC, Ye H, Wu H. 2002. All TRAFs are not created equal: com-
mon and distinct molecular mechanisms of TRAF-mediated signal transduction.
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
36
Manuscripts

Thrombospondin-1 switches nitric oxide

function from inhibitory to stimulatory for gene
expression of human endothelial tumor markers
CELL BIOLOGY

DAYAN LI1,2, JEFFREY S. ISENBERG1, DAVID D. ROBERTS1

1
Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD 20892; and 2Harvard University,
Cambridge, MA 02138
Angiogenesis, the growth of new blood capillaries from existing vessels, is essential for cancer progres-
sion. Selective inhibition of such growth has tremendous therapeutic potential. Thrombospondin-1 (TSP1)
is one of the most potent angiogenesis inhibitors and interacts with nitric oxide (NO), which can be both
inhibitory and stimulatory for blood vessel formation. Several tumor marker genes, namely four matrix
JHQHVFROODJHQV,ơ,ơ,,,ơDQG7(0DQGWKUHHEDVHPHQWPHPEUDQHSURWHLQJHQHVFROODJHQ,9ơ
ODPLQLQơDQGODPLQLQơDUHUHJXODWHGE\76312FURVVWDON,QWKLVVWXG\TXDQWLWDWLYH573&5ZLWK
KLJKVSHFLÀFLW\VHQVLWLYLW\DFFXUDF\DQGSUHFLVLRQZDVGHYHORSHGWRVLPXOWDQHRXVO\PRQLWRUP51$WUDQ-
VFULSWLRQRI WKHVHJHQHVLQKXPDQXPELOLFDOYHLQHQGRWKHOLDOFHOOV+89(&V&RPSDUHGWRWKHEDVHOLQH
KRXVHNHHSLQJJHQHK\SR[DQWKLQHJXDQWLQHSKRVSKRULERV\OWUDQVIHUDVH+357DOOIRXUPDWUL[JHQHVFRO-
ODJHQV,ơ,ơ,,,ơ7(0DQGRQHEDVHPHQWPHPEUDQHSURWHLQJHQHODPLQLQơZHUHIROG
XQGHUH[SUHVVHGZKLOHWKHRWKHUWZREDVHPHQWPHPEUDQHJHQHVODPLQLQơDQGFROODJHQ,9ơZHUH
DQGIROGRYHUH[SUHVVHGUHVSHFWLYHO\7UDQVFULSWLRQP51$OHYHOVRI DOOWKHWXPRUPDUNHUJHQHVZHUH
WRGRZQUHJXODWHGE\H[SRVXUHWRH[RJHQRXV763Q0DQGWRGRZQUHJXODWHGE\H[SR-
VXUHWR12GRQRU'(7$12Ƭ0:KHQ+89(&VZHUHWUHDWHGZLWKERWK763DQG12763OLNHO\
VZLWFKHG 12 IXQFWLRQ IURP LQKLELWRU\ WR VWLPXODWRU\ DV P51$ H[SUHVVLRQ RI  WKH JHQHV UHERXQGHG WR
FRQWUROOHYHOV7RRXUNQRZOHGJHWKLVLVWKHÀUVWUHSRUWRI D763LQGXFHG12IXQFWLRQDOVZLWFKLQKXPDQ
endothelial cells. Such a discovery will be useful in understanding the TSP1-NO crosstalk during angio-
JHQHVLVUHJXODWLRQDQGLQÀQGLQJFRQYHUJHQFHSRLQWVEHWZHHQSURDQJLRJHQLFDQGDQWLDQJLRJHQLFVLJQDO-
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about their potential to be regulated in matrix synthesis, cancer progression, and cancer treatments.
____________________________________________________

INTRODUCTION
Angiogenesis, the growth of new blood capillaries from
existing vessels, contributes to tumor growth and metastasis1
through the degradation of extra-cellular matrices, endothelial
cell adhesion, migration, proliferation, and re-differentiation into
patent vessels.2 It is regulated by signals received from soluble
growth factors and components of the extra-cellular matrix.3
WXPRU UHVSRQVH WR RWKHU 763 FRQVWUXFWV KDV EHHQ UHSRUWHG10
Nitric oxide (NO) is also an important modulator of endothelial
angiogenesis,11 acting via a guanylate cyclase-dependent pathway
and extracellular signal-regulated kinase (ERK) phosphorylation.
Depending on the redox environment, NO concentration, and
duration of NO exposure, cancer cells have a biphasic response
to NO.122XUSUHYLRXVVWXGLHVKDYHLGHQWLÀHGGLVFUHWHWKUHVKROG
concentrations of NO required for stabilizing signaling proteins
7KURPERVSRQGLQV D IDPLO\ RI  ÀYH H[WUDFHOOXODU JO\SURWHLQV LPSRUWDQWLQWXPRUELRORJ\/RZFRQFHQWUDWLRQVƬ0RI 12
secreted in matrices of various tissues,4 have diverse effects induce an angiogenic response, while high concentrations (1000
on cell adhesion, motility, proliferation, and survival.5 Among
WKH ÀYH 763 LV D SRWHQW LQKLELWRU RI  DQJLRJHQHVLV WKDW SOD\V
an important role in limiting neovascularization of tumors in
transgenic mice.7 Antiangiogenic fragments and peptides de-
ULYHGIURP763FDQLQKLELWDQJLRJHQHVLVDQGWXPRUJURZWKLQ
animals.8$WKLJKFRQFHQWUDWLRQV763LQKLELWVSUROLIHUDWLRQRI 
both endothelial and vascular smooth cells.9 However, adverse
37 w w w . T H U R J . o r g T H U R J V o l
Ƭ0 DUH LQKLELWRU\13 In this catalytic mechanism, NO regulates
DQJLRJHQHVLVWKURXJKDIXQFWLRQDOVZLWFKLQYROYLQJ76312
6HYHUDOVWXGLHVKDYHLQGLFDWHGWKDW12VLJQDOLQJUHJXODWHV763
activity.12, 14, 15 +RZHYHU LW LV OHVV FOHDU ZKHWKHU 763 FDQ LWVHOI 
UHJXODWH12VLJQDOLQJLQHQGRWKHOLDOFHOOV763H[SUHVVLRQLQHQ-
dothelial cells is reduced by hypoxia or inhibition of NO synthe-
sis.16&RQYHUVHO\763LQKLELWHGHQGRWKHOLDOFHOOUHVSRQVHVWR12
I I s s u e 1 S p r i n g 2 0 0 8

YLDDF*03GHSHQGHQWPDQQHU13 Recombinant type 1 repeats of
763DQG&'ELQGLQJSHSWLGHVZHUHVXIÀFLHQWZKLOH&'ZDV
necessary to inhibit NO-stimulated responses in both endothelial
and vascular smooth muscle cells.14 The antiangiogenic function
RI 763DQGLWVLQWHUDFWLRQZLWK12DUHGHSHQGHQWRQQRWRQO\
various cell surface receptors but also metalloproteinases secreted
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   6HYHUDO LQWHJULQ UHFHSWRUV ZHUH UHSRUWHG WR PHGLDWH 763
HIIHFWV RQ HQGRWKHOLDO FHOOV 2I  WKHVH FROODJHQV W\SH ,ơ  ơ
W\SH ,,,ơ DQG W\SH ,9ơ DUH WXPRU HQGRWKHOLDO PDUNHUV 7\SH
I collagen is a molecular target for inhibition of angiogenesis
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Manuscripts
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research is to spawn more sweeping therapies against cancer, data
on human cells is crucial in catapulting the research towards bio-
logically viable drugs. Furthermore, a more comprehensive study
of all the matrix protein and basal lamina genes together would
SURYLGHYDOXDEOHLQVLJKWLQWRWKH76312FURVVWDON·VUHJXODWLRQ
of the tumor markers as a whole. Therefore, to study the com-
CELL BIOLOGY
SOH[LW\LQFHOOUHVSRQVHVWRWKHFURVVWDONEHWZHHQ763DQG12
GXULQJDQJLRJHQHVLVZHXVHGT573&5WRVLPXOWDQHRXVO\TXDQ-
tify gene expression of tumor markers and major basement mem-
brane proteins in human endothelial cells. Our results showed
WKDW IRXU WXPRU HQGRWKHOLDO PDUNHUV FROODJHQV ,ơ ,ơ ,,,ơ
DQG ,,,ơ ZHUH GRZQUHJXODWHG  E\ WUHDWPHQW RI  763 DQG7(0DQGWKUHHEDVDOODPLQDJHQHVFROODJHQ,YơODPLQLQ
QXOO PLFH FHOOVZLWK ƬJP/ RI  H[RJHQRXV 763 IRU  KRXUV
1RWDEOHUHJXODWLRQRI FROODJHQJHQHH[SUHVVLRQE\763ZDVDOVR
reported in angiogenic endothelium isolated from human tumor
vasculature.18 However, studies on the collagen breakdown path-
way showed neither collagenase expression nor collagenase pro-
tein expression was affected by the NO donor s-nitroso-N-acetyl-
penicillamine.19
/DPLQLQVLQFRQMXQFWLRQZLWKFROODJHQ,9DUHPDMRUFRP-
SRQHQWVRI EDVHPHQWPHPEUDQHVRI EORRG YHVVHOV/DPLQLQVơ
 GUDPDWLFDOO\LPSDFWHQGRWKHOLDOFHOOEHKDYLRUE\LQWHUDFWLQJ
with the integrin receptors on the surface of endothelial cells.20
ơDQGODPLQLơZHUHGLIIHUHQWLDOO\H[SUHVVHGLQWKHSUHVHQFHRI 
H[RJHQRXV76312RUERWKWRJHWKHU7KHTXDQWLWDWLYHJHQHH[-
pression modules formulated throw light upon the genetic basis
RI DQJLRJHQHVLVDQGWKHFRPSOH[FHOOXODUUHVSRQVHVWR76312
interactions in addition to facilitating the screening of anti-an-
JLRJHQLFGUXJVIRUFDQFHUWUHDWPHQWV0RVWLPSRUWDQWO\ZHKDYH
IRXQG WKDW D ORZ FRQFHQWUDWLRQ RI  H[RJHQRXV763FDQVZLWFK
NO function from inhibitory to stimulatory for the gene expres-
VLRQRI WXPRUPDUNHUJHQHVLQKXPDQHQGRWKHOLDOFHOOV7KLVÀQG-
ing paves the way for the discovery of convergence points be-
tween pro-angiogenic and anti-angiogenic signaling.
+XPDQ XPELOLFDO YHLQ HQGRWKHOLDO FHOOV +89(&V PDLQWDLQHG
in vitro VHFUHWHG D ODPLQLQơ VXEXQLW * GRPDLQ21 7(0 RQH
RI QLQHWXPRUHQGRWKHOLDOPDUNHUV7(0VGLVFRYHUHGE\6$*( MATERIALS AND METHODS
analysis of human colon cancer,18 also plays a principal role in
PRGXODWLQJ763LQGXFHGDQJLRJHQHVLVWKURXJKDQH[WUDFHOOXODU Cells and Reagents. +89(&VDQGHQGRWKHOLDOJURZWKPHGLD
domain which preferentially binds to collagen.22 7(0 ZDV XS (*0 ZHUH SXUFKDVHG Irom Cambrex Biotechnology (Walkers-
regulated in angiogenic endothelial cells undergoing differentia- YLOOH 0' 'LHWK\OWULDPLQH 1212DWH '(7$12 ZDV SUR-
tion and tube formation on collagen.22 YLGHG E\ -RVHSK 6DDYHGUD DQG /DUU\ .HHIHU 1DWLRQDO &DQFHU
While previous research qualitatively studied such genes in
mice, a more quantitative analysis of gene expression is needed
TABLE 1. Target genes and their respective empirically-de-
signed primer pair sequences
,QVWLWXWH )UHGHULFN 7KURPERVSRQGLQ 763 ZDV REWDLQHG
from human platelets from the National Institutes of Health
EORRGEDQN3ULPHUV3ODWLQXP®6<%5®*UHHQ6XSHU0L[8'*
DQG6XSHU6FULSW70,,,)LUVW6WUDQG6\QWKHVLV6\VWHPIRU573&5
kits were purchased from Invitrogen (Carlsbad, CA).
Primer design. 3ULPHUSDLUVHTXHQFHVZHUHGHVLJQHGHPSLUL-
cally to standardize primer and amplicon sizes, GC content, and
7P VR WKDW VLPLODU 3&5 DPSOLÀFDWLRQ VHQVLWLYLW\ DQG HIÀFLHQF\
could be obtained. Each sequence was 20-21 base pairs long, with
DPSOLFRQVRI DERXWEDVHSDLUVD*&FRQWHQWRI DERXW
and a melting temperature of around 60°C (Table 1).
Cell culture. +89(&V SDVVDJHV  ZHUH JURZQ LQ (*0
supplemented with epidermal growth factor, bovine brain extract,
VWUHSWRP\FLQJHQWDP\FLQ)%6SHQLFLOOLQDQGFRUWLVRQHDQG
LQFXEDWHGDWƒ&ZLWK&23ULRUWRH[SHULPHQWDWLRQFHOOV
ZHUHZDVKHGZLWK3%6WU\SVLQL]HGDQGWKHQSODWHGDWDGHQVLW\
of one million cells per 100-mm tissue culture dish and main-
tained for 24 hours in additive-free endothelial basal medium with
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Treatments. +89(&V DW  FRQÁXHQFH ZHUH WUHDWHG RQ

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
38
 Manuscripts

C ollagen I α 1 C ollagen I α 2
CELL BIOLOGY

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FIGURE 1. Quantitative RT-PCR plots with melting curves for each FIGURE 2. Effects of TSP1/NO treatments on mRNA expression
of the studied genes. Curves with different colors represent differ- levels for each gene - different letters on bar graphs represent sta-
ent treatments (TSP1, NO, TSP1 + NO) and the three repetitions WLVWLFDOO\VLJQL¾FDQWFKDQJHVGDWD

FPFXOWXUHGLVKHVLQP/(%0ZLWKERYLQHVHUXPDOEX- WRWDOP51$ZLWK6XSHU6FULSW70,,,)LUVW6WUDQG6\QWKHVLV6\VWHP
PLQ%6$)RXUFRQGLWLRQVZHUHVHOHFWHG2QHEDWFKZDVWUHDWHG IRU573&5NLW2OLJRG720ZDVXVHGDVWKHSULPHU51$SULPHU
ZLWKQ0763DQRWKHUZLWKƬ0'(7$12DQRWKHUZLWK mixture incubated at 65°C for 5 minutes, then immediately placed
Q0763DQGƬ0'(7$12DQGWKHFRQWUROUHFHLYHGQR on ice for 1 minute. cDNA synthesis mix was then added. The
WUHDWPHQWV&HOOVZHUHLQFXEDWHGLQ&2DWƒ&,QFXEDWLRQ entire mixture was incubated at 50°C for 1 hour, 85°C for 5 min-
WLPHV ZHUH KUV DQG KUV +89(&V LQ P/ (%0 ZLWK 
%6$ZHUHWUHDWHGZLWKƬ0'(7$12DQGLQFXEDWHGLQ
CO2 at 37°C for 0hrs, 1hr, 2hrs, 4hrs, 8hrs, and 24hrs. Within
HDFKWLPHIUDPHRQHEDWFKRI FHOOVZDVWUHDWHGZLWK'(7$12
while another was left untreated as control.
Total mRNA extraction. After treatment, cells were washed
XWHVDQGWKHQFKLOOHGRQLFH51DVH+Ƭ/ZDVDGGHGEHIRUHWKH
PL[WXUHZDVLQFXEDWHGDWƒ&IRUPLQXWHV7KHÀQDOSURGXFW
was stored at -70°C.
Quantitative Real-Time PCR. cDNA obtained from control
ZDVGLOXWHGWHQIROGZLWK'(3&WUHDWHGZDWHUWRWKH-7 dilution
to establish sample standard curves for each of eight gene expres-
ZLWK 3%6 VFUDSHG RII  QR WU\SVLQL]DWLRQ DQG WUDQVIHUUHG WR VLRQVƬ/RI XQGLOXWHGF'1$IURPWUHDWHGDQGXQWUHDWHGFHOOV
15mL conical tubes. Cells in each dish were counted with a hemo- ZDVDPSOLÀHGLQWULSOLFDWHVLQÀQDOYROXPHVRI Ƭ/FRQWDLQLQJ
cytometer before mRNA extraction. Equal numbers of treated Ƭ/RI 6<%5*UHHQPDVWHUPL[DQGQ0HDFKRI VSHFLÀF
FHOOV§[6 per extraction) were lysed with TRIzol reagent.
Chloroform was used for phase separation and isopropyl alcohol
IRU51$SUHFLSLWDWLRQ51$SHOOHWVZHUHZDVKHGZLWKHWKD-
QROUHVXVSHQGHGLQ'(3&WUHDWHGZDWHUTXDQWLÀHGLQDVSHFWUR-
photometer, and stored in –70°C until further use.
Reverse Transcription. F'1$ZDVV\QWKHVL]HGIURPƬJRI 
39 w w w . T H U R J . o r g T H U R J
SULPHUV7DEOHLQD0-5HVHDUFKUHDOWLPH3&5PDFKLQH7KH
cycling program was as follows: 95°C for 2 minutes, 95°C for 10
seconds, 60°C for 10 seconds, 72°C for 30 seconds, (repeat steps
2-4 50 times more), 72°C for 5 minutes, and 28°C for 1 minute.
Statistical Analysis. 'DWDIURPUHDOWLPH3&5ZDVH[SRUWHG
from DNA Engine Opticon© software for the calculation of
V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

TABLE 2. Regression analysis of sample standard curves SUHVVLRQRQ+89(&VSDVVDJH7DEOHDQG)LJXUH7(0

IURPVHULDOVXFFHVVLYHGLOXWLRQV
expression was only detectable by immunohistochemical analy-
VLVZHVWHUQEORWWLQJDQGFRQYHQWLRQDO3&5E\SDVVDJHEHFDXVH
LW ZDV ORVW TXLFNO\ DV FHOOV DJHGGLIIHUHQWLDWHG in vitro.22 These
UHVXOWV FRQÀUPHG WKDW TXDQWLWDWLYH 573&5 LV DW OHDVW IROG
PRUH VHQVLWLYH WKDQ FRQYHQWLRQDO 3&5 %DVHG RQ VHULDO GLOX-
tions of cDNA used to establish standard curves for eight gene
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CELL BIOLOGY
OLPLWRUWKHGHWHFWDEOHHQGSRLQWGLOXWLRQIRURXUT573&5ZDV
estimated to be at least one log concentration of the target cDNA
SHUUHDFWLRQDVZDVLQRWKHU573&5DVVD\V25 According to this
sensitivity, standard curves were used to estimate gene expression
in mRNA copy numbers (Figure 2).
Endothelial tumor marker genes express differentially.
Based on standard curves (Table 2), the expression levels of col-
TABLE 3. Mean threshold values from qRT-PCR: qRT- PCR ODJHQV,ơ,ơ,,,ơ,YơODPLQLQVơơ7(0DQG+357
F\FOHQXPEHUVDWZKLFK'1$¿XRUHVFHQFHUHDFKHGDWKUHVK- were 0.26, 3.14, 3.47, 3651.04, 9.29, 971.61, 9.13, and 116.11
old level (calculated by a computer algorithm). Experiment FRSLHV RI  P51$FHOO UHVSHFWLYHO\ LQ +89(&V in vitro under
was performed in triplicates. QRUPDO FRQWURO FRQGLWLRQV 7ZR PDWUL[ JHQHV FROODJHQ ,ơ DQG
FROODJHQ ,,,ơ H[SUHVVHG DW WKH VDPH OHYHO 0DWUL[ JHQH 7(0
H[SUHVVHGWKUHHWLPHVPRUHWKDQFROODJHQ,ơDQGFROODJHQ,,,ơ
EXWPDWUL[JHQHFROODJHQ,ơH[SUHVVHGDERXWWLPHVOHVVWKDQ
FROODJHQ,ơDQGFROODJHQ,,,ơ$PRQJWKHWKUHHEDVHPHQWPHP-
EUDQHJHQHVODPLQLQơH[SUHVVHGDWWKHVDPHOHYHODVPDWUL[JHQH
7(0ODPLQLQơH[SUHVVHGDERXWWLPHVPRUHWKDQODPLQLQ
ơDQGFROODJHQ,9ơH[SUHVVHGDERXWWLPHVPRUHWKDQODPLQLQ
ơ7KHH[SUHVVLRQOHYHOVRI DOORI WKHVHHQGRWKHOLDOWXPRUPDUNHU
JHQHVZHUHGHWHFWDEOHXSWRSDVVDJHE\RXUT573&5
NO curtails human endothelial tumor gene expres-
sion.NO plays a major role in the regulation of the synthesis of
PHDQ &W YDOXHV DQG VWDQGDUG GHYLDWLRQ 6WDQGDUG OLQHDU UHJUHV- extracellular matrix components, including type I collagens. To
26

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LHV<YHUVXVWKHPHDQ&WYDOXHV;ZHUHGHYLVHGZLWKD7H[DV VDJHZHUHH[SRVHGWRDQGƬ0RI WKHGRQRU'(7$
,QVWUXPHQWV7,3OXVFDOFXODWRU3&5DPSOLÀFDWLRQHIÀFLHQF\ 12IRUKUV7DEOHDQGWRƬ0RI WKHGRQRU'(7$12
(AE) was calculated from the slopes of the standard curves us- for 4, 8 and 24 hrs (Table 5). All the mRNA levels of collagen
LQJWKHHTXDWLRQ$( -slope – 1.24 When necessary, analysis of ,ơFROODJHQ,,,ơFROODJHQ,9ơODPLQLQVơDQGơDQG7(0
YDULDQFH$129$ZDVSHUIRUPHGDQGZKHQVLJQLÀFDQWWKLVZDV
IROORZHGE\D7XNH\·VPHDQVFRPSDULVRQWHVWXVLQJDFRQÀGHQFH
OHYHORI 3 
TABLE 4. Regulation (%)* of transcription mRNA levels of
tumor marker genes by exposure to different concentration
RI'(7$12IRUKUV

RESULTS

Endothelial tumor maker gene expressions are quanti-

¾HG$VH[SHFWHGDOOSULPHUSDLUVZHUHVSHFLÀFWRFHUWDLQJHQHV
The melting curves clearly showed no primer dimers for any of
eight genes evaluated (Figure 1). Based on regression analysis of
standard curves established with serial dilutions of cDNA, the
accuracy (r2 > 0.99) and precision (most standard deviations <
F\FOHVRI 3&5ZHUHKLJKIRUDOOJHQHH[SUHVVLRQV7KH3&5
DPSOLÀFDWLRQHIÀFLHQF\ZDVDERXWIRUDOOWDUJHWJHQHVVKRZ-
LQJVLPLODUT573&5VHQVLWLYLW\+LJKVHQVLWLYLW\RI WKHT573&5
FDQEHVHHQIURPGHWHFWDELOLW\DQGTXDQWLÀFDWLRQRI 7(0H[-

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
40
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ZHUH GRZQUHJXODWHG E\ H[SRVXUH WR '(7$12 DW GLIIHUHQW TABLE 6. TSP1-NO cross-talk effects* on transcription
FRQFHQWUDWLRQVIRUKUVZLWKH[FHSWLRQRI FROODJHQ,ơZKLFK mRNA levels of tumor marker genes in human endothelial
ZDVXSUHJXODWHGE\H[SRVXUHWR'(7$12DWWKHORZ- cells
HVWFRQFHQWUDWLRQƬ07DEOH$OWKRXJKWKHUHZHUHQRVLJ-
QLÀFDQWGLIIHUHQFHVLQH[SUHVVLRQUHJXODWLRQRI WKHWXPRUPDUNHU
JHQHVDPRQJGLIIHUHQWH[SRVXUHWLPHVRI Ƭ0'(7$12
KRXUH[SRVXUHWRƬ0'(7$12PRUHHIIHFWLYHO\VXSSUHVVHG
expression of the tumor marker genes (Table 5). The results agree
CELL BIOLOGY

partially with early activation of type I collagen genes in mouse

tissues by NO inhibition26 and are analogous to the downregula-
tion of the extracellular matrix synthesis in the model of murine
lupus nephritis,27 LQFOXGLQJ FROODJHQ , ,,, DQG ,9 ODPLQLQ DQG
proteoglycan, by a receptor antagonist of the endothelin, which
mediates in the increased collagen I gene expression induced by
NO inhibition.26 As expected, the mRNA level of the housekeep-
LQJJHQH+357ZDVQRWUHJXODWHGE\WKH12WUHDWPHQW
TSP1 downregulates gene expression of human en-
dothelial tumor markers. 0DWUL[ DQG PHPEUDQH JHQHV DUH TSP1 switches NO functions to regulate endothelial
WZRJURXSVRI NQRZQPROHFXODUWDUJHWVRI 76317 To study the tumor marker gene expression. Based on its concentrations,
HIIHFWV RI  763 RQ JHQH H[SUHVVLRQ RI  WKH WZR WDUJHW JURXSV
12VZLWFKHVLWVWULSKDVLFLQÁXHQFHVRQ763SURWHLQH[SUHVVLRQ12
+89(&[SDVVDJHZHUHWUHDWHGZLWKQ0RI H[RJHQRXV763
and biphasic modulation on endothelial cell proliferation.13 To
IRUDQGKUV0DUNHGUHJXODWLRQRI P51$OHYHOVRI 
EHWWHUXQGHUVWDQGWKHSRWHQWLDOFURVVWDONEHWZHHQ763DQG12
these genes was detectable only when the cells were treated up to
VLJQDOLQJ DW WKH JHQH WUDQVFULSWLRQ OHYHO +89(&V ZHUH WUHDWHG
24hrs. The mRNA levels of all the tumor maker genes, collagens
VLPXOWDQHRXVO\ZLWKQ0RI H[RJHQRXV763DQGƬ0RI WKH
,ơ,ơ,,,ơ,9ơODPLQLQVơDQGơDQG7(0ZHUHWR
12GRQRU'(7$12IRUKUVWRVWXG\WKHLUMRLQWIXQFWLRQV
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on endothelial tumor marker gene expression. The results showed
RXV VWXG\ P51$V RI  FROODJHQ ,ơ DQG ,ơ ODPLQLQơ DQG
FOHDUO\WKDWWKHMRLQWHIIHFWRI 763DQG12ZDVQRWPHUHO\WKH
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VXPRI WKHLQÁXHQFHVWKH\KDYHDVLQGLYLGXDOPRGXODWRUVRI WKH
GRZQUHJXODWHGDQGWKDWRI ODPLQLQơUHPDLQHGXQFKDQJHGLQWKH
gene expression (Table 6).
763QXOOPLFHPXVFOHH[SODQWV763VWLPXODWHGGRZQUHJXODWLRQ
$VVKRZQDERYHERWK763DQG12KDGVLJQLÀFDQWQHJDWLYH
RI FROODJHQ,9ơDQGODPLQLQơLQ+89(&VLQWKLVVWXG\LVFRQ-
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sistent with the upregulation of these two basement membrane
PDUNHUJHQHH[SUHVVLRQZKHQ+89(&VZHUHWUHDWHGVHSDUDWHO\
SURWHLQJHQHVLQ+89(&VXQGHUJRLQJFDSLOODU\PRUSKRJHQHVLV
ZLWKH[RJHQRXV763DQG12DWWKHGRVHVLQWKHH[SHULPHQWDO
in 3D collagen gels.28$VH[SHFWHG763WUHDWPHQWGLGQRWFKDQJH
conditions. When the two mediators of extracellular matrix syn-
WKHP51$OHYHORI WKHKRXVHNHHSLQJJHQH+357 thesis were used together in the same conditions, at least one of
them switches its negative function to a positive one in regulating
TABLE 5. Down-regulation* (%) of transcription mRNA lev- endothelial tumor marker gene expression (Figure 2). It is unlikely
HOVRIWXPRUPDUNHUJHQHVE\H[SRVXUHWRț0'(7$12 WKDW736VZLWFKHVIURPLQKLELWLQJWRVWLPXODWLQJWXPRUPDUNHU
at different treatment times
JHQHH[SUHVVLRQEHFDXVH763LVDZHOONQRZQDQJLRJHQHVLVLQ-
hibitor. However, it is possible for NO to switch its functions
because it can both suppress and induce angiogenesis.29 To our
NQRZOHGJHWKLVLVWKHÀUVWUHSRUWRQ12IXQFWLRQDOVZLWFKDWWKH
VDPHORZGRVDJHFDXVHGE\H[RJHQRXV763DWDGRVDJHLQWKH
PHDQYDOXHVRI HQGRJHQRXV763UHSRUWHGLQSDWLHQWVZLWKFRORQ
carcinomas.30

DISCUSSION

Angiogenesis regulation is key for cancer progression, mak-

ing inhibition of blood vessel formation pivotal for cancer
treatment.15,31 An array of approaches have been successfully
used to investigate phenotypic and functional changes, including
41 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

outgrowth, proliferation, adhesion, migration, and differentia- tion, and differentiation of endothelial cells in vitro and angio-
tion of endothelial cells undergoing angiogenesis or in response genesis in vivo.29 Distinct threshold NO concentrations induce
WR DQJLRJHQHVLV PRGXODWRUV VXFK DV 763 DQG 12   RSSRVLQJ763UHVSRQVHV13 A 24-hour exposure of NO donor
26 Immunohistochemistry, immunoprecipitation, Northern and
:HVWHUQ EORWV HQ]\PH DFWLYLW\ DQG FRQYHQWLRQDO 3&5 EDQG LQ-
tensities all have been used to monitor the angiogenesis regulation
of gene transcriptional and translational levels.12, 13, 17, 19, 21, 22, 26, 32
,Q FRQWUDVW ZH KDYH GHYHORSHG TXDQWLWDWLYH 573&5 SURWRFROV
to precisely monitor endothelial tumor gene expression during
DQJLRJHQHVLV:HGHVLJQHGVSHFLÀFSULPHUVIRUT573&5ZKLFK
showed very high assay accuracy, precision, sensitivity, and cDNA
DPSOLÀFDWLRQ HIÀFLHQF\ *LYHQ VXFK FKDUDFWHULVWLFV T573&5
methodology promises to be a very useful tool for the evaluation
of angiogenesis regulation, especially matrix synthesis regulation
at the gene transcriptional level.
In previous qualitative methods, the expression of some of the
Ƭ0PHGLDWHVDWULSKDVLFUHVSRQVHLQ763WUDQVODWLRQDO
OHYHOVGHFUHDVLQJDWƬ0UHERXQGLQJDWƬ0DQGGHFUHDV-
LQJ DJDLQ DW Ƭ0 ,Q WKLV VWXG\ Ƭ0 RI  '(7$12 ZDV
FKRVHQWRWUHDW+89(&V in vitro because the selected concen-
WUDWLRQRSWLPDOO\UHJXODWHV763WUDQVODWLRQDOOHYHOV&RQVLVWHQW
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ZLWKSUHYLRXVUHVXOWVDKUH[SRVXUHWRƬ0RI 12GRQRU
'(7$12 VXSSUHVVHG P51$ OHYHOV RI  DOO +89(& WXPRU
marker genes. The results show that NO could be used as an an-
giogenesis antagonist at the early stage of cancer treatment.
7KHƬ012XSUHJXODWLRQRI FROODJHQ,ơP51$LVSUH-
VXPDEO\GXHWRWKHJHQH·VH[WUHPHO\ORZWUDQVFULSWLRQDOOHYHOXQ-
GHUQRUPDOFRQWUROFRQGLWLRQV3RWHQWDQJLRJHQLFDQWDJRQLVWVOLNH
763FDQIXUWKHUGRZQUHJXODWHFROODJHQ,ơP51$H[SUHVVLRQ
genes studied were reported to be negligible in certain tissues un-
der normal conditions26 and undetectable in cell cultures at high
passages.22,QWKLVVWXG\ZHKDYHVKRZQIRUWKHÀUVWWLPHWKDWDOO
of these matrix and membrane protein genes are simultaneously
expressed in human endothelial cells in vitro. Our results showed
WKDWFROODJHQ,ơZDVaIROGOHVVH[SUHVVHGWKDQFROODJHQ,ơLQ
+89(&Vin vitro under normal control conditions. However, ma-
WXUHFROODJHQ,LVDWULSOHKHOL[PROHFXOHFRQWDLQLQJWZRơDQGRQH
ơKHOLFHV267KLVLQGLFDWHVWKHSRWHQWLDORI FROODJHQ,ơWUDQVFULS-
tion to be increased under pathological conditions. This potential
KDVEHHQFRQÀUPHGE\aDQGIROGUHJXODWLRQRI FROODJHQ,ơ
mRNA level in bovine aortic endothelial cells32 and mouse affer-
ent arterioles,26UHVSHFWLYHO\*LYHQVXFKLQVLJKWWKHTXDQWLÀFDWLRQ
of differential gene expression levels may provide invaluable in-
formation on the potential of the selected genes to be regulated
in cancer progression and treatments. To consummate such pre-
dictive knowledge, matrix synthesis balance and gene expression
patterns should be further investigated by microarrays.
Although none of the expression pathways of the seven genes
in question is completely understood, they are indubitably impli-
cated in angiogenesis.17, 21, 22, 32 ([RJHQRXV 763 D ZHOONQRZQ
angiogenesis inhibitor,12 markedly suppresses the transcription
mRNA level of all these genes. The transcription downregulation
of these endothelial tumor marker genes is consistent with the
PRUSKRORJLFDOWHUDWLRQVLQFHOOXODUSKHQRW\SHLQGXFHGE\76312,
13
 ,Q RXU SUHYLRXV VWXG\ WKH P51$ OHYHO RI  FROODJHQ ,ơ ZDV
aDQGWKDWRI FROODJHQ,ơZDVaXSUHJXODWHGLQ763
null mouse explants compared with explants with normal endog-
HQRXV76317%DVHGRQWKHHIIHFWVRQHQGRWKHOLDOFHOOVRI 763
we have observed in our previous studies, a unique but effective
IURPLWVQRUPDOO\ORZOHYHORI H[SUHVVLRQ7DEOH12
DWGRVDJHVRI DQGƬ0FDQDOVRGRZQUHJXODWHFROODJHQ,ơ
P51$H[SUHVVLRQIURPWR+RZHYHUDWLWVORZGRVDJH
RI Ƭ012VZLWFKHGLWVIXQFWLRQIURPLQKLELWRU\WRVWLPX-
ODWRU\ IRU WUDQVFULSWLRQ P51$ OHYHO RI  FROODJHQ ,ơ SRVVLEO\
through an alternative pathway or a special promoter in the syn-
thesis chain of type I collagen.26
:HKDYHVKRZQIRUWKHÀUVWWLPHWKDW763DWDORZFRQFHQ-
WUDWLRQ RI  Q0 FDQ VZLWFK 12 IXQFWLRQ IURP LQKLELWRU\ WR
stimulatory for transcription of endothelial tumor marker genes
FROODJHQ ,ơ FROODJHQ ,,,ơ 7(0 FROODJHQ ,9ơ ODPLQLQơ
DQGODPLQLQơ7KH12IXQFWLRQDOVZLWFKDWWKHJHQHWUDQVFULS-
WLRQOHYHOFODULÀHVLWVFRQWUDGLFWRU\HIIHFWVRQDGKHVLRQPLJUDWLRQ
proliferation, and differentiation of endothelial cells in vitro, and
angiogenesis in vivo %HFDXVH RI  WKLV 763LQGXFHG 12 IXQF-
WLRQDOVZLWFKWKH763EDVHGGUXJVQRZLQGHYHORSPHQWFRXOG
EHKDUPIXOUDWKHUWKDQEHQHÀFLDOZKHQWKH\DUHJLYHQWRSDWLHQWV
who have the low endogenous NO concentrations typical of an
HDUO\VWDJHRI FDQFHUSURJUHVVLRQ:KHQ763IURPDGPLQLVWHUHG
drugs mixes with NO already present in cancer patients, it may
be able to unexpectedly switch NO function from being anti-
angiogenic to pro-angiogenic, thus promoting tumor formation
and cancer progression. This functional switch may be triggered
naturally at this angiogenic stage, because the mean endogenous
763FRQFHQWUDWLRQVLQSDWLHQWVZLWKFRORQFDUFLQRPDVDUH
Q030ZKLFKFRYHUVWKH763FRQFHQWUDWLRQDQDO\]HGLQWKLVVWXG\
Ultimately, the NO functional switch prompts a re-evaluation of
763GHULYHGGUXJVVRWKDWWKH\FDQEHPDGHVDIHUDQGPRUHHI-
ÀFLHQW
GRVDJHQ0RI 763ZDVHPSOR\HGWRHYDOXDWHUHVSRQVHVRI 
YDULRXVJHQHVWRWKHDQJLRJHQHVLVLQKLELWRU6LQFHHDFKJHQHKDV
its own promoter and reporter genes that possess variable sensi-
WLYLWLHVWR763WKHHIIHFWVRI GLIIHUHQW763GRVDJHVRQJHQH
expression should be further tested.
8QOLNH WKH DQWLDQJLRJHQLF 763 12 GRQRUV KDYH EHHQ
shown to both stimulate and inhibit the proliferation, immigra-
ACKNOWLEDGEMENTS
:HH[SUHVVRXUWKDQNVWRWKH25:+1,+)$(6SURJUDPIRU
rewarding Dayan Li an internship opportunity to work on this
VWXG\LQDQG6SHFLDOWKDQNVJRWR'U(OL]DEHWK3HU-
UXFFLRDQG'U7LNYD9RJHOIRUWHFKQLFDODVVLVWDQFH

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
42
 Manuscripts

migration by regulating cell-matrix interactions on collagen. Exp.Cell Res., 305,

133-144.
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DQG*SURWHLQVLJQDOLQJ-&HOO6FL
WUXPRI SGHÀFLHQWPLFH$P-3DWKRO 29. %ODLVH*$*DXYLQ'*DQJDO0 $XWKLHU61LWULFR[LGHFHOO
8. 7ROVPD669ROSHUW29*RRG'-)UD]LHU:$3ROYHULQL3-%RXFN signaling and cell death. Toxicology, 208, 177-192.
13HSWLGHVGHULYHGIURPWZRVHSDUDWHGRPDLQVRI WKHPDWUL[SURWHLQ 30. <DPDVKLWD<.XURKLML77XV]\QVNL*36DNDL7 6KLUDNXVD7
thrombospondin-1 anti-angiogenic activity. J. Cell. Biol, 122, 497-511. 3ODVPDWKURPERVSRQGLQOHYHOVLQSDWLHQWVZLWKFRORUHFWDOFDUFLQRPD&DQFHU
9. Isenberg, J. Wink, D. A., Roberts, D. D. (2006). Thrombospondin-1 antagoniz- 82, 632-638.
es nitric oxide-stimulated vascular smooth muscle cell responses. Cardiovasc 31. 0RUDELWR$6DUPLHQWR5%RQJLQHOOL3 *DVSDULQL*$QWLDQJLR-
Res., 71, 785-93. genic strategies, compounds, and early clinical results in breast cancer. Crit.
10. 'H )UDLSRQW ) .HUDPLGDV 0 (O $WLÀ 0 &KDPED] ( 0 %HUJHU ) Rev. Oncol. Hematol., 49, 91-107.
Feige, J. J. (2004). Expression of the thrombospondin 1 fragment 167-569 in 32. )RXVHU/,UXHOD$ULVSH/%RUQVWHLQ36DJH(+7UDQVFULSWLRQDO
C6 Glioma cells stimulates tumorigenicity despite reduced neovascularization. $FWLYLW\ RI  WKH ơ,&ROODJHQ 3URPRWHU ,V &RUUHODWHG ZLWK WKH )RUPDWLRQ
Oncogene, 23, 3642-3649. RI &DSLOODU\OLNH6WUXFWXUHVE\(QGRWKHOLDO&HOOVLQ9LWUR-%LRO&KHP
11. 0DULQRV56=KDQJ::X*.HOO\.$DQG0HLQLQJHU&-$P 18345-48351.
-3K\VLRO++
12. 5LGQRXU / $ ,VHQEHUJ - 6 (VSH\ 0 * 7KRPDV ' ' 5REHUWV '
D., Wink, D. A. (2005). Nitric oxide regulates angiogenesis through a func-
WLRQDOVZLWFKLQYROYLQJWKURPERVSRQGLQ3URF1DWO$FDG6FL86$
13147-13152.
13. ,VHQEHUJ-6&DO]DGD0-=KRX/*XR1/DZOHU-:DQJ;)UD]LHU
W. A., Roberts D.D. (2005). Endogenous thrombospondin-1 is not necessary
for proliferation but is permissive for vascular smooth muscle cell responses to
SODWHOHWGHULYHGJURZWKIDFWRU0DWL[%LRORJ\
14. ,VHQEHUJ-65LGQRXU/$'LPLWU\-)UD]LHU:$:LQN'$5REHUWV
D. D. (2006). CD47 is necessary for inhibition of nitric oxide-stimulated vascu-
lar cell responses by thrombospondin-1. J. Biol. Chem., 281, 26069-26080.
15. &DO]DGD0- 5REHUWV''1RYHOLQWHJULQDQWDJRQLVWVGHULYHGIURP
WKURPERVSRQGLQV&XUU3KDUP'HV
16. 'H\1%%RHUWK1-0XUSK\8OOULFK-(&KDQJ3/3ULQFH&: 
/LQFROQ70&\FOLF*03GHSHQGHQWSURWHLQNLQDVHLQKLELWVSVWHR-
pontin and thrombospondin production in rat aortic smooth muscle cells. Circ.
Res., 82, 139-146.
17. =KRX/,VHQEHUJ-6&DR=5REHUWV''7\SH,FROODJHQLVD
molecular target for inhibition of angiogenesis by endogenous thrombospon-
din-1. Oncongene, 25, 536-545.
18. 6W&URL[%5DJR&9HOFXOHVFX97UDYHUVR*5RPDQV.(0RQWJRP-
HU\(/DO$5LJJLQV*-/HQJDXHU&9RJHOVWHLQ%.LQ]OHU.:
*HQHVH[SUHVVHGLQKXPDQWXPRUHQGRWKHOLXP6FLHQFH
19. :LWWH0%7KRUQWRQ)-(IURQ'7%DUEXO$(QKDQFHPHQWRI 
ÀEUREODVWFROODJHQV\QWKHVLVE\QLWULFR[LGH1LWULF2[LGH
20. *RQ]DOH]$0*RQ]DOHV0+HUURQ*61DJDYDUDSX8+RSNLQVRQ6
B., Tsuruta, D., Jones, J. C. (2002). Complex interactions between the laminin
alplpha 4 subunt and integrins regulate endothelial cell behavior in vitro and
DQJLRJHQHVLVLQYLYR3URF1DWO$FDG6FL86$
21. 'H+DKQ.&*RQ]DOHV0*RQ]DOH]$0+RSNLQVRQ6%&KDQGHO
16%UXQHOOH-.-RQHV-&57KHơODPLQLQVXEXQLWUHJXODWHV
endothelial cell survival. Exp. Cell Res., 294, 281-289.
22. +RWFKNLVV.$%DVLOH&06SULQJ6&%RQXFHOOL*/LVDQWL037HU-
PDQ%,7(0H[SUHVVLRQVWLPXODWHVHQGRWKHOLDOFHOODGKHVLRQDQG

43 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

Bright, luminescent silicon nanoparticles

for biological applications
ALEXANDRA COURTIS

Department of Chemistry, University of California, Davis, CA 95616

acourtis@fas.harvard.edu
In the last two decades, there has been increased interest in the utility of quantum scale semi-
FRQGXFWRUV 7KHVH ÁXRUHVFHQW VLQJOH FU\VWDOV FDQ EH DV VPDOO DV  QP LQ GLDPHWHU DQG WKHLU VL]H

CHEMISTRY
and shape has been shown to be controlled by the duration, temperature, and ligand molecules
XVHG LQ WKHLU V\QWKHVLV 4XDQWXP GRWV SURYLGH FOHDU EHQHÀWV RYHU WKH RUJDQLF G\HV FXUUHQWO\
used for tracking biological processes. Yet, as the production of quantum dots is often very cost-
O\ WKH VHDUFK FRQWLQXHV IRU ÀQGLQJ DQ LQGXVWU\UHDG\ V\QWKHVLV IRU D TXDQWXP VFDOH VHPLFRQ-
ductor which would have high yield, optimal durability, high luminescence, and a broad absor-
bance range. Silicon, in particular, has been of great interest as it is the second most abundant
element on the Earth’s crust and is generally a bioinert and electrochemically stable element.
We report the synthesis of water-soluble, luminescent silicon nanoparticles with potential ap-
plications to bioimaging. Through a solution state top-down approach, the synthesis of hydrogen
capped silicon nanoparticles was achieved in various organic solvents. The surface of the nanopar-
ticles was capped with the functional organic molecules rendering the Si-QDs both air and water
stable. Cell studies performed with our silicon nanoparticles and human monocytes show the di-
rect applications these particles could have for tracking biological processes and the progression
of cancer in the human body. In attempt to shift the luminescence of these particles, alterations
of experimental methodology was also explored in the areas of reaction solvent and heating time.
Through these changes, shape control of silicon nanoparticles was achieved in the form of silicon
QDQRURGV 7KH V\QWKHVLV RI  WKLV QHZ VKDSH RI  VLOLFRQ DW WKH TXDQWXP VFDOH ZDV FRQÀUPHG E\ XO-
traviolet spectroscopy, photoluminescence, and transmission electron microscopy. The results
of this study indicate that the use of silicon nanocrystals for biomedical applications is feasible.
____________________________________________________

INTRODUCTION As the production of quantum dots is often very costly, the

VHDUFKFRQWLQXHVWRÀQGDQLQGXVWU\UHDG\V\QWKHVLVIRUDTXDQ-
In the last two decades, there has been increased interest in tum scale semiconductor that would have high yield, optimal du-
QDQRVFDOHVHPLFRQGXFWRUVRUTXDQWXPGRWV4'V7KHVHÁXRUHV-
cent single crystals can be as small as 1 nm in diameter, and their
properties can be controlled by the duration, temperature, and
ligand molecules used in their syntheses.1 Quantum dots, which
EHKDYHOLNHVLQJOHDWRPVSURYLGHFOHDUEHQHÀWVRYHUWKHRUJDQLF
dyes currently used for tracking biological processes. On average,
a quantum dot or nanoparticle is 20-30 times brighter than a single
dye molecule. Furthermore, nanoparticles resist photobleaching, a
SURFHVVE\ZKLFKG\HVLQVROXWLRQUDSLGO\ORVHWKHLUÁXRUHVFHQFH2 As
documented by Michalet et al., researchers have begun to explore
the wide applicability of the quantum dots’ luminescence for in
vivo imaging, tumor targeting, diagnostics, and even as photosen-
sitizers of active oxygen in the photodynamic therapy of cancer.3
w w w . T H U R J . o r g T H U R J V
o l I I s s u e 1
rability, high luminescence, and a broad, tunable emission range.
Notably, the “top-down” process (Figure 1) has shown promise
for developing such a semiconductor. The “top-down” method
involves starting with a bulk solid and obtaining a nanostructure
by structural decomposition.47KLVPHWKRGFDQEHPRUHFRVWHIÀ-
FLHQWWKDQWKH´ERWWRPXSµPHWKRGZKLFKLQYROYHVÀUVWLVRODWLQJ
individual atoms or small molecules of an element and then com-
bining them to make a nanoparticle or nanoclusters. The “bot-
tom-up” approach was elegantly demonstrated by the synthesis
of CdSe nanoparticles in 1993 with the work of Murray et al.5
In this study, novel silicon nanoparticles were synthesized
employing a “top-down” solution state approach. The optimal
methodology addressed the four aforementioned aspects of
an industry ready synthesis of a quantum sized semiconductor,
S p r i n g 2 0 0 8
44
Manuscripts

et al. In a drybox, elemental sodium and elemental silicon were

placed in a 1:1 ratio inside acid-etched tantalum tubes that had
been previously crimped and welded on one end. The other ends
were crimped and welded in an argon arc welder. The tubes were
subsequently sealed in evacuated quartz ampules. The sealed re-
action vessels were heated in a programmable furnace at a rate of
60°C/hr until the temperature reached 650°C. At 650°C, the reac-
tion was left to run for 80 hours. The reaction vessels were then
cooled at a rate of -60°C/hr to room temperature, and were sub-
sequently opened inside a drybox to yield black, chunky solids.
$IWHUJULQGLQJWKHSURGXFWWRDÀQHEODFNSRZGHUWKHVXFFHVV-
IXOV\QWKHVLVRI 1D6LZDVFRQÀUPHGE\SRZGHU;UD\GLIIUDFWLRQ
using an INEL diffractometer (Co K° radiation). It was estimated
based on the relative intensities of expected and extraneous peaks
CHEMISTRY

WKDWWKH1D6LZDVDSSUR[LPDWHO\SXUH6RPHXQUHDFWHGVLOL-
con likely existed in the product, but they were unreactive in sub-
FIGURE 1. Schematic of the top-down method sequent reaction steps. Due to its extreme sensitivity to air, the
namely high yield, optimal durability, high luminescence, and a compound was stored in a drybox under a nitrogen atmosphere.
suitable emission range in the synthesis of water soluble silicon N,N-dimethylformamide (DMF). The solvent, N,N-dime-
QDQRSDUWLFOHV7KHV\QWKHVLVKDVSURYHGVXFFHVVIXODQGVLJQLÀFDQW thylformamide, was dried to remove water using 3Å molecular
on two counts: a) the propylamine capped silicon nanoparticles VLHYHV7KHVLHYHVZHUHDFWLYDWHGLQDP/VWRUDJHÁDVNFRQ-
Figure 2. Reaction setup and heating scheme of NaSi synthesis
emit at longer wavelengths than those described in prior literature
Sealed
on the solution state syntheses of silicon nanoparticles;6 and b) Na + Si NaSi
Quartz
the quantum dots are both water-soluble and non-toxic to live Elemental Si 650qC , 3-4 days
cells. Furthermore, during experimentation, the ability to control Powder
60qC /hr 60qC /hr
the shape of the silicon nanoparticles was realized in the syn- Heating Scheme

WKHVLVRI RFWDQHWHUPLQDWHGVLOLFRQQDQRURGV7KLVZDVDVLJQLÀ- Na Metal

FDQWEUHDNWKURXJKVLQFHWKHVROXWLRQVWDWHV\QWKHVLVRI ÁXRUHV- (cut pieces)

cent silicon nanorods has never before been reported through Acid-etched Nb tubing
such a solution-state approach. The silicon nanorods may have (crimped and arc welded)
enhanced opportunity for biological applications, as their thin- FIGURE 2. Reaction setup and heating scheme of NaSi synthesis
ner diameter, relative to spherical nanoparticles, may be advanta-
geous in minimizing quantum dot surface-cell membrane inter- taining a stir bar, which was placed under evacuation in a hot oil
DFWLRQVZKLFKFRQWULEXWHWRQRQVSHFLÀFFOHDUDQFHLQYLYR7KH EDWKDWÜ&IRUKRXUV7KHÁDVNZDVUHPRYHGIURPWKHRLO
cell studies show that the particles are bright enough for poten- bath and left to cool. DMF was put in a separate round bottom
tially tracking biological processes, and they may have applications DQGDLUZDVEXEEOHGRXWRI WKHÁDVNE\SXWWLQJRQHQHHGOHWXUQHG
in monitoring the progression of cancer in the human body. to high argon pressure through the septum and below the solu-
tion level. The other needle was put through the septum only and
EXPERIMENTAL SECTION provided an outlet for the gas purge. After 30 minutes, the DMF
ZDVWUDQVIHUUHGWRWKHÁDVNZLWKWKHPROHFXODUVLHYHVYLDFDQQXOD
Materials and Methods. Ammonium bromide was acquired 7KHVWRUDJHÁDVNZLWKWKHVLHYHVDQG'0)ZDVVWLUUHGRYHUQLJKW
IURP )LVKHU 6FLHQWLÀF VLOLFRQ SRZGHU  ZDV REWDLQHG 7KHGULHG'0)ZDVNHSWLQWKHVWRUDJHÁDVNXQGHUDUJRQWRSUR-
from Aldrich; sodium was purchased from Alfa Aesar; N,N-Dim- tect it from air and water contamination.
HWK\OIRUPDPLGHZDVDFTXLUHGIURP6LJPD$OGULFK'LRF- Dioctyl Ether (DOE). DOE was degassed via the “freeze-
W\O(WKHUZDVDFTXLUHGIURP$OGULFK2FWHQHZDV SXPSWKDZ PHWKRGµ $ RQHQHFN  P/ URXQG ERWWRP ÁDVN
REWDLQHGIURP$OGULFKDOO\ODPLQHDQGWKHFDWDO\VWD ZDVÀOOHGWRKDOI YROXPHZLWK'2(7KHVWRSFRFNRI WKHÁDVN
M solution of H2PtCl6‡+2O, were generously donated by Dr. ZDVFORVHGDQGWKHÁDVNZDVKRRNHGXSWRD6FKOHQNOLQHWXUQHG
;LDRPLQJ =KDQJ 7KH VSHFLÀF H[SHULPHQWDO SURFHGXUHV IRU WKH WRYDFXXP7KHÁDVNZDVLPPHUVHGLQDOLTXLGQLWURJHQ'HZDU
synthesis of the starting materials and of the silicon nanoparticles When the DOE was completely frozen, the stopcock was opened
are given below. All samples were air and water sensitive unless to vacuum for a few minutes. The stopcock was closed and the
otherwise stated. ÁDVN ZDV OHIW DW URRP WHPSHUDWXUH XQWLO WKH '2( PHOWHG EDFN
Sodium Silicide (NaSi). The salt NaSi was prepared by a into liquid. This procedure was repeated until no bubbles emerged
PRGLÀFDWLRQ RI  WKH SURFHGXUH VHW IRUWK E\ :LWWH7 and Mayeri from the frozen DOE as it thawed.
45 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Ammonium Bromide (NH4Br). NH4Br was dried for four
hours in a two-neck round bottomed under evacuation. The oil
EDWKWHPSHUDWXUHZDVVWDELOL]HGDWÜ&$VDPPRQLXPEURPLGH
KDVDQH[WUHPHDIÀQLW\WRZDWHULWZDVVWRUHGLQDQLWURJHQÀOOHG
drybox after drying.
Propylamine Terminated Silicon Nanoparticles. In a
drybox, sodium silicide and ammonium bromide were placed in
a 1:2 ratio (excess NH4Br was added to ensure all NaSi reacted)
Manuscripts
JRQRYHUQLJKWLQDÜ&VLOLFRQRLOEDWK7KHK\GURSKRELFVROX-
WLRQZDVVWRUHGLQDVWRUDJHÁDVNIRUVXEVHTXHQWFKDUDFWHUL]DWLRQ
Photoluminescence spectroscopy (PL), Transmission Electron
Microscopy, and Ultraviolet -visible spectroscopy (UV-vis) were
done to characterize the properties of the nanorods.
Cell Study. Human monocytes (U937, American Type Cul-
ture Collection), plated at 500,000 cells/mL, were incubated for
48 hours in 150 nM PMA (phorbol 12-myristate 13-acetate; Sigma
inside a two-neck round bottomed with a stirbar. Via cannula,
100 mL of dried, degassed N,N-dimethylformamide (DMF) was
DGGHGWRWKHÁDVNDQGWKHUHDFWLRQYHVVHOZDVKHDWHGRYHUQLJKW
DW UHÁX[ XQGHU DUJRQ LQ DQ DSSUR[LPDWHO\ Ü& RLO EDWK 7KH
PL[WXUHIROORZLQJWKHUHDFWLRQZDVQRWDEO\FORXG\$IWHUÀOWHULQJ
RII WKHEODFNSUHFLSLWDWHLQDQDLUIUHHÀOWUDWLRQV\VWHP—/RI 
3DWž&&22 to activate the cells. Activation media
was carefully removed, washed 3 times with RPMI-1640 (Ameri-
can Tissue Culture Collection, #30-2001), and the silicon nano-
particle solution was added to each Petri dish and incubated for
KRXULQD&22 atmosphere. Cells were washed 3x with
media, 1mL CO2 independent media (Gibco, #18045-088) was

CHEMISTRY
0.072 M H2PtCl6‡+2O solution was added by anaerobic syringe added, and the cells were imaged by confocal microscopy (LSM
followed by 2 mL allylamine, also added by anaerobic syringe. 5 Pascal, Zeiss). Please refer to Figure E (Attachment I) for a de-
The solution was stirred and heated under argon overnight in a tailed graphic outlining the experimental design of the cell study.
Ü&VLOLFRQRLOEDWK7KHVROYHQWZDVWKHQGLVWLOOHGRII XQGHU Instrumentation. All X-ray diffractometer data was taken
vacuum (or under argon) using a standard distillation-apparatus on the INEL Powder X-Ray Diffractometer; photoluminescence
on an air-free Schlenk line. The resulting air-stable powder was (PL) scans were carried out on a John Horiba Fluoromax instru-
resuspended in nanopure water. The solution was yellow to deep ment, photographs of the silicon nanoparticles in solution were
yellow in color, depending on the concentration of the particles. taken using a UV-light lamp (365 nm), and UV-vis data was taken
Figure 4. Basic Reaction Scheme for the Synthesis of Octane Terminated Silicon Nanorods
Photoluminescence spectroscopy (PL), High Resolution Trans- using a PharmaUV instrument.
mission Electron Microscopy (HR-TEM), and Ultraviolet-visible Refluxing in H H H Oct Oct Oct
DOE
spectroscopy (UV-Vis) were performed to characterize the prop- NaSi + NH4Br Si H
1-octene
Oct Si Oct
H
erties of the nanoparticles. Please refer to Figures A-D (Attach- -NaBr
-NH3
ment I) for detailed graphics outlining the experimental design of H H H Oct Oct Oct

these experiments. FIGURE 4. Basic Reaction Scheme for the Synthesis of Octane
Octane Terminated Silicon Nanorods. In a drybox, sodi- Terminated Silicon Nanorods
um silicide and ammonium bromide were placed in a 1:2 ratio (ex-
RESULTS AND DISCUSSION
cess NH4Br was added to ensure all NaSi reacted) inside a three-
QHFNURXQGERWWRPHGÁDVNFRQWDLQLQJDVWLUEDU9LDFDQQXOD
Sodium Silicide. The experimental powder X-ray diffrac-
P/RI GHJDVVHG'2(ZDVWUDQVIHUUHGWRWKHUHDFWLRQÁDVNRQD
tion data for NaSi produced in this report are compared with the
6FKOHQNOLQH8VLQJDKHDWLQJPDQWOHWKHUHDFWLRQÁDVNZDVKHDWHG
calculated data (shown in Figure 5). Patterns (b) and (c) are scans
WRUHÁX[IRUKRXUV$IWHUÀOWHULQJRII WKHEODFNSUHFLSLWDWHXV- Figure 5. Experimental Powder X-Ray Diffraction Patterns of
of different
Prepared NaSiexperiments
Samples (b andfollowing
c) Compared thetoaforementioned
Calculated synthesis.
LQJDQDLUIUHHÀOWUDWLRQV\VWHPP/RI RFWHQHZDVDGGHGE\
7KHSDWWHUQVDUHDJRRGÀWWRWKHFDOFXODWHGSDWWHUQIURPVLQJOH
Pattern Figure
(a) from
2. Crystal Data Diffraction of Calculated NaSi versus
Powder X-Ray
anaerobic syringe. The solution was stirred and heated under ar-
crystal Experimental
data in terms of peak position and relative intensity, indi-
Results

H cating the presence of NaSi as the major phase. Similarities in the

H H
DMF
1) NaSi + NH4Br Si
-NaBr,
(a) NaS i
-NH3 H H calculated
H 16 24 32 40 48 56 64 72 80

NH2 (b)

H NH2 16 24 32 40 48 56 64 72 80

H H NH2
2) DMF Si (c)
Si NH2
capping 16 24 32 40 48 56 64 72 80

H H agents NH2
H Degrees 2 theta

NH2 Figure 5. Experimental Powder X-Ray Diffraction Patterns of Pre-

FIGURE 3. Basic Scheme of Silicon Nanoparticle Reaction and pared NaSi Samples (b and c) Compared to Calculated Pattern (a)
Surface Termination from Crystal Data
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
46
 Manuscripts

patterns (b and c) indicate the reproducibility of the synthesis. All
peaks are almost identical in the powder patterns (b) and (c) in
regards to both position and relative intensity. In comparison with
the calculated pattern, both experimental powder patterns exhibit
all eight major peaks, which indicate that NaSi was produced suc-
cessfully. The lattice structure of NaSi (Figure 6a) is comprised
of both sodium ions and Si44- tetrahedron units (Figure 6b). This
CHEMISTRY
route, due to the high level of the particles’ monodispersity and
homogeneity. If nanoparticles are to be considered for low-cost
industrial applications, the increase in yield of one single synthesis
LVFULWLFDO%H\RQGWKHHIÀFLHQF\DQGFRVWHIIHFWLYHQHVVRI WKHKLJK
yield, the TEM results are also indicative of the product’s homo-
geneity. The nanoparticles on the above grid are consistently in
the range of 5 nm. The ability to produce such monodisperse
particles could be essential if large-scale synthesis of these nano-
particles is to be attempted for biomedical or photovoltaic ap-
plications.
As shown by photoluminescence spectroscopy (Figure 8a), the
silicon nanoparticles exhibit luminescence in the 480 to 510 nano-
PHWHU UDQJH 7KH QDQRSDUWLFOHV PDLQWDLQHG WKHLU KLJK ÁXRUHV-
cence after 3 weeks in solution. Holmes and coworkers presented

theoretical evidence that 1 nm silicon quantum dots with carbon

surface termination have direct band gap optical transitions that
FIGURE 6A. Crystal lattice structure of NaSi, generated from single
crystal data.
lead to photoluminescence in the UV/blue-green region of the
electronic spectrum.6,107KHÀQGLQJVRI WKLVUHSRUWH[SDQGXSRQ
their theory on band gap optical transitions to carbon terminated

FIGURE 6B. Computer

Figure generated
7. High-resolution model
transmissionof the Si44- tetrahedron
electron
microscopy (HRTEM) image of high yield
in NaSi. propylamine terminated silicon quantum dots

FIGURE 8A. Photoluminescence scan of propylamine capped sili-

con nanoparticles
FIGURE 7. High-resolution transmission electron microscopy (HR-
TEM) image of high yield propylamine terminated silicon quantum
dots
visualization of the crystalline structure of NaSi was generated in
agreement with the crystallography results of the NaSi synthesis
and with the results of Busman et al.9ZKRÀUVWVHWIRUWKWKHFU\V-
talline structure of NaSi. Since special precautions were taken to
ensure that the reacted NaSi did not come in contact with air, no
oxidation product was observed in the powder X-ray diffraction
pattern.
Propylamine Terminated Silicon Nanoparticles. FIGURE 8B. UV-Vis Scan of Propylamine Terminated Silicon Nano-
Propylamine terminated silicon nanoparticles were successfully particles
V\QWKHVL]HG ZLWK VXLWDEOH ÁXRUHVFHQFH VROXELOLW\ DQG VL]H KR-
mogeneity for potential biological applications. Figure 7 shows Silicon nanoparticles 5 nm in diameter. UV-Vis absorption data
a Transmission Electron Microscopy (TEM) image of quantum (Figure 8b) show that the nanoparticles absorb light in the range
dots dispersed on a carbon-coated copper grid. The average size of 300 to 400 nanometers. Corresponding Figure A (Attachment
of the particles was determined by analysis of TEM results to II) shows the blue-green luminescence the propylamine termi-
be approximately 5 nanometers. The importance of this image is nated silicon nanoparticles exhibit in nanopure water when placed
highlighted in contrast to work of Warner et al.6 , who prepared under a UV lamp. A notable aspect of Figure A is the relatively
propylamine capped silicon nanoparticles through a different low level of light scattering in the solution.
47 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Octane Terminated Silicon Nanorods7RVKLIWWKHÁXR-
rescence of the nanoparticles to the 600-700nm range when ex-
cited with UV light, and thus generate a product more appropriate
for biological applications, the heating time and temperature were
DOWHUHG,WZDVK\SRWKHVL]HGWKDWE\KHDWLQJWKHUHDFWLRQÁDVNIRUD
longer duration, crystallinity will improve, causing the wavelength
of emission to shift into the red, commensurate with particle size.
Manuscripts
Cell Study. In view of the optimal properties of the pro-
pylamine capped silicon nanoparticles presented in this report,
the utility of these quantum dots for both diagnostics and bio-
imaging was explored. The cell study, performed on monocytes,
demonstrated the high uptake rate of the particles into live cells.
After 1 hour, the monocytes had uptaken the allylamine capped
silicon nanoparticles and began to glow brightly (Figure B-At-
Although no substantial change was seen in photoluminescence
scans (Figure 9) relative to the previous samples, The UV-vis of
these octane-capped particles was quite remarkable. As shown in
Figure 10, two peaks are observed in the UV-vis spectrum. The
two absorption peaks may be indicative of the formation of nan-
orod shaped particles because the peaks represent absorbance on
the horizontal and vertical axes of the rods. Transmission Elec-
tron Microscopy images (Figure 11) further indicate the forma-
tion of silicon nanorod structures.
FIGURE 9. PL Scan of Silicon Nanorods
demonstrated the high uptake rate of the particles into live cells.
After 1 hour, the monocytes had uptaken the allylamine capped
silicon nanoparticles and began to glow brightly (Figure B-At-
WDFKPHQW,,FOHDUO\GHÀQLQJDOOWKHFHOOVRQWKHFHOOSODWH)XU-
thermore, in Figure C (Attachment II), small vacuoles were visible
around the nanoparticles, offering evidence that the nanoparticles
were indeed uptaken by the monocytes.
CONCLUSION
CHEMISTRY
Through a top-down solution state approach, the facile syn-
thesis of water-soluable propylamine capped silicon nanoparticles
was demonstrated. The formation of these nanoparticles was con-
ÀUPHGE\89YLV3/VSHFWURVFRS\DQG7(07KHQDQRSDUWLFOHV
KDYHRSWLPDOÁXRUHVFHQFHDUHKRPRJHQRXVDQGWKHLUV\QWKHVLV
provides for high yield. Cell studies on monocytes have shown
that the particles were non-toxic to cells, and that they also caused
WKHPWRÁXRUHVFHDIWHUWKH\ZHUHXSWDNHQLQKRXU7KLVH[WHQGV
the utility of the particles beyond inorganic chemistry and into
biology and medicine as potential candidates for bio-imaging.
Future work investigating the role the silicon nanoparticles put
forth by this report could relate to cancer diagnosis. Previous stud-
ies have proposed that by capping CdSe nanoparticles with ZnS
and active cancer targeting ligands, the nanoparticles could target
and destroy tumor cells. Although these particles are effective in
killing malignant cells, they cannot play a role in cancer detection
and imaging because of their high toxicity. It thus remains of
JUHDWLQWHUHVWWRFDSÁRUHVFHQWQRQWR[LFQDQRSDUWLFOHVLQDZD\
that would allow their uptake into cancerous cells. Since these pre-
WDFKPHQW,,FOHDUO\GHÀQLQJDOOWKHFHOOVRQWKHFHOOSODWH)XU-
thermore, in Figure C (Attachment II), small vacuoles were visible
around the nanoparticles, offering evidence that the nanoparticles
were indeed uptaken by the monocytes.
FIGURE 10. UV-Vis Scan of Silicon Nanorods
FIGURE 11. TEM image indicating the formation of silicon nano-
rods
w w w . T H U R J . o r g T H U R J
VHQWHGVLOLFRQQDQRSDUWLFOHVDUHQRQWR[LFKLJKO\ÁXRUHVFHQWDQG
water-soluble, capping them with active cancer detectors could
make them a novel tool in cancer detection and tumor imaging.
If this course of investigation continues to progress successfully,
we hope to test these results in advanced cytotoxicity studies, live
cancer cell studies and eventually in animal studies.
ACKNOWLEDGMENTS
I would like to gratefully acknowledge Professors Susan Kauzla-
rich and Angelique Louie at the University of California at Da-
vis for their support and for allowing me to conduct research in
their laboratories. I am especially thankful to Dr. Andrea Goforth,
postdoctoral fellow at UC Davis, for her mentorship and assis-
tance in Schlenk line technique training. In addition, I would like
to thank Ben Jarrett, Ph.D. candidate at UC Davis, for training
me in cell study techniques, and Shizhong Wang for the TEM
micrographs presented herein. Finally I would like to thank the
National Science Foundation and the University of California at
Davis for funding my research.
48
V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

open to
FOOTNOTES Ar 0.072 M H23W&O4‡+2O Allylamine
FIGURE C.
Capping of
1. Alvisatos, A.P., Science, 1996, 271, 933. Propylamine
2. Wiesner,U., Nanoparticle News, 2005,8,3. + 40 ML + 2 mL
Terminated Sili-
3. Timoshenko, V.Y.; Kudryavtsev, A.A.Osminkina, L.A.; Vorontsov, A.S.; Ryab- DMF + QDs
con Quantum
chikov, Y.V.; Belogorokhov, I.A.; Kovalev,D.; Kashkarov, P.K. JETP Letters,
2006, 9, 423-426. Dots
4. Koch, C.C., Rev. Adv. Mater. Sci., 2003, 5, 91-99.
5. Murray, C.B.; Norris, D.J.; Bawendi, M.G., J. Am. Chem. Soc., 1993, 115,
8706.
After addition of allylamine and
6. Warner, J.H.;Hoshino, A.;Yamamoto, K.;Tilley, R.D., Angew.Chem. Int. Ed., Pt catalyst, the reaction flask is
2005, 44, 4550-4554. heated in an oil bath system at
Û&RYHUQLJKW
7. Witte, J.; von Schnering, H.G. Z. Anorg. Allg. Chem., 1964, 327, 260.
8. Mayeri, D.; Phillips, B.L.; Augustine, M. P.; Kauzlarich, S. M. Chem. Mater., to H2O
Schlenk:
2001, 13, 765-770. closed tap
to H2O
sink
9. Busmann, V.E.Z., Anorg. Allg. Chem. 1961, 327, 260-272
10. Holmes, J.D.; Ziegler, K.J.; Doty, C.; Pell, L.E.; Johnson, K.P.; Korgel, B.A. J.
CHEMISTRY

Am. Chem. Soc., 2001, 123, 3748. Open

vac. to
Schlenk

REFERENCES DMF + Si-QDs

DMF
Ice-Water
1. Alvisatos, A.P. “Semiconductor Clusters, Nanocrystals and Quantum Dots.” Bath
Science 271 (1996): 933-937.
2. Busmann, V.E.Z. “Die Kristallstruktur von NaSi und NaGe.” Zeitschrift für
FIGURE D. Schlenk Line Dynamic Distillation System of Propylam-
anorganische und allgemeine Chemie 324(1964): 260-274.
3. Holmes, J.D.; Ziegler, K.J.; Doty, C.; Pell, L.E.; Johnson, K.P.; and B.A. Korgel, ine Terminated Silicon Quantum Dots
B.A. “Highly Luminescent Silicon Nanocrystals with Discrete Optical Transi- &HOO6WXG\6HWXS
tions.” Journal of the American Chemical Society 123.16 (2001): 3748-3738.
4. Koch, CC. “Top-down synthesis of nanostructured materials: mechanical PMA+cells
and thermal processing Methods.” Reviews on Advanced Material Science C ells are plated @ 500,000 cells /mL in 150 nmol
phorbol 12-myris tate 13-acetate (P MA) and
5( 2003): 91-99. incubated at 37 C in a 5% C O 2 atmosphere for 48
5. Mayeri, D, Phillips, B.L., Augustine, M. P., and S.M. Kauzlarich. “NMR Study hours to activate the cells .

of the Synthesis of Alkyl-Terminated Silicon Nanoparticles from the Reac-

tion of SiCl4 with the Zintl Salt, NaSi.” Chemistry of Materials 13.3 (2001):
765-770.
6. Murray, C.B.; Norris, D.J.; Bawendi, M.G. “ Synthesis and Characterization of Si-QDs +cells
P MA is removed and plates were was hed three times
Nearly Monodisperse CdE ( E= S,Se,Te) Semiconductor Nanocrystallites.” with R P MI-1640. S i-QD s olution is added to each P etri
Journal of the American Chemical Society 115 (1993): 8706. dis h and incubated for 1 hour in a 5% C O 2
atmos phere.
7. Timoshenko, V.Y.; Kudryavtsev, A.A.Osminkina, L.A.; Vorontsov, A.S.; Ryab-
chikov, Y.V.; Belogorokhov, I.A.; Kovalev,D.; Kashkarov, P.K. “Silicon Nano-
crystals as Photosensitizers of Active Oxygen for Biomedical Applications.”
JETP Letters 83.9 (2006): 423-426. &RQIRFDO C ells were was hed three times with media, 1
mL C O 2 independent media was added and
8. Warner, J.H.; Hoshino, A.; Yamamoto, K.; Tilley, R.D. “Water-Soluble Photo- Microscope:
the cells were imaged by confocal
LSM 5 Pascal,
luminescent Silicon Quantum Dots.” Angewandte Chemie International Edi- Zeiss micros copy (ins trumentation, right).
tion 44.29 (2005): 4550-4554.
9. Witte, J.; von Schnering, H.G. “ Die Kristallstrukturen von KSi, RbSi, CsSi,
KGe. RbGe und CsGe.” Zeitschrift für anorganische und allgemeine Chemie
FIGURE E. Cell Study Setup: Human Monocytes
324 (1964): 260-274.

ATTACHMENT I: FIGURES A-E

ATTACHMENT II: FIGURES A-B
to Schlenk FIGURE A.
line
Experimental/
used H2O Schlenk Line to Schlenk:
to sink
Set closed tap

Open to
vac.
H2O

Û&
Oil
Bath
DMF

NH4Br, NaSi, FIGURE A. Fluorescence

teflon-coated
stirbar FIGURE B. Air Free Filtration of from Propylamine
Propylamine Terminated Silicon Terminated Silicon
Quantum Dots Quantum Dots in FIGURE B. Monocytes after 1 hour In-
Nanopure Water Excited cubation with Propylamine Terminated
with a UV Lamp Silicon Nanoparticles

49 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

0LFURÁXLGLFFHOOXODUGRXEOHHQFDSVXODWLRQLQD
SRO\PHUVKHOO
ALEXANDER BICK AND PATRICK TABELING
Ecole Supérieure de Physique et de Chimie Industrielles
10 rue Vauquelin, 75231 Paris, France
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EXIIHUVROXWLRQZLWKLQDVROLGVKHOORI 73*'$SRO\PHU7KHSODWIRUPLVÁH[LEOHLQWKDWLWFDQSURGXFH
GRXEOH HPXOVLRQV RI  YDU\LQJ VL]HV DQG FHOO FRQFHQWUDWLRQV :H FDQ FRQWURO WKH VL]H DQG VKDSH RI  WKHVH
HPXOVLRQV WKURXJK PDWKHPDWLFDO PRGHOLQJ RI  GLIIHUHQW ÁRZ UDWH SDUDPHWHUV 7KH FHOO FRQFHQWUDWLRQ LQ
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____________________________________________________
INTRODUCTION MATERIALS AND METHODS

A key challenge in biological research today is the manipulation
of cells on an individual or small group level. Single-cell
manipulation provides a basis for complex tissue engineering
challenges,1 advanced cell culturing,2 and cell cryopreservation,3
ENGINEERING
To form micro-channels of controlled geometry, a variation
of the standard PDMS soft lithography procedure was used.14
SU-8 2100 Photoresist (Microchem, Newton, MA) was added to
the wafer using a P6700 Spincoater (Specialty Coating Systems,
ZKLOHVLQJOHFHOODQDO\VLVHQDEOHVVLJQLÀFDQWDGYDQFHVLQWKHDUHDV
of stem cell differentiation,4 cell signaling,5 high-throughput
screening,6 and disease diagnosis.7 &HUWDLQ DSSOLFDWLRQVSHFLÀF
approaches have been developed, but there is currently no general
SODWIRUPÁH[LEOHHQRXJKWRHQDEOHDOORI WKHVHDSSOLFDWLRQV
7KH GRPDLQ RI  PLFURÁXLGLF HPXOVLRQV SUHVHQWV D URXWH WR
VROYLQJWKLVSUREOHP:KHQWZRLPPLVFLEOHÁXLGVDUHPL[HGWKH\
FUHDWHHPXOVLRQVFRQVLVWLQJRI GURSOHWVRI RQHÁXLGGLVSHUVHGLQ
the second.80L[LQJWKHVHÁXLGVLQPLFURVFDOHFKDQQHOVRI VSHFLÀF
geometries creates droplets of controllable size and shape.9 These
PLFURÁXLGLFGURSOHWVKDYHSUHYLRXVO\EHHQGHPRQVWUDWHGWRKDYH
DZLGHYDULHW\RI XVHVLQÀHOGVUDQJLQJIURPIRRGSURGXFWLRQ10
to cosmetics and pharmaceuticals.11 Double emulsions have also
EHHQ SURGXFHG LQ PLFURGHYLFHV E\ VXFFHVVLYH HPXOVLÀFDWLRQV
HJZDWHUGURSOHWVDUHIRUPHGLQRLOZKLFKLVLQWXUQHPXOVLÀHG
LQ ZDWHU RU D WKLUG LPPLVFLEOH ÁXLG SURGXFLQJ FRUHVKHOO OLTXLG
structures.12 Recent work has demonstrated that cell encapsulation
ZLWKLQVXEVHTXHQWO\VROLGLÀHGPLFURÁXLGLFGURSOHWVRI ELRJHOVLV
possible.13
In this communication, we synthesize these previous advances
and demonstrate the encapsulation of micro-bead particles within
D GRXEOH HPXOVLRQ GURSOHW :H SUHVHQW D ÁH[LEOH PLFURÁXLGLF
platform that controllably produces a single or several beads
VXVSHQGHG LQ D OLTXLG GURSOHW LQVLGH D VROLG SRO\PHU VKHOO 7KH
size and composition of the resulting object is controllable by
DGMXVWLQJ ÁRZ UDWHV 7KH HQFDSVXODWLRQ PHFKDQLVP PDLQWDLQV
FRPSOHWHVHSDUDWLRQRI WKHOLTXLGDQGSRO\PHUSKDVHVWKURXJKRXW
the encapsulation process. The resulting novel platform directly
HQDEOHVÁH[LEOHFHOOFXOWXULQJDSSOLFDWLRQVHDVLHUFHOOPDQLSXODWLRQ
and single-cell studies.
w w w . T H U R J . o r g T H U R J V o l
,QGLDQDSROLV,1DWWUPLQFUHDWLQJDƬPWKLFNOD\HU
7KHSKRWRUHVLVWZDVWKHQH[SRVHGXVLQJDSUHGHÀQHGSKRWRPDVN
pattern and the MJB4 mask aligner (SUSS Microtech, Garching,
Germany). Photoresist was chemically developed using the SU-8
wet etch Developer (Microchem, Newton, MA).
7RFUHDWHWKH3'06PLFURÁXLGLFGHYLFH6\OJXDUG3'06
was mixed with the curing agent in a 10:1 ratio (Dow Corning,
Midland, MI), poured on top of the silicon wafer mold at
70°C for 45 minutes, and removed from the wafer. Holes were
mechanically punched. The same PDMS-curing agent mixture
ZDVDSSOLHGLQDƬPWKLFNOD\HURQDJODVVVOLGHXVLQJD'
spin coating apparatus (SUSS Microtech, Garching, Germany)
and partly cured in the oven for 25 minutes at 70°C. The fully
cured PDMS with the pattern imprinted on it was placed on top
of the partly cured PDMS on a glass slide, and the entire device
was placed back in the oven for 12 hours to allow for complete
curing and binding of the two PDMS layers.
In this process, the device design and function is dependent on
the shape of the photoresist, which can be adjusted based on the
photomask pattern and the thickness of the resist layer. Figure 1a
GLVSOD\VDVFKHPDWLFRI WKHÁRZIRFXVLQJGHYLFHGHVLJQZHXVHG
IRURXUVWXG\(DFKLQSXWFKDQQHOFRQWDLQVDVLQJOHÁXLGZLWKWKH
LQQHUPRVWLQSXWEHLQJWKHLQQHUOLTXLGSKDVHRI WKHGRXEOHGURS
the middle input the middle phase (the shell), and the outer phase
FRQVLVWLQJRI DQRLOÁXLGSKDVH7KHÀUVWLQSXWFKDQQHOUDQJHGLQ
ZLGWK IURP  ƬP 7KH PLGGOH SKDVH FKDQQHO UDQJHG IURP
ƬPDQGWKHRXWHUSKDVHFKDQQHOIURPƬP
In our system the inner phase (phase-1) consisted of a Dulbecco’s
Phosphate Buffered Saline solution (PAA, Pasching, Austria)
I I s s u e 1 S p r i n g 2 0 0 8
50
 Manuscripts
FIGURE 1A. A schematic
LOOXVWUDWLRQ RI D GRXEOH ¿RZ
IRFXVLQJPLFUR¿XLGLFV\VWHP7KH
circles represent the locations of
WKH WKUHH ¿XLG LQ¿RZV DQG RQH
¿XLG RXW¿RZ 7KH VROLG DUURZV
VKRZ GLUHFWLRQ RI ¿XLG ¿RZ
The PBS/sucrose solution is
hydrodynamically focused by the
73*'$ ¿RZ FUHDWLQJ HPXOVLRQV
DW WKH ¾UVW MXQFWLRQ 7KH 3%6
sucrose in TPGDA is in turn
hydrodynamically focused at
MXQFWLRQE\WHWUDGHFDQHOHDGLQJ
to double emulsions. The devices
UDQJHG IURP  FP ORQJ DQG
water in a TPGDA drop suspended in tetradecane should occur
under the condition S1 < 0, S2 < 0, and S3 > 0. The measured
LQWHUIDFLDOWHQVLRQVEHWZHHQWKHWKUHHÁXLGVDUHDQG
mN/m for the TPGDA/tetradecane, TPGDA/PBS-sucrose, and
tetradecane/PBS-sucrose interfaces, respectively; hence S1 < 0, S2
< 0, and S3 > 0, yielding stable double emulsions. We assume
WKHVHFULWHULDIRUVWDEOHGRXEOHHPXOVLRQVDWHTXLOLEULXPWRKROG
LQÁRZFRQGLWLRQV
In order to mathematically model the double encapsulation using
RXUFXUUHQWXQGHUVWDQGLQJRI VLQJOHHQFDSVXODWLRQVZHUHTXLUHG
WKHLQQHUDQGPLGGOHÁXLGVWRKDYHWKHVDPHYLVFRVLW\7KLVDOORZV
us to treat the single encapsulations at the second junction as a
VLQJOH ÁXLG 7KLV DVVXPSWLRQ LV H[WHQVLYHO\ XVHG DQG VLPSOLÀHV
calculations.12,13 73*'$KDVDYLVFRVLW\RI P3D‡V,QLWLDOZRUN
FKDQQHO ZLGWKV UDQJHG IURP  ZLWKZDWHUYLVFRVLW\P3D‡VDVWKHLQQHUSKDVH\LHOGHGLQIHULRU
FIGURE 1B. The chemical emulsions. A water-sucrose solution with 25% sucrose by mass at
formula for the polymer room temperature yielded the desired viscosity.15
middle phase, a mixture
(PXOVLRQ 3URGXFWLRQ The performance characteristics
of TPGDA (90%) and
alpha-hydroxycyclohexyl generated by our device are comparable to those produced by
ENGINEERING

phenylketone (10%) a previously described microcapillary device.12%\DGMXVWLQJÁRZ

linking agent. rates of the inner phase and middle phases (sucrose solution and
73*'$ UHVSHFWLYHO\ GULSSLQJ DQG MHWWLQJ ÁRZ UHJLPHV ZHUH
SRVVLEOH%RWKUHJLPHVUHVXOWHGLQGRXEOHHPXOVLRQV,I WKHÁRZ
mixed with 25% w/w sucrose. For the middle phase (phase-3) rate Q1RI WKHLQQHUSKDVHZDVVPDOOHUWKDQWKHÁRZUDWH43 of
we used the monomer tripropylene glycol diacrylate (TPGDA, the middle phase by at least a factor of 2, then a dripping regime
Aldrich) mixed with 10% w/w 1-Hydroxycycloheyl phenyl ketone UHVXOWHG DW WKH ÀUVW MXQFWLRQ )LJ D ,Q WKLV UHJLPH LQGLYLGXDO
(Aldrich) as a photoinitiator (Figure 1b). The outer phase (phase-2) GURSVRI VXFURVHVROXWLRQZHUHIRUPHGZLWKLQWKH73*'$ÁRZ
FRQVLVWHGRI WHWUDGHFDQH$FURV5HGÁXRUHVFHQWEHDGVƬPLQ $VLJQLÀFDQWDPRXQWRI 73*'$ZDVVSDFHGEHWZHHQWKHZDWHU
GLDPHWHU 'XNH 6FLHQWLÀF 3DOR $OWR &$ ZHUH PL[HG ZLWK WKH GURSOHWV$WWKHVHFRQGÁRZIRFXVLQJMXQFWLRQFRPSOHWHGRXEOH
inner phase at concentrations ranging from 5 to 10% w/w. All three emulsions resulted, interspersed at regular intervals with single
phases were immiscible and interfacial tensions were measured TPGDA droplets (Fig. 2b). Despite the poly-disperse character
using a K9 KRUSS ring tensiometer. Flowing the inner phase into of this process, it led to the greatest variety of drop sizes and shell
WKHPLGGOHSRO\PHUSKDVHUHVXOWHGLQHPXOVLÀFDWLRQ)LJXUHD WKLFNQHVVHV6KHOOWKLFNQHVVUDQJHGIURPƬPRURI 
7KHGURSOHWVZLWKLQWKHPLGGOHSKDVHÁRZHGLQWRWKHRXWHUSKDVH WKHGURSOHWUDGLXV)LJF'URSVZHUHSURGXFHGDWIUHTXHQFLHV
again emulsifying and resulting in double encapsulations (Figure
E 7KH PRYHPHQW RI  ÁXLGV ZDV FRQWUROOHG ZLWK FRPSXWHU FIGURE 2 FIGURE 3
driven syringe pumps (New Era NE-500 Syringe Pump Systems,
Farmingdale, NY). The UV crosslinking of the TPGDA shell
ZDVDFFRPSOLVKHGXVLQJDHET:0HUFXU\ODPS&XUWLV
Milwaukee, WI) focused through the 10x C Plan Leica lens to
produce a measured power of 106 W/cm2 at 365 nm wavelength.

RESULTS AND DISCUSSION

)OXLG 6HOHFWLRQ Both empirical observations and theoretical FIGURE 2A.7KH¾UVWMXQFWLRQRI3%6VXFURVHLVK\GURG\QDPLFDOO\

XQGHUVWDQGLQJ LQIRUPHG RXU ÁXLG VHOHFWLRQ 6SHFLÀFDOO\ WKH
ÁXLGV ZH XVHG ZHUH VFUHHQHG IRU DSSURSULDWH VXUIDFH HQHUJ\
characteristics and viscosity.
7RU]D DQG 0DVRQ KDYH VKRZQ WKDW WKH HTXLOLEULXP
FRQÀJXUDWLRQV RI  WKH WZR LPPLVFLEOH OLTXLGV GURSV VXVSHQGHG
LQDWKLUGLPPLVFLEOHÁXLGDUHJLYHQE\FRPSDULQJWKHVSUHDGLQJ
FRHIÀFLHQWVSi = ƣjk – (ƣij + ƣik), where ƣij are the interfacial energies
EHWZHHQ WKH WKUHH OLTXLGV  )RU RXU V\VWHP VWDEOH HQJXOÀQJ RI 
51 w w w . T H U R J . o r g T H U R J V o l
¿RZ IRFXVHG E\ 73*'$ GULSSLQJ UHJLPH FIGURE 2B. The
73*'$ ZLWK 3%6VXFURVH GURSOHWV DUH K\GURG\QDPLFDOO\ ¿RZ
focused by tetradecane. FIGURE 2C.$ZLGHYDULHW\RIVL]HDQG
shape emulsions are possible using this device. Scale bars are 100
țP FIGURE 3A. 3%6VXFURVH LV K\GURG\QDPLFDOO\ ¿RZ IRFXVHG
E\73*'$ DW WKH ¾UVW MXQFWLRQ ,Q WKH MHWWLQJ UHJLPH VKRZQ KHUH
WKLVLVDFRQVWDQW¿RZFIGURE 3B.7KH73*'$ZLWK3%6VXFURVH
GURSOHWVLVK\GURG\QDPLFDOO\¿RZIRFXVHGE\WHWUDGHFDQHFIGURE
3C. $ YDULHW\ RI VL]H DQG VKDSH GRXEOH HPXOVLRQV DUH SRVVLEOH
I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

ranging from 3-190 Hz. parameters. The average number of beads per double encapsulation
The second regime occurred when the inner phase rate was DWHDFKÁRZUDWHUDQJHGIURPWRZLWKVWDQGDUGGHYLDWLRQV
larger than the middle phase rate by at least a factor of 2. In this ranging from 0.93 to 2.96. Figure 5a contains pictures of these
UHJLPHWKHLQQHUSKDVHMHWGLGQRWEUHDNLQWRGURSOHWVDWWKHÀUVW double encapsulations, and Figure 5b displays the distributions of
MXQFWLRQ)LJDUHVXOWLQJLQFRD[LDOÁRZRI ZDWHUDQG73*'$
DIWHU WKH ÀUVW MXQFWLRQ 7KH VLJQLÀFDQWO\ ODUJHU ÁRZ UDWH DW WKH
second junction caused the drops to pinch off (Fig. 3b), thus
producing thin-shelled double emulsions. In this regime, shell
WKLFNQHVV UDQJHG IURP  ƬP RU  RI  WKH GURS UDGLXV
This second double emulsion regime was most similar to the one
previously described using a microcapillary device.12
0RGHOLQJ 'RXEOH (PXOVLRQV With several small
PRGLÀFDWLRQV ZH IRXQG WKH PRGHO GHULYHG E\ 8WDGD HW DO12 to
accurately describe the double emulsions we generated using our
PLFURÁXLGLFGHYLFH7KH8WDGDPRGHOZDVGHYHORSHGIRUWKHFDVH
of drop breakup in a radially symmetric microcapillary device. It
GHVFULEHVWKHGURSUDGLXVDVDIXQFWLRQRI WKHRXWHUSKDVHÁRZ
rate (Qof):
(1)
The model is shown to be consistent with the empirical results
relating Rdrop = 1.87Rjet. As our devices were created with soft
lithography and were rectangular, not circular, we used the stan-
GDUGFDOFXODWLRQIRUHTXLYDOHQFHUDGLXV18:
(2)
where A is the cross-sectional area and P is the wetting perimeter.
$VRXURULÀFHZDVƬPZLGHDQGƬPKLJKWKHHTXLYDOHQFH
UDGLXV ZDV  ƬP 3ORWWLQJ WKLV PRGHO DJDLQVW WKH REVHUYHG
data yields a good agreement among observations, theoretical
calculations, and prior work,12ZLWKDFRUUHODWLRQFRHIÀFLHQW52)
beads per double encapsulation. The number of beads per double
encapsulation was adjusted by changing the bead concentration
LQ WKH LQQHU SKDVH VROXWLRQ DQG E\ FKDQJLQJ WKH ÁRZ UDWH UDWLR
for the inner and middle phases. The higher the inner phase bead
concentration, the more beads were observed encapsulated in
the resulting double encapsulation. Decreasing the ratio of inner
SKDVHÁRZUDWHWRPLGGOHSKDVHÁRZUDWHLQFUHDVHGWKHQXPEHURI 
beads per double encapsulation. Finally, as discussed previously,
LQFUHDVLQJ WKH RXWHU SKDVH ÁRZ UDWH LQFUHDVHG GURSOHW YROXPH
which increased the number of beads per droplet as well.
89 'RXEOH 'URSOHW &URVVOLQNLQJ To crosslink the double
HQFDSVXODWLRQVWKHFHOOVÁRZLQJLQWKHH[LWFKDQQHOZHUHH[SRVHG
WR89OLJKW7KHH[SRVXUHWLPHGHSHQGHGRQWKHUDWHRI ÁRZLQWKH
PLFURÁXLGLFFKDQQHODVWKHOLJKWVRXUFHZDVÀ[HG7RHYDOXDWHWKH
HQFDSVXODWLRQVZHLQVSHFWHGWKHPXVLQJÁXRUHVFHQFHPLFURVFRS\
ENGINEERING
to ensure that the objects were in fact double encapsulations. We
then dried the encapsulations, placed them on a glass slide, added
a coverslip, and applied pressure to crack the shells. After cracking
WKHVKHOOVQRÁXRUHVFHQWEHDGVZHUHREVHUYHGLQVLGHWKHREMHFWV
DQGVLJQLÀFDQWÁXLGZDVREVHUYHGRQWKHPLFURVFRSHVOLGH)LJXUH
6). We concluded that we had obtained the desired cross-linked
objects.
CONCLUSION
In this study we developed a novel platform for encapsulating
microbeads suspended in a biocompatible buffer solution within
DVROLGVKHOORI 73*'$7KHSODWIRUPLVÁH[LEOHLQWKDWLWFDQ
produce double emulsions of varying size and cell concentration.
HTXDOWR)LJXUH We can control the size and shape of these emulsions through
0RGHOLQJ&HOOXODU(QFDSVXODWLRQ:HXVHGÁXRUHVFHQWƬP PDWKHPDWLFDO PRGHOLQJ RI  GLIIHUHQW ÁRZ UDWH SDUDPHWHUV 7KH
SRO\VW\UHQHEHDGV'XNH6FLHQWLÀFWRDQDO\]HFHOOHQFDSVXODWLRQ cell concentration in the double emulsions may be controlled by
FIGURE 4 FIGURE 5A FIGURE 5B

FIGURE 4. 'RXEOHHPXOVLRQUDGLXVYHUVXV¿RZUDWHVLQWKHMHWWLQJUHJLPH7KHGRWWHGOLQHLQGLFDWHVWKHSUHGLFWHGYDOXHDWHDFKSRLQW
based on our model. Squares represent the observed outer radius and circles represent the observed inner radius. For all experiments
WKHVXPRILQQHUPLGGOHDQGRXWHUSKDVH¿RZUDWHV4if4mf4ofZDV¾[HGDWț/PLQDQG4LI4PIZDV¾[HGDW3ORWWLQJWKHPRGHO
DJDLQVWWKHREVHUYHGGDWD\LHOGVDJRRGDJUHHPHQWEHWZHHQHPSLULFDOREVHUYDWLRQVDQGWKHRUHWLFDOFDOFXODWLRQV
FIGURE 5A.5HSUHVHQWDWLYHSLFWXUHVRIGRXEOHHQFDSVXODWLRQVZLWK¿XRUHVFHQFHIURPWULDOVDQGIRUFRPSDULVRQSXUSRVHV7KHVFDOH
EDUVDUHțPFIGURE 5B. A histogram of the percentage of beads per double encapsulation at each trial.

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
52
 Manuscripts
TABLE 1. The rate of PBS + sucrose/TPGDA/tetradecane and the grams
RI 3%6EHDGVVXFURVH LQ HDFK VROXWLRQ DV ZHOO DV WKH PHDQ DQG VWDQGDUG
deviation in each trial
DGMXVWLQJ WKH ÁRZ UDWH SDUDPHWHUV DQG FRQFHQWUDWLRQ :H KDYH
GLVFXVVHGDQDSSURDFKWRVHOHFWLQJDSSURSULDWHÁXLGVWKDWIXQFWLRQ
within this platform. We have also provided a comparison to the
PLFURFDSLOODU\DSSURDFKWRGRXEOHHQFDSVXODWLRQVDQGÀQGWKDWLW
is similarly capable.
Although beads were used in place of cells, this is a standard
SUDFWLFH LQ WKH PLFURÁXLGLF OLWHUDWXUH13,17,19 Much prior work
VXJJHVWVWKDWLQDPLFURÁXLGLFV\VWHPFHOOVRI FRPSDUDEOHVL]HDQG
density to the given microbeads will behave in the same manner.
Microbeads are accepted as a model to simplify experimental
procedures and minimize cost, since the physics governing the
ENGINEERING
system is the same.
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work12,18EHFDXVHLWDOORZVIRULQWHJUDWLRQZLWKRWKHUPLFURÁXLGLF
systems that have been previously developed using standard
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FIGURE 6A. &URVVOLQNHG GRXEOH HQFDSVXODWLRQV ZHUH H[WUDFWHG
from the device and placed onto a glass slide. A glass cover slip
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photographed.
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RIWKHFDSVXOHVLQGLFDWLQJWKDWWKHGRXEOHHPXOVLRQVZHUHLQGHHG
completely broken.
T cell antigen receptor signaling thresholds and intraclonal heterogeneity for
individual cytokine responses of CD4+ T cells,” The Journal of Experimental
Medicine 186 (5), 757-766 (1997).
6. '1%UHVODXHU3-/HHDQG/3/HH´0LFURÁXLGLFVEDVHGV\VWHPVELRORJ\µ
Molecular BioSystems 2 (2), 97-112 (2006).
7. Y. H. Cho, N. Takama, T. Yamamoto et al., “MEMS-based biochip for the
characterization of single red blood cell,” Microtechnology in Medicine and
Biology, 2005. 3rd IEEE/EMBS Special Topic Conference, 60-63 (2005).
8. S. Anna, N. Bontoux, and H.A. Stone, “N of Dispersions Using “Flow
Focusing” in Microchannels,” Applied Physics Letters 82 (3), 364-366 (2003).
9. D. R. Link, S. L. Anna, D. A. Weitz et al., “Geometrically Mediated Breakup of
designed cell analysis systems19 and cell manipulation systems20
could be integrated with this device to enable novel functionality
within a larger system. Also, it allows for recovery of the cells
from the double encapsulation, via a second polymer crosslinking
agent that can break to permit cell recollection. Future work
with this platform could enable cell culturing of individual
or very few cells at a time within a microcapsule or single-cell
drug screening experiments. Beyond the life sciences domain,
generating controlled polymer shells may be of interest to lab-on-
DFKLSFKHPLVWU\ZRUNZKHUHVPDOOTXDQWLWLHVRI UHDJHQWVFRXOG
'URSVLQ0LFURÁXLGLF'HYLFHVµ3K\VLFDO5HYLHZ/HWWHUV
10. Y. H. Cho and J. Park, “Evaluation of process parameters in the O/W/O
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68 (2), 534-538 (2003).
11. H. A. Stone, A. D. Stroock, and A. Ajdari, “Engineering Flows in Small Devices
0LFURÁXLGLFV7RZDUGD/DERQD&KLSµ$QQXDO5HYLHZRI)OXLG0HFKDQLFV
36, 381-411 (2004).
12. A. S. Utada, E. Lorenceau, D. R. Link et al., “Monodisperse Double Emulsions
Generated from a Microcapillary Device,” Science 308 (5721), 537 - 541
(2005).
13. + =KDQJ ( 7XPDUNLQ 5 3HHUDQL HW DO ´0LFURÁXLGLF 3URGXFWLRQ RI
Biopolymer Microcapsules with Controlled Morphology,” J. Am. Chem. Soc.
128 (37), 12205-12210 (2006).
14. D. C. Duffy, J. C. McDonald, O. J. A. Schueller et al., “Rapid Prototyping
EHUHDFWHGZLWKLQWKHLQQHUOLTXLGSKDVHRI WKHHPXOVLRQDQGODWHU RI 0LFURÁXLGLF 6\VWHPV LQ 3RO\GLPHWK\OVLOR[DQHµ $QDO &KHP  
recovered from within the polymer shell. 4974-4984 (1998).
15. M. Mathlouthi and J. Génotelle, in Sucrose Properties and Applications, edited
by M. Mathlouthi and P. Reiser (Blackie Academic & Professional 1995), pp.
ACKNOWLEDGEMENTS 126-154
16. 1LFRODV 3DQQDFFL LQ &RQJUqV *pQpUDO GH OD 6RFLpWp )UDQoDLVH GH 3K\VLTXH
(Grenoble, France, 2007).
The author thanks Hervé Willaime and Vincent Studder for 17. N.-T. Nguyen and S.T. Wereley, Fundamentals and applications of
their support and the assistance of Howard Stone for his many PLFURÁXLGLFVQGHG$UWHFK+RXVH%RVWRQSS[LLLS
fruitful conversations. 18. J. W. Kim, A. S. Utada, A. Fernandez-Nieves et al., “Fabrication of
0RQRGLVSHUVH*HO6KHOOVDQG)XQFWLRQDO0LFURJHOVLQ0LFURÁXLGLF'HYLFHVµ
Angew Chem. Int. Ed. Engl. (2007).
19. $5:KHHOHU:57KURQGVHW5-:KHODQHWDO´0LFURÁXLGLF'HYLFHIRU
REFERENCES Single-Cell Analysis,” Electrophoresis 22, 283-288 (2001).
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development,” International Surgery 91 (5 Suppl), S55-62 (2006).
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Synthesis of a bioresorbable electrospun guide

tube for spinal cord regeneration
AMAREEP SINGH GREWAL1

MIT Institute for Soldier Nanotechnologies, 77 Massachusetts Avenue, Cambridge, MA 02139, USA,1

A novel electrospun nerve guide tube has recently been synthesized for spinal cord nerve regeneration.
The guide tube was created from the bioresorbable polymers poly(E-caprolactone) (PCL) and poly(D-
lactic-co-glycolic acid) (PLGA). To optimize the strength and elasticity of the guide tube, various
concentrations and blends of PCL and PLGA have been mixed and electrospun. Mechanical testing
was conducted to determine the modulus, toughness, and the stress and strain at the fracture point
of the tubes. These values were compared amongst the various tubes, and then compared against
guide tubes that had been annealed to promote crystallization. The degree of crystallinity present
within the electrospun tubes was determined by differential scanning calorimetry. The annealed guide
tube created by a blend of 5% PCL and 5% PLGA solution in 3:1 chloroform:methanol showed the
greatest strength and elasticity. The morphological studies that were performed showed the changes
that occurred within the scaffold while in tension and in a hydrated environment. In vitro and in vivo
tests in mice suggest that the tubular scaffold allows for cellular migration and attachment. This nerve
guide tube shows great promise for soldiers who are injured while at war, or for civilians who suffer
from spinal cord trauma.

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____________________________________________________
INTRODUCTION inhibitor of regeneration in the injured spinal cord. Scar tissue,
ZKLFKLVULFKLQP\RÀEUREODVWVDOVRFRQWDLQVPROHFXOHVNQRZQDV
Each year over ten thousand new cases of spinal cord nerve FKRQGURLWLQVXOIDWHSURWHRJO\FDQVZKLFKGHOD\QHUYHÀEHUJURZWK
injuries are reported in the United States alone.1 Physical trauma to by forming neuromas and nerve stumps at the site of injury.2
the mammalian spinal cord initiates a series of pathophysiological It has recently been suggested that the difference in successful
events that result in progressive tissue loss and the formation of regeneration between the CNS and the PNS is due to anatomical
large cavities at the site of injury. In humans, these cavities often differences in the two systems. The endoneurial sheaths of the
span the entire diameter of the spinal cord, leaving behind only a PNS, which are credited for nerve regeneration within the PNS,
small rim of white neuronal matter. Therefore, long descending are absent in the CNS. Axons present at the edge of a nerve stump
and ascending supraspinal pathways are interrupted, leading become misaligned within twenty-four hours after injury and lack
to a loss in locomotive and processing functions.2 Defects in the ability to elongate substantial distances in a straight line3. For
VSLQDOFRUGQHUYHVUHPDLQDFKDOOHQJLQJDQGGLIÀFXOWSUREOHPLQ this reason, it has been hypothesized that spinal axon elongation
reconstructive surgeries. fails to occur in the CNS because axons lack appropriate guiding
Injuries to the spinal cord are often caused by shattered tracks needed for the relocation and fusion of a neuroma and
vertebral bones or intervertebral disks that puncture the spinal nerve stump.
cord. Such injuries vary in severity, and may result in paraplegia, Current methods used to heal spinal cord injuries involve the
tetraplegia, or full body paralysis.2 Unlike the tissues of the use of tissue grafts. These grafts may have arrived from another
peripheral nervous system (PNS), tissues of the central nervous location in a patient’s body, or from another person altogether. In
system (CNS) are highly resistant to regeneration. It is believed the latter case, a patient is more susceptible to both rejection and
that scar tissue, formed after an injury to the CNS, is a major LQÁDPPDWRU\UHVSRQVH3 However, even with autologous grafts, it
is impossible to recreate a damaged nerve completely. Autologous
_______________________________ grafts often lead to nerve failure in the tissue’s area of origin.
* Corresponding author. Mailing Address: 7350 Coldspring A newly discovered alternative to tissue grafts is the insertion
/DQH :HVW %ORRPÀHOG 0,  (PDLO DJUHZDO#IDVKDU- of a tube into the spinal cord area to connect the injured nerve
YDUGHGX3KRQH stumps. The use of a tubular scaffold in the injured spinal cord
area improves axonal regeneration by limiting the buildup of scar
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
54
 Manuscripts

tissue. The tube-like structure may also promote proper neural

regeneration by guiding a neuroma and nerve stump toward one FIGURE 3.
another. Electrospinning
Apparatus. The
Synthetic guide tubes can be made from polymeric materials
image above
and may be non-resorbable or resorbable. Non-resorbable tubes shows different
remain present as foreign bodies around the regenerated nerve components
and require a second surgery for removal. By contrast, resorbable of the
tubes naturally degrade by hydrolytic degradation. However, re- electrospinning
sorbable tubes that degrade too quickly may not provide enough apparatus,
including
time and adequate protection for nerve regeneration. If nerve
the voltage
guides break down at an early stage, scar tissue can form within a supply, ground
tube and impair further maturation of the regenerating nerve. collector, and
Ideally, a nerve guide should be composed of a biocompatible syringe. (Image
and bioresorbable material that degrades at a controlled rate Source: http://
in accordance with the rate of axonal growth and maturation, www.mtu.edu/
electrospin)
while also maintaining mechanical continuity and lumen stability.
Two biodegradable polymers that have the potential to be permeable one.
incorporated in nanodevices, such as tube conduits, are poly(D- 2QHPHWKRGRI GHYHORSLQJSRURXVÀEURXVWXEHVIURP3/*$
lactic-co-glycolic acid) (PLGA) and poly(E-caprolactone) (PCL). and PCL is electrospinning. Such tubes are formed by applying a
PLGA, a copolymer of glycolic and lactic acids, is used in a host high voltage to a PLGA or PCL polymer solution. The high volt-
of therapeutic devices due to its biodegradability. age overcomes the surface tension of a polymer solution within a
Beyond biodegradability, it is important to make sure that a nerve syringe, resulting in the ejection of the solution from the spinneret
conduit does not induce cellular toxicity. One way of achieving of the syringe. Upon ejection, a series of whipping events occur
this is by ensuring that the byproducts of the polymer in question within a region known as the band of instability, causing all sol-
are naturally metabolized by the body, which is the case for PLGA, vent within the polymer solution to evaporate. The result of this
which degrades into carbon dioxide and water [5]. For this reason, DFWLRQLVWKHIRUPDWLRQRI ÀEHUVWKDWDUHQDQRPHWHUVWRPLFURP-
eters in diameter.10 7KHVH ÀEHUV FROOHFW RQ D QHJDWLYHO\ FKDUJHG
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there is little, if any, systematic toxicity after the introduction of

PLGA to the body. In vitro tests have also demonstrated that no VXUIDFHDQGFDQEHPDGHWRPLPLFWKHQDWXUDOÀEHUVIRXQGZLWKLQ
toxicity occurs upon the hydrolytic degradation of PCL’s ester the extracellular matrix in size and orientation since they form a
linkages in the human body. unique non-woven mesh. Hence, this method represents a favor-
Current synthetic guide tubes are often impermeable. While they DEOHDSSURDFKWRZDUGVWKHIDEULFDWLRQRI ÀEURXVQHUYHFRQGXLWV
do aid in the elongation of myelinated axons, they often prohibit for spinal cord regeneration.
the migration of growth factors and essential nutrients into the Although an electrospun conduit nerve tube offers greater
injury site. The lack of neuronal growth factors at the site of strength than a solid polymer tube, it is still susceptible to tear-
injury may delay axonal elongation, thus increasing recovery time.7 ing and crushing during fabrication, implantation, and recovery.
Impermeable guide tubes also show increased rates of degradation Ideally, a nerve guide should be able to extend in case of muscle
due to the increased polymer content, and upon degradation, movements which cause the tube to move. At the same time, if an
these tubes release acidic byproducts into the extracellular matrix, organ suddenly applies a large force on a nerve guide, not allowing
LQGXFLQJDQLQÁDPPDWRU\UHVSRQVH Favorable aspects of porous for extension time, the nerve guide should be able to manage the
tubes include the possibility for metabolic exchange, and the heavy load. This paper explores ways to engineer an electrospun
retention of trophic factors secreted from nerve stumps by creating nerve conduit with the properties of elasticity and strength. Prop-
more sites for HUWLHV LQFOXGLQJ ZDOO WKLFNQHVV ÀEHU VL]H SRO\PHU FRPSRVLWLRQ
growth factor and crystallinity are investigated to characterize the synthesized
attachment. nerve conduit both mechanically and morphologically. In addi-
Great effort tion, in vitro and in vivo tests are used to determine the conduit’s
has been functionality and nerve growth host properties. These tests help
exerted in engineer a more robust and optimized nerve conduit to aid civil-
studying ians and soldiers suffering from spinal cord nerve trauma.
methods to
FIGURE 1. Molecular Structure of PLGA convert an MATERIALS AND METHODS
imper meable
p o l y m e r Preparation of Electrospinning Solutions. Polycaprolac-
material into WRQH 3&/ RI  PROHFXODU ZHLJKW  JPRO DQG SRO\ODFWLF
FIGURE 2. Molecular Structure of PCL a porous and FRJO\FROLFDFLG3/*$RI PROHFXODUZHLJKW²JPRO
were obtained from Sigma-Aldrich. A solvent solution comprised
55 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

of one liter of methanol and three liters of chloroform was then ïRQWKH6(0DVZHOO$QDO\VLVVRIWZDUHE\6RIW,PDJLQJ6\V-
mixed. To create solutions of varied concentrations (12% PLGA, tems was used to determine the exact diameter of individual elec-
 3/*$  3&/  3&/  3&/ 3/*$  WURVSXQÀEHUV7RGHWHUPLQHWKHH[DFWWKLFNQHVVRI WKHZDOORI 
3&/ 3/*$  3&/ 3/*$ WKH DSSURSULDWH
amounts of PLGA and PCL were weighed on an analytical bal-
ance and incorporated into the solvent solution using a magnetic
stirrer.
Electrospinning Nerve Regeneration Tubes. To prepare
the electrospinning apparatus, a PHD 2000 syringe pump (Har-
vard) was loaded with a syringe containing polymer solution, and
WKHV\ULQJHZDVWKHQFRQQHFWHGWRDVSLQQHUHWQHHGOHXVLQJ7HÁRQ
WXELQJ,QVXEVWLWXWLRQIRUDJURXQGFROOHFWRUSODWHDJDXJH
cm bare copper wire was placed approximately 35 cm from the
spinneret. To begin the electrospinning, the syringe pump was set
WRDÁRZUDWHRI P/PLQDQGEHWZHHQDQGNLORYROWV
were applied to the ground plate. The nerve tubes were formed
E\URWDWLQJWKHFRSSHUZLUHXQGHUWKHVSLQQLQJÀEHUVIRUDSUHGH-
the conduits, samples of bare copper wire were viewed under the
SEM to determine their diameter. The guide tube wall thickness
was found by subtracting the bare copper wire diameter from the
GLDPHWHURI WKHFRSSHUZLUHZLWKWKHÀEURXVWXEHRQLWDQGWKHQ
dividing by two.
Annealing Electrospun Samples. To increase the strength
of electrospun guide tubes, some samples that were electrospun
IRUVHFRQGVZHUHDQQHDOHGDWÜ&IRUKRXUV%HFDXVHWKH
PHOWLQJ SRLQW RI  3&/ LV Ü& WKH WKHUPDO HQHUJ\ ZDV DEOH WR
convert some amorphous segments of PCL into a crystalline
phase. The crystalline phase of a polymer is believed to increase
the toughness and strength of the polymer by rearranging mo-
lecular bonds into an organized lattice. These annealed samples
were characterized by differential scanning calorimetry and UTM
WHUPLQHGWLPHSHULRGRUVHFRQGVLQRUGHUWRFUHDWHD
uniform scaffold around the entire circumference of the wire.
Separation of Nerve Regeneration Tubes. After elec-
WURVSLQQLQJZDVFRPSOHWHGWKHÀEURXVWXEHZDVUHPRYHGIURP
the copper wire by pinching one end of the copper wire while
VWHDGLO\PRYLQJWKHÀEURXVWXEHXSZDUGVXQWLOWKHHQWLUHWXEHZDV
separated from the wire. This removal method introduced crin-
kles into the polymer nerve conduit, so samples were stretched
back to their original lengths.
Hydrating Nerve Guide Tubes. Upon removal from the
copper wires, some nerve regeneration tubes were placed in dis-
tilled water for a period of 2 weeks. Hydration of the scaffolds
was used to simulate the hydrated internal environment of the hu-
man body. After two weeks, scaffolds were removed, lyophilized
and freeze-dried for Scanning Electron Microscopy.
Scanning Electron Microscopy. Samples of tubes still
mounted on copper wire and lyophilized tubes were viewed using
Nano-Mechanical testing.
Mechanical Testing. Mechanical testing was performed to
obtain stress and strain curves, which were then used to determine
the ultimate force and strain required to break the electrospun
guide tubes. The graphs and data charts provided by the software
for the testing system also provided important information re-
garding the modulus of the sample, which quantitatively scores
the elasticity and strength of that sample. Toughness is calculated
by measuring the area under the stress and strain curve. Samples
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for tensile testing were mounted on cardboard sample holders.
Epoxy was applied to the ends of the tubes on the cardboard, and
VDPSOHVZHUHDOORZHGWRGU\IRUKRXUVEHIRUHWHVWLQJ7HQVLOH
testing was conducted using a MTS Nano-UTM Testing System
to stretch the nerve conduit. Data was recorded by the UTM-
%ORZ 6WDQGDUG 7RHFRPS 4XDVLVWDWLF SURJUDP RQ 7HVW :RUNV 
6RIWZDUH7HVWLQJSDUDPHWHUVLQFOXGHGDVWUDLQUDWHRI ï-3
PPVHFXSWRDPD[LPXPVWUDLQRI PPPP
D-(2/-606FDQQLQJ(OHFWURQ0LFURVFRSH6(06OLGHV
were prepared by applying samples to a circular aluminum holder.
Samples were then coated with platinum nanoparticles for 70 sec-
onds using a Polaron Range Sputter Coater to make the surface
conductive. After the platinum coating was applied, samples were
YLHZHGXQGHUQHDWKWKH6(0DWYDULHGPDJQLÀFDWLRQV7KHUHODWLYH
GHJUHHV RI  ÀEURXV DQG EHDG SKHQRW\SHV LQ WKH QHUYH FRQGXLWV
ZDVGHWHUPLQHGE\LPDJHDQDO\VLVDWPDJQLÀFDWLRQVRI ïDQG
FIGURE 4.
Setup Used
in Procedure.
To electrospin
Differential Scanning Calorimetry. Differential scanning
calorimetry was performed to obtain information regarding the
change in crystallinity of the annealed samples. The melting point
and the enthalpy of fusion were determined by a Q100 Auto
Modulated Differential Scanning Calorimeter (TA Instruments)
VHW WR D KHDWLQJ UDWH RI  Ü&PLQXWH $OO VDPSOHV ZHUH KHDWHG
IURPÜ&WRÜ&FRROHGGRZQEDFNWRÜ&DQGWKHQWKHSUR-
cess was repeated once again.
In vitro Cell Seeding. Nerve regeneration scaffolds were
sterilized and injected with neuron promoting growth factor,
Schwann cells (Invertigen) and self-assembling peptides. Nerve
cells from a fetal rat medulla cell line (Invertigen) were maintained
nerve con- LQ'XOEHFFR·V0RGLÀHG(DJOHV0HGLXP'0(0VXSSOHPHQWHG
duits, a copper ZLWK)HWDO%RYLQH6HUXPIRUKRXUVFRQÁXHQWQHX-
wire was held URQVZHUHVXVSHQGHGLQZYDJDURVH&DPEUH[)LVKHU6F
between the
3$ VROXWLRQ LQ 6)'0(0 WR REWDLQ  [  FHOOVPO &HOOV
spinneret and
collector. This were then transferred to the ends of the nerve regeneration tubes
image depicts XVLQJ D PLFURSLSHWWH ƬO RI  6)'0(0 ZHUH DGGHG WR HDFK
the setup used RI WKHVDPSOHVDQGVDPSOHVZHUHWKHQLQFXEDWHGDWÜ&6XSHU-
to create the QDWDQWVROXWLRQZDVFKDQJHGHYHU\KRXUVDQGDIWHURQHZHHN

w w w . T H U R J . o r g
nerve conduits
T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
56
 Manuscripts
FHOOV ZHUH À[HG E\ DGGLQJ ƬO  PHWKDQRO 6DPSOHV ZHUH
WKHQH[SRVHGWRLPPXQRÁRXUHVFHQWG\HVIRUPLFURVFRS\DQGLP-
aging.
In vivo Testing. In vivo tests were performed in Sprague-Dawley
rats weighing 225-275 g (Charles River). Sodium pentobarbital
FIGURE 5. 6(0,PDJHVRI1HUYH&RQGXLWV7KH¾UVWVHWRILPDJHV
are taken at !50, and shows the entire tube diameter. The bottom
VHWLVDWíDQGVKRZVWKH¾EURXVPHVK7KHSRO\PHUFRPSRVL-
tion of the tubes is seen in the upper left.
beads serve no purpose in a nerve conduit, as they take up
essential space where neurons could potentially migrate and
attach. The tubes for 12% PLGA, 7.5%:2.5% PLGA:PCL and
3/*$3&/KDYHÀEHUVRI GLIIHUHQWVL]HVWKDWGRQRW
form a uniform three dimensional matrix. The tubes need the
ÀEHUVWRKDYHDFRQVLVWHQWÀEHUVL]HDFURVVWKHWXEHVRWKDWLWLV
possible to maintain a relatively constant degree of porosity. The
3&/JXLGHWXEHVODFNHGSRO\PHUEHDGVKRZHYHUWKHÀEHUV
of the tube form an extremely porous structure which could allow
LQÁDPPDWRU\ FHOOV WR SHQHWUDWH WKH ZRXQG VLWH ,QÁDPPDWRU\
cells interrupt neuronal migration, thus prolonging regeneration
time.3 The conduit produced by a 1:1 ratio of PCL to PLGA,
KRZHYHUVDWLVÀHVWKHSUHYLRXVO\RXWOLQHGPRUSKRORJLFDOFULWHULD
necessary for a successful guide tube, including porosity and a
lack of beads.
Fiber Diameter of Electrospun Guide Tubes. Fiber di-
ameter was investigated based on the concentration of polymer
SUHVHQWZLWKLQDQHUYHFRQGXLW7KHÀEHUGLDPHWHUZLWKLQDWXEH
ultimately regulates the porosity, strength and elasticity of that
PJNJZDVXVHGDVDJHQHUDODQHVWKHWLFLQDOOSURFHGXUHV
Maintaining aseptic technique, microsurgical exposure of the
spinal cord was undertaken through a dorsal incision. A 3-cm
segment of the spinal cord was resected from each animal and
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proximal and distal nerve ends of different nerve stumps were
inserted into the optimized nerve conduit. The skin was then
FORVHGXVLQJ'H[RQVXEFXWLFXODUVXWXUHV
Microscopy and Imaging. Images of the cross sections of
the in vitro nerve tubes with nerve cells were obtained by using
RSWLFDOOLJKWPLFURVFRS\DWPDJQLÀFDWLRQVRI ïDQGï)RXU
weeks after the surgical procedure was performed in the rats,
sutures were opened, and photographs were taken of the spinal
cord nerve conduits. Images were used to determine vascular
growth and tube compatibility within the rats. The implanted
QHUYH FRQGXLWV ZHUH WUDQVHFWHG DQG ZHUH À[HG ZLWK  P/ RI 
1.5% methanol. In their place, a new nerve conduit was implanted
within the rats. The assessment of the innervation process in the
in vivoPRGHOZDVYLVXDOL]HGE\WKHLPPXQRÁRXUHVFHQWVWDLQLQJRI 
neurons in migration and in growth. Samples were viewed using a
Leica Confocal Microscope.
RESULTS AND DISCUSSION
Morphological Analysis of Nerve Conduits. Viewing the
nerve guide tubes using SEM allowed for the characterization of
WKHYDULRXVWXEHVEDVHGRQSRURVLW\ÀEHUVL]HDQGWKHSUHVHQFH
of a three dimensional network and polymer beads. The desired
phenotype was one in which the tube would form a three
GLPHQVLRQDOÀEURXVQHWZRUNZLWKSRUHVWKDWZRXOGDOORZH[WHUQDO
growth factors to diffuse into the conduit. Analyzed SEM images
are displayed in Figure 5. These images show that some of the
nerve guide tubes, including the 7% PCL and 20% PLGA, have
polymer beads present in their matrices in abundance. Polymer
57 w w w . T H U R J . o r g T H U R J V o l
WXEH7KHDYHUDJHÀEHUVL]HZLWKLQWKHGLIIHUHQWQHUYHFRQGXLWV
FDQEHVHHQLQ)LJXUH'LIIHUHQWFRQFHQWUDWLRQVRI WKHVDPH
SRO\PHU FUHDWHG HOHFWURVSXQ ÀEHUV RI  YDULHG GLDPHWHUV $ JHQ-
eral trend that the data exhibit is that for any given polymer, as
WKH VROXWLRQ FRQFHQWUDWLRQ LQFUHDVHV VR GRHV WKH DYHUDJH ÀEHU
diameter. For PLGA samples, when the solution concentration
LQFUHDVHGE\WKHDYHUDJHÀEHUGLDPHWHULQFUHDVHGE\PL-
crometers. However, for PCL samples, there is a greater change
LQÀEHUGLDPHWHUDVWKHFRQFHQWUDWLRQLQFUHDVHVIURPWR
This difference may be attributed to the molecular structure of
PLGA and PCL. Due to the extra oxygen atom it possesses in
comparison with PCL, PLGA is more electrically conductive than
PCL. As PLGA is charged during electrospinning, it is more likely
WRVHOIUHSHODQGVSOD\LQWRVPDOOHUÀEHUV3&/ZLOOQRWVHOIUHSHO
DWDVKLJKDGHJUHHDQGWKHUHIRUHIRUPVODUJHUÀEHUVDVLWVFRQ-
centration increases. The blended scaffolds also exhibit this phe-
nomenon. The blends of PCL and PLGA that have higher con-
FHQWUDWLRQVRI 3/*$KDYHVPDOOHUÀEHUVRQDYHUDJH7KHVDPSOH
RI 3/*$DQG3&/LQDUDWLRKDGDQDYHUDJHÀEHUVL]HRI 
PLFURPHWHUVZKLOHIRUWKHVROXWLRQLQDUDWLRWKHDYHUDJHÀ-
EHUVL]HZDVPLFURPHWHUV$ORQJZLWKHOHFWULFDOFRQGXFWLYLW\
other properties that may cause this difference are the viscosity
and elasticity of the solutions, and the dielectric properties of the
solvent and polymer.
$IWHU GHWHUPLQLQJ WKH ÀEHU VL]HV RI  YDULRXV VDPSOHV LW ZDV
QHFHVVDU\WRVHHZKLFKÀEHUVL]HVRSWLPL]HGWKHWRXJKQHVVRI WKH
electrospun guide tubes. To compare the samples, the toughness
of each tube was obtained by UTM Nano-Mechanical Testing;
WKHVH YDOXHV DUH VHHQ LQ )LJXUH  )RU ERWK SRO\PHUV DV ÀEHU
size increased, so did the toughness of each corresponding
nerve conduit. The toughness, which is represented by the area
underneath a stress and strain curve, indicates how much force
I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

Toughness of PLGA and PCL Guide Tubes at

can be applied to a tube along with the elasticity of the tube Varying Fiber Sizes
before it fractures. A higher toughness directly corresponds to a 4 P LG A F ibers
3.5
more elastic tube that can withstand high forces. The data appear P C L F ibers

Tougness (MPa)
3
WRÀWDQH[SRQHQWLDOFXUYHDVGHWHUPLQHGIURPÀQGLQJWKHEHVWÀW 2.5
FXUYHVDQGFDOFXODWLQJWKHFRUUHODWLRQYDOXHVRI DQG 2
Nerve Guide Wall Thickness. %HVLGHV LQGLYLGXDO ÀEHU GL- 1.5
1
ameter, it was important to determine how the wall thickness of 0.5
nerve guides would affect the overall toughness of the guide it- 0
self. Calculations from Image Analysis Software showed that tube 0 1 2 3 4

wall thickness and electrospinning time directly correlate with one Diameter of Fibers (micrometers)

another. However, it was still possible to obtain samples with var-

ied wall thicknesses by altering polymer concentrations. Differ- FIGURE 7. Toughness of Nerve Conduits Based on Fiber Size.
ent trends were observed for PCL and PLGA samples. For PCL 7KH GDWD IRU WXEH WRXJKQHVV EDVHG RQ ¾EHU VL]H DSSHDUV WR ¾W
exponential curves (as seen in the dashed lines). Data show that
samples, as the guide tube wall thickness increased, the resulting
DVWKHDYHUDJHGLDPHWHURI¾EHUVSUHVHQWZLWKLQDWXEHLQFUHDVHVR
toughness decreased in an exponential manner, as depicted in Fig- does the toughness of the same tube.
XUH  +RZHYHU IRU 3/*$ VDPSOHV DV WKH WXEH ZDOO WKLFNQHVV
WKHFRSSHUZLUHZLWKWKHÀEURXVWXEHRQLWDQG:LVWZRWLPHV
increased, the toughness also increased. The overall trends for
the tube wall thickness. After the value for A was obtained, the
ERWK SRO\PHUV DUH GLIIHUHQW SHUKDSV GXH WR WKH ÀEHU QHWZRUNV
diameter could be determined by inserting A into the equation
within the tube walls. For the PLGA tubes, the wall is made up of
G $ư:KLOH3/*$LVDSRO\PHUWKDWFDQZLWKVWDQGKLJK
PDQ\ÀEHUVDQGIHZSRO\PHUEHDGV$VWKHWKLFNQHVVRI WKHZDOO
forces and PCL is an elastic polymer, neither PLGA nor PCL
LQFUHDVHVWKHUHLVDOVRDQLQFUHDVHLQWKHQXPEHURI ÀEHUVSUHV-
in isolation possesses both of these properties. For this reason,
ent, thus increasing the strength of the tube. However, in many
different blends of PLGA and PCL were formed. It was also
PCL tubes, the presence of polymer beads decreases the number
EHOLHYHG WKDW DQQHDOLQJ WKH VDPSOHV DW Ü& ZRXOG PDGH 3&/
FIGURE 6. Average Fiber Size Within Guide Tubes. The average more crystalline, allowing for greater stress and strain before the
¾EHU GLDPHWHU ZLWKLQ WKH GLIIHUHQW QHUYH FRQGXLWV DUH GHSLFWHG LQ conduits fracture. The stress and strain at fracture for the annealed
WKHEDUJUDSKDERYH7KH3&/IRUPHG¾EHUVZLWKWKHODUJHVW and non-annealed conduits are shown in Table 1. The ultimate

diameter, 2.99 µm.
Average Fiber Size Within Guide Tubes
PL Diameter of Fiber (micrometers)
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strain for the annealed and unannealed samples was lowest for the
PLGA samples and highest for the blended and PCL samples. As
a more elastic polymer, PCL can be strained to a greater degree

3 than PGLA can be. When PCL was present in a blended solution
2.99
2.5 1.98 at either 50% or 75%, the resulting guide tube was able to sustain
2 1.35
1.5
higher levels of strain. These results suggest the importance of
0.76
1 0.48 0.445
0.61 PCL in a nerve guide tube. Although the 7% PCL and PLGA:PCL
0.5 in a ratio of 1:3 reached high strain values, the morphology of
0
the sample immediately prior to breaking had no representation
7%

%
%

%
15
12

20

/5

FIGURE 8. Toughness of Nerve Conduits Based on Wall Thick-

.5

.5
L
PC

5%

/2

/7
A

PC
A
G

5%

5%
G

ness. The data for tube toughness based on wall thickness appear
PL

7.

2.
P
A:

L
C

C
G

WR¾WH[SRQHQWLDOFXUYHVDVVHHQLQWKHGDVKHGOLQHV'DWDVKRZ

P

P
PL

A:

A:
G

G
PL

PL

that for PLGA, as wall thickness increases, toughness increases,

Concentration of Polym er
whereas for PCL, as wall thickness increases, tube toughness
RI ÀEHUVSUHVHQWZLWKLQWKHWXEHZDOO2QHVLGHRI D3&/WXEH decreases. At 40 micrometers, where the two data sets intersect,
may be weaker than the other if it contains more polymer beads, PLGA data has a rising trend, while PCL tube toughness begins
ultimately leading to the tube’s failure. decreasing.
Stress and Strain of Nerve Guide Tubes. Along with Toughness for Guide Tubes
of Various Wall Thickness
providing measurements for toughness, nano-mechanical testing 3
also showed the force and amount of displacement that samples PLGA
2.5
could withstand before fracturing. These values are key points to PCL
Toughness (MPa)

2
be optimized for a successful nerve guide. Since the nerve guide
tubes were of different thicknesses and diameters, force and 1.5

displacement were normalized into the values of stress and strain, 1

respectively. One required input value for the nano-mechanical 0.5

tester was the conduit diameter. To calculate this value, the guide 0
tube was treated as a rectangular prism. The cross sectional area 0 20 40 60 80 100 120 140
Guide Tube Wall Thickness (m icrom eters)
RI WKHUHFWDQJXODUSULVPLV$ /ï:ZKHUH/LVWKHGLDPHWHURI 

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
58
 Manuscripts

TABLE 1. Stress and Strain of Annealed and Unannealed Samples. Tensile testing provided data for the stress and strain at
the fracture point for annealed and unannealed nerve conduits. A general trend seen in the data is that annealed tubes had
higher stress and strain values at their fracture points than the unannealed control tubes.

Stress and Strain at Fracture Point for Annealed and Unannealed Samples
Stress Strain
Sample Unannealed St. Dev. Annealed St. Dev. Unannealed St. Dev. Annealed St. Dev
(MPa) (MPa) PPPP PPPP
3/*$V    2.22 0.37   
3/*$V  .55 --- ---   --- ---
3&/V  .01   1.05 .10  .17
3/*$3&/V  .15   1.52 .15  
3/*$3&/V  .05 --- ---  .03 --- ---
3/*$3&/V  .11 2.25   .05 1.72 .20

of a nerve guide tube. Instead, the sample was deformed and was molecules gradually begin to align themselves in the direction of
EHLQJVWUHWFKHGDORQJDIHZÀEHUV7KLVPRUSKRORJ\LVLQDGHTXDWH any applied force. This internal reorganization results in a material
for the application of a nerve guide tube, as it is necessary for the that requires greater stress to stretch, which is a favorable quality
conduit to maintain its tubular structure to protect elongating and for a nerve conduit to possess.
maturing axons. There was no set strain value, however, beyond Toughness and Moduli of Nerve Guide Tubes. The
which the nerve conduits deformed. Although the PCL conduits modulus of a polymer is a characteristic that can be used to
exhibited high strain values, their stress values were lower than classify unknown molecules. On a stress and strain curve, the
the values for the PLGA conduits. The pure PLGA samples were modulus is represented by the slope of the graph in its elastic
able to maintain the highest stress values at their breaking points. region. The modulus should be consistent for polymers with
These were followed by the blended scaffold in a 1:1 ratio. This similar physical morphology. Therefore, a solid piece of either
blended scaffold has an ultimate stress value that was higher PCL or PLGA will have a different modulus from an electrospun
than the 20% PLGA sample, suggesting that the blend exhibits sample of PCL or PLGA. The same was found to be true for
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the properties of both strength and elasticity. The stress values DQDQQHDOHGVDPSOHDQGXQDQQHDOHGVDPSOHDVVHHQLQ)LJXUH
at the breaking point for the annealed samples are greater than For all conduits, the modulus increased in annealed samples, as
those values for the unannealed samples, for all tubes tested. The seen in the data tables, signifying that the annealed samples are
difference in stress values may be due to the increased crystallinity stronger than the unannealed samples. Compared to the values
within the tubes after annealing. In a crystalline polymer, the of the recorded values of the moduli for PCL (120 MPa) and

FIGURE 9. Modulus and

Toughness Comparison
for Annealed and
Unannealed Nerve
Conduits. The tables
above compare the
toughness and moduli for
annealed and unannealed
nerve conduits composed
of one polymer. The two
bar graphs represent the
same data for tubes made
of PLGA and PCL blends.
The annealed samples
have higher values for
toughness and modulus,
indicating that annealing
the conduits makes
them stiffer and stronger.
These properties will
allow the tubes to
withstand sudden and
prolonged forces applied
by neighboring organs
and muscles.
59 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

FIGURE 10. Percent Crystallinity of Annealed Nerve Conduits. The table below shows data that were used to determine the percent
FU\VWDOOLQLW\ZLWKLQWKHDQQHDOHGQHUYHFRQGXLWV7KHFRPSXWHUJHQHUDWHGYDOXHIRUHQWKDOS\RIIXVLRQZDVPRGL¾HGE\VXEWUDFWLQJDQ\
area for the glass transition for PLGA (shaded in red in the DSC image to the right).

3/*$  03D LQ OLWHUDWXUH WKH PRGXOL IRU WKH XQDQQHDOHG
DQG DQQHDOHG VDPSOHV ZHUH YHU\ GLIIHUHQW 7KH ÁXFWXDWLRQV LQ
WKH PHDVXUHG YDOXHV PD\ EH GXH WR WKH ÀEURXV TXDOLW\ RI  WKH
tubes and the degree of crystallinity present in the samples. The
toughness of the annealed samples also increased for all annealed
tubes. Changes that occurred in the annealed samples appear to
EHEHQHÀFLDODQGVXJJHVWWKDWDQRSWLPL]HGQHUYHFRQGXLWVKRXOG
be annealed to induce crystallinity. This change will improve the
ability of the nerve guide tube to extend under forces exerted on
it and accept loads that are quickly applied on it.
Crystallinity of Annealed Samples. The percent of
crystallinity in the unannealed and annealed samples was found
using differential scanning calorimetry and calculated by dividing
the enthalpy of fusion of the sample by the published value for
fusion, as more heat must be applied to break apart the crystal
lattice. Often, certain parts of a polymer chain can align, while
other parts are tangled to such an extent that it is impossible for
chains to align themselves with one another. The DSC curves also
FRQÀUPHGWKDWOLWWOHLI DQ\3/*$FU\VWDOOL]HGGXULQJWKHKHDWLQJ
process. These results suggest that annealing a nerve conduit can
VLJQLÀFDQWO\LQFUHDVHWKHFU\VWDOOLQLW\WKXVHQKDQFLQJWKHVWUHQJWK
and elasticity for in vivo use.
Morphology of Nerve Conduits after Tensile Testing
and Hydration. The lyophilized samples of the optimized
scaffold gave a view of what a nerve guide tube may look like
once expanded within the moist environment of the human body.
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MEDICAL
WKH HQWKDOS\ RI  IXVLRQ IRU 3&/  -J11 The enthalpy of
IXVLRQIRUWKHVDPSOHVZDVPRGLÀHGE\VXEWUDFWLQJWKHHQWKDOS\
required for the glass transition of PLGA. Since PLGA has no
PHOWLQJWUDQVLWLRQQHDUÜ&LWZDVDVVXPHGWKDWWKHFU\VWDOVWKDW
did form during annealing were all PCL crystals. In the blended
VDPSOHVWRÀQGWKHHQWKDOS\RI IXVLRQIRU3&/RQO\WKHHQWKDOS\
RI IXVLRQJLYHQE\WKHFRPSXWHUZDVPXOWLSOLHGE\>3/*$@
[PCL]). Percent crystallinity was then calculated by dividing
the enthalpy of melting, which was determined experimentally,
Not only did the porosity of the scaffold greatly increase once it
ZDVLQWURGXFHGWRDK\GUDWHGHQYLURQPHQWEXWPDQ\ÀEHUVIXVHG
together and many polymer beads disappeared, as seen in Figure
11. SEM Images of a stretched nerve conduit also show what
would happen to the guide tube if a muscle applied a heavy load
onto the tube once in was implanted in the human body. Apart
IURPWKHIHZÀEHUVWKDWEUHDNDSDUWWKHÀEURXVQHWZRUNUHDOLJQV
itself in the direction of the applied force, as shown in Figure 12.
7KH WXEH VWLOO KDV XQLIRUP ÀEHUV DQG IRUPV D SRURXV QHWZRUN
E\ WKH SXUH HQWKDOS\ RI  PHOWLQJ IRU 3&/ RQO\  -J $OO
annealed samples had an increased percent crystallinity compared
to the unannealed control nerve conduits. The experimentally
determined enthalpies of fusion were all smaller than the pure
value due to the fact that not all of the polymers present in the
guide tubes crystallized. Crystallization increases the enthalpy of
FIGURE 11. SEM Images of Hydrated Nerve Conduits. Image A
shows a nerve conduit (1:1 PLGA:PCL) before being introduced
into a hydrated environment. Image B shows the same tube one
week after being immersed in distilled water. Both images are at a
PDJQL¾FDWLRQRIí
w w w . T H U R J . o r g T H U R J V o l
which would continue to allow for nutrient exchange across the
tube wall.
Preliminary in vitro and in vivo results. Results from
preliminary in vitro and in vivo tests show that the guide tubes induce
OLWWOH WR QR LQÁDPPDWRU\ UHVSRQVH DQG DUH QRW UHMHFWHG in vivo.
Images of the nerve guide in vivo (Figure 13) showed that the tube
FIGURE 12. SEM Images of Stretched Nerve Conduits. Image A
shows a nerve conduit (1:1 PLGA:PCL) before being mechanically
tested. Image B shows the same tube after mechanical testing.
%RWK LPDJHV DUH DW D PDJQL¾FDWLRQ RI í 7KH VWUHWFKHG WXEH
KDV¾EHUVUXQQLQJSDUDOOHOWRRQHDQRWKHU\HWVWLOOFRQWDLQVDKLJK
degree of porosity.
I I s s u e 1 S p r i n g 2 0 0 8
60
 Manuscripts

can be formed by using a 5% PCL and 5% PLGA solution and

annealing the resulting tube. Images from a SEM show that the
tubes created by these parameters have qualities that regenerative
scaffolds should possess. Tensile testing has also shown this tube to
have a high toughness, modulus, strain at breaking point, and stress
at breaking point. These attributes will allow a regenerative tube to
withstand sudden and prolonged loads from neighboring organs
or muscles. Differential scanning calorimetry has shown that the
FIGURE 13. In vivo images of Nerve Guide Tube. Both images, annealing process has crystallized the PCL molecules, making the
which were taken three months after implantation, show the tube
tube stiffer and stronger. In vitro and in vivo tests have shown that
still connecting the transected spinal cord nerves.
myelinated axons can migrate throughout the conduit, and that the
ÀOOHGXSZLWKOLTXLGDQGWKDWLWZDVVWLOOFRQQHFWLQJWKHWZRVSLQDO conduit has the potential to lead transected axons together. Further
cord nerve stumps. The many nerve tubes that were inserted into investigations include determining whether it is possible to tether
the spinal cord were all still intact after four weeks, indicating that RQOLJDQGVDQGQHUYHJURZWKIDFWRUVRQWR3/*$DQG3&/ÀEHUV
the nerve conduit did not degrade too quickly. A cross sectional in Such an adaptation could potentially expedite nerve regeneration.
vitro model of the electrospun nerve guide shows injected peptides,
WRZKLFK6FKZDQQFHOOVDQGQHXURQVFDQDWWDFK)LJXUH7KH
dark ring on the outside represents the electrospun tube, while the
dark circles inside the guide tube illustrate migrating myelinated
axons. The spread of axons within the guide tube suggests that
the cells can migrate and attach throughout the entire conduit,
FIGURE 14. Optical Microscope Image
of Conduit. The image above shows an
SCIENCES
MEDICAL
in vitro nerve conduit. The image shows
myelinated axons migrating through the
nerve tube, which is marked by the dark
REFERENCES
1. Barnes, Deborah. “Spinal Cord Injuries.” NIH OSE Research in the News:
6SLQDO &RUG ,QMXULHV 1DWLRQDO ,QVWLWXWH RI  +HDOWK  -XO\  KWWS
VFLHQFHHGXFDWLRQQLKJRYKRPHQVI!
2. Lanza, Robert. Principles of Tissue Engineering. Ed. Langer R and Vacanti J.
ed. 2 New York: Academic Press, 2000. pgs. 112-231.
3. 'HOORQ$/DQG0DFNLQQRQ6($QDOWHUQDWLYHWRWKHFODVVLFQHUYHJUDIW
IRUPDQDJHPHQWRI WKHVKRUWQHUYHJDS3ODVW5HFRQVWU6XUJ
 Kiyotani T, Teramachi M, Takimoti Y, Nakamura T, Shimizu Y, Endo K
1HUYHUHJHQHUDWLRQDFURVVDPPJDSEULGJHGE\DSRO\JO\FROLFDFLG
collagen tube: a histological and electrophysical evaluation of regenerated
QHUYHV%UDLQ5HVHDUFK
5. Lu L, Meter S J, Lyman M D, Lai H L, Leite S M, Tamada J A, Uyama S,
Vacanti J P, Langer R and Mikos A G 2000 in vitro and in vivo degradation of
ring surrounding the cells and peptides.
7KHLPDJHLVDWDPDJQL¾FDWLRQRIVHWLV
at !10.
FIGURE 15. Fluorescent Image of in vitro nerve conduit.
The red within the image represents all neuronal growth
within the nerve tube after four weeks. Neurons were
able to invade and migrate successfully throughout
the entire diameter of the nerve guide. Mag: !8
SRURXVSRO\'/ODFWLFFRJO\FROLFDFLGIRDPV%LRPDWHULDOV
 Ratner, Buddy and Hoffman, Allan. Biomaterials Science: An Introduction to
0DWHULDOVLQ0HGLFLQHHG1HZ<RUN$FDGHPLF3UHVVSJV
7. 5RGUtJXH] ) *RPH] 1 3HUHJR * 1DYDUUR ;  +LJKO\ SHUPHDEOH
polylactide-caprolactone nerve guides enhance peripheral nerve regeneration
WKURXJKORQJJDSV%LRPDWHULDOV
 Loo S, Ooi C, Wee S, Boey Y 2005 Effect of isothermal annealing on
hydrolytic degradation rate of poly(lactide-co-glycolide) (PLGA) Biomaterials.

 /L:/DXUHQFLQ&&DWHUVRQ(7XDQ5.R)(OHFWURVSXQQDQRÀEURXV
VWUXFWXUH $ QRYHO VFDIIROG IRU WLVVXH HQJLQHHULQJ %LRPHG 0DWHU 5HV 

10. <DQJ)0XUXJDQ5:DQJ65DPDNULVKQD6(OHFWURVSLQQLQJRI QDQR
PLFURVFDOHSRO\/ODFWLFDFLGDOLJQHGÀEHUVDQGWKHLUSRWHQWLDOLQQHXUDOWLVVXH
allowing for greater nerve regeneration. The myelinated axons also HQJLQHHULQJ%LRPDWHULDOV
suggest the regenerative potential of the synthesized guide tubes. 11. )LVFKHU(:6WHU]HO+-:HJQHU*,QYHVWLJDWLRQRI WKHVWUXFWXUHRI 
7KH ÁXRUHVFHQW PLFURVFRSH LPDJH RI  WKH QHUYH FRQGXLW VKRZV solution grown crystals of lactide copolymers by means of chemical reactions
3RO\PHUH
the in vivo model in which spinal cord nerve stumps were added
to the ends of the guide tube (Figure 15). The neuronal growth
DIWHUZHHNVDVLQGLFDWHGE\WKHUHGLVVHHQWKURXJKRXWWKHJXLGH
tube, thus illustrating that the nerve conduit allows for cellular
attachment and migration. These promising results indicate that
an electrospun nerve conduit has the potential to lead spinal cord
nerve stumps together.

CONCLUSION
We determined that an optimized nerve regeneration guide

61 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

NADPH oxidase-derived free radicals are

required for cortex-controlled motor activity
QI YU

Weill Medical College of Cornell University, 411 East 69th St.,

New York, NY 10021-5608

The ionotropic glutamate receptor N-methyl-D-aspartate (NMDA) receptor, a primary receptor in the
cerebral cortex, plays an important role in cortex-mediated locomotor activity. Reactive oxygen species
(ROS) have been found to serve as cellular messengers in pathological conditions of neurogenerative
diseases. However, the molecular and cellular mechanisms underlying the ROS-mediated functions
in the cerebral cortex remain unclear. First, immunostaining results show that NR1 NMDA recep-
tors and gp91 (Nox2) subunits of NADPH oxidase are expressed in cultured mouse cortical neurons.
6HFRQG526SURGXFWLRQLQFRUWLFDOQHXURQVZDVVWXGLHGXVLQJWKHÁXRUHVFHQFHG\HGLK\GURHWKLGLXP
NMDA triggered substantial ROS production, an effect blocked by the NMDA receptor antagonist
MK801, the ROS scavenger MnTBAP, the NADPH oxidase inhibitor apocynin, and the Nox2 NADPH
oxidase inhibitor gp91ds-tat peptide. Third, a patch-clamp study demonstrated that NMDA triggers
an inward ion current in the cortical neurons, an effect inhibited by the NADPH oxidase inhibitor
apocynin. Fourth, NMDA increased ROS production in cortical neurons from adult wild-type mice
EXWQRWLQWKRVHRI JSNQRFNRXWPLFH)LQDOO\DEHKDYLRUDO<PD]HWHVWGHPRQVWUDWHGDVLJQLÀFDQW
decrease in the locomotor activity of adult gp91 null mice when compared to wild-type mice.
____________________________________________________

INTRODUCTION unclear.
Growing evidence indicates that increased ROS production
The cerebral cortex of humans plays a central role in sensory,

cognitive, memory, and motor functions. In all vertebrates, the ce-
rebral cortex is fundamental to voluntary locomotor movement.
0DQ\VSHFLÀFDUHDVLQFOXGLQJWKHSULPDU\PRWRUFRUWH[SUHPR-
tor cortex, posterior parietal cortex, prefrontal lobe, and dorsolat-
eral prefrontal cortex, participate in execution or modulation of
1, 2
locomotor activity.
A large body of evidence suggests that the excitatory amino
acid glutamate is a neurotransmitter critical for cerebral corti-
cal functions. Glutamate evokes locomotor activity through N-
methyl-D-aspartate (NMDA) receptors,3 and genetic manipula-
tion of NMDA receptors is detrimental to the coordination of
mice.4 Moreover, activation of NMDA receptors increases levels
of reactive oxygen species (ROS), creating oxidative stress. In the
human central nervous system, NMDA receptors are involved in
oxidative stress of the cerebral endothelium and hippocampus.5,
6
Since ROS exert their damaging effects on lipids, proteins, and
DNA by peroxidation and/or crosslinking, prolonged exposure
to NMDA results in the excitotoxicity of cultured cortical neu-
rons.7 Despite the established role of NMDA receptors in physi-
ological and pathological functions of cerebral cortex, the source
of NMDA-induced ROS in cerebral cortical neurons remains
w w w . T H U R J . o r g T H U R J
NEUROSCIENCE
is attributable to nicotinamide adenine dinucleotide phosphate-
oxidase (NADPH oxidase) in many areas of the brain. NADPH
oxidase is an important source of ROS production essential to
normal hippocampal function6 as well as ischemic dysfunction.8
Furthermore, NADPH oxidase is considered to be a source of
superoxide (O2î, the major type of ROS) for NMDA receptor-de-
pendent activation by extracellular signal-regulated kinase (ERK)
and memory in the hippocampus.9
The NADPH oxidase enzyme is a complex composed of the
membrane-bound subunits, gp91phox and p22phox, and the cytosolic
subunits, which include p47phox, p40phox, and p67phox. The mem-
brane-bound subunits are stabilized by forming a heterodimer.
The cytoplasmic subunits translocate towards the membrane-
bound subunits, and their binding activates the enzyme and initi-
ates production of ROS.9 The gp91phox VXEXQLW ÀUVW GLVFRYHUHG
and described in phagocytes, is also termed Nox2 and has sev-
eral homologues, Nox1, Nox3, Nox4, and Nox5, the location of
ZKLFKLVFHOOW\SHVSHFLÀF10
Under normal physiological conditions, tightly regulated ROS
are used as biochemical messengers. In the CNS, ROS are consid-
ered necessary for long-term potentiation (LTP), a type of syn-
aptic plasticity that has been proposed as a biological basis for
62
V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts
hippocampal learning and memory.6 ROS are also required for a
number of cellular signaling mechanisms,9 including mediation of
nonsynaptic communications between neurons and glia.11 Taken
together, the previous studies suggest that ROS derived from the
Nox2 NADPH oxidase may serve as signaling molecules in cere-
bral cortical neurons and play a role in cortex-mediated locomo-
tor activity.
Using wild-type and Nox2 knockout mice in immunohistochem-
days before use. In cortical cultures obtained by this procedure,
the major cell types were neurons, and under 10% percent of the
cells showed characteristics of glial cells.
Acute dissociation of adult mice neurons. Male mice at
3 months and 12 months of age, both wild-type and Nox2 null,
were utilized in intracellular ROS detection. Mice were anesthe-
tized with CO2 and decapitated. Brains were quickly collected
DQGVWRUHGLQDFKDPEHUFRQWDLQLQJLFHFROGVXFURVHDUWLÀFLDOFH-
istry, ROS imaging, and a behavioral Y-maze test, this study aimed UHEURVSLQDOÁXLGV$&6)P01D+&23, 1 mM NaH2PO4,
to investigate the following questions: (1) whether cortical neurons 3 mM KCl, 5 mM MgSO4, 0.5 mM CaCl2, 10 mM glucose, and
express NMDA receptors or NADPH oxidase subunit Nox2; (2) 248 mM sucrose, oxygenated with 95% O2 and 5% CO2, pH 7.4.
whether activation of NMDA receptors induces ROS production The cortex was separated from the rest of the brain, sliced with
LQFRUWLFDOQHXURQVLI VRZKHWKHU1$'3+R[LGDVHLVDVLJQLÀ-
cant contributor to NMDA receptor-mediated ROS production;
and (4) whether NADPH oxidase-derived ROS are instrumental
to the excitability of cortical neurons or cortex-linked functions,
such as locomotor activity in vertebrates.
MATERIALS AND METHODS
All protocols and procedures were performed in compliance
with the guidelines of the Institutional Animal Care and Use
Committee at Weill Medical College of Cornell University.
Materials. Goat polyclonal antiserum against Nox2 (gp91phox)
and mouse monoclonal antiserum against NMDA NR1 recep-
tors were purchased from Santa Cruz Biotechnology (CA). Anti-
mice Texas Red and anti-goat FITC secondary antibodies were
purchased from Jackson ImmunoResearch (PA). Apocynin and
Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP)
were purchased from Calbiochem (La Jolla, CA). NMDA, MK-
NEUROSCIENCE
801, HEPES free acid, PIPES, proteinase K, and trypsin were
purchased from Sigma-Aldrich (St. Louis, MO). The polypep-
WLGHJSGVWDWDFHOOSHUPHDEOHVSHFLÀFLQKLELWRUIRU1R[FRQ-
taining NADPH oxidase,12, 13 was synthesized by Bio-Synthesis
(Lewisville, TX). Dihydroethidium (DHE) was purchased from
Molecular Probes. C57BL/6J mice were used as wild-type (Nox2
+/+) controls and mice lacking the gene for the gp91phox com-
ponent (Nox2 null) of NADPH oxidase were obtained from an
in-house colony.9
Cell culture. E16-18 embryos were obtained from female wild-
type mice. Primary cortical neurons were isolated and cultured
according to the protocol developed by Kawano et al. (2006).14
The cerebral cortex was dissected in Hanks’ balanced salt solution
(HBSS) without Ca2+ or Mg2+ and digested in trypsin (1 mg/ml)
for 15 min at room temperature. After neutralization with trypsin
inhibitor for 2 min (1 mg/ml of trypsin inhibitor in HBSS with-
a vibratome, and incubated at room temperature in lactic acid
(l)-ACSF: 124 mM NaCl, 26 mM NaHCO3, 5 mM KCl, 1 mM
NaH2PO4, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 4.5
mM lactic acid, aerated with 95% O2 and 5% CO2, pH 7.35. Corti-
cal slices were then digested and placed in PIPES buffer: 115 mM
NaCl, 5 mM KCl, 20 mM PIPES free acid, 1 mM CaCl2, 4 mM
MgCl2, and 25 mM dextrose, pH 7.0, oxygenated with 100% O2
and 0.2 mg/ml proteinase K at 30°C for 5 minutes. Slices were
then incubated in PIPES buffer with 1 mg/ml trypsin type XI at
30°C for 30 minutes. Finally, slices were rinsed 4-5 times in PIPES
buffer. Neurons were dissociated in ice-cold lactic acid-ACSF by
WULWXUDWLRQXVLQJÁDPHSROLVKHG3DVWHXUSLSHWWHV&HOOVZHUHSODWHG
in glass-bottom 35-mm Petri dishes and bathed in HEPES Mg2+-
free buffer for optimal ROS imaging.15
,PPXQR¿XRUHVFHQW ODEHOLQJ The cultured mice cortical
QHXURQVZHUHÀ[HGLQSDUDIRUPDOGHK\GHLQ0SKRVSKDWH
buffer (PB), washed in 0.1 M PB, and stored in 30% sucrose buf-
IHUDWƒ&,QSUHSDUDWLRQIRULPPXQRÁXRUHVFHQWODEHOLQJQHX-
rons were washed in 0.1 M phosphate buffered saline (PBS) and
permeabilized in 0.01% Triton X-100 in PBS. Neurons were then
washed in 0.05% Tween20 in PBS and treated with 3% BSA, fol-
lowed by incubation in PBS containing primary mice anti-NR1 re-
ceptor (1:500) or goat anti-Nox2 (1:100). Afterward, the neurons
were incubated in PBS containing anti-mice Texas Red or anti-
goat FITC antisera (Jackson ImmunoResearch, PA), respectively,
for one hour.13 Fluorescent labeling of the neurons was visualized
on a Nikon Diaphot 300 microscope.
Detection of intracellular ROS. ROS production of both
cultured embryonic neurons and acutely dissociated adult neu-
rons was assessed using DHE because the oxidized products of
DHE bind to DNA.16 The isolated neurons were incubated for
30 minutes with 2 ƬM DHE, and the vehicle containing 2 ƬM
DHE was continuously present throughout experiments. Time-
UHVROYHGÁXRUHVFHQFHZDVPHDVXUHGHYHU\VHFRQGVXVLQJWKH
out Ca2+, Mg2+WKHFRUWH[ZDVGLVVRFLDWHGZLWKÀUHSROLVKHG3DV-
teur pipettes. Cell suspensions were transferred to 6-well plates
coated with polyethylenimine (PEI; 1:10,000 dilution in borate
buffer, pH 8.0) and were cultured in minimum essential medium
(MEM) supplemented with 10% fetal bovine serum at 37°C for 4
hrs. The culture medium was replaced with Neurobasal medium
supplemented with 2% B27, and the cells were cultured for 11
63 w w w . T H U R J . o r g T H U R J
image analysis software IPLab (Scanalytics Inc). Recordings were
started after a stable baseline with control buffer (vehicle) was
achieved.127KHUDWLR)W)RZDVSUHVHQWHGDVUHODWLYHÁXRUHVFHQFH
LQWHQVLW\ZKHUH)WLVÁXRUHVFHQFHDWDQ\JLYHQWLPHIROORZLQJWKH
application of NMDA in a given neuron, and Fo is the baseline
ÁXRUHVFHQFHRI WKHVDPHFHOOLPPHGLDWHO\EHIRUHDSSOLFDWLRQRI 
NMDA.
V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

Whole-cell patch clamp. Whole-cell recordings were con- VLGHUHGVWDWLVWLFDOO\VLJQLÀFDQWDQGOHVVWKDQYHU\VWDWLVWLFDOO\

ducted from cultured mice cortical neurons using an Axopatch VLJQLÀFDQW
$DPSOLÀHUDQGS&ODPSVRIWZDUH$[RQ,QVWUXPHQWV)RVWHU
&LW\&$3DWFKHOHFWURGHVZHUHÀOOHGZLWKDVROXWLRQFRQWDLQLQJ
140 mM CsF, 5 mM NaCl, 10 mM TEA-Cl, 1 mM MgCl2, 5 mM
EGTA, 1 mM ATP, 0.2 mM cAMP, and 10 mM HEPES-CsOH,
pH 7.3. The Mg2+-free solution perfusing the neurons contained
121 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.01 mM glycine, 1
mM Na-pyruvate, 17 mM HEPES-NaOH, 20 mM glucose, and
3 mM NaCO3, pH 7.4. The drug solutions were driven by gravity
RESULTS
&RUWLFDO QHXURQV H[SUHVV ERWK 10'$ 15 UHFHSWRUV
and Nox2. To determine expression of NMDA receptors or
Nox2 in cultured mice cortical neurons, immunoreactivities for
NMDA NR1 receptor and Nox2 were examined using immunos-
taining. As shown in Fig. 1, of forty-three total neurons in the
through a fast perfusion system. NMDA-elicited inward currents ÀHOG)LJ$WKLUW\VHYHQQHXURQVZHUHLPPXQRUHDFWLYHWR15
were induced at holding potential of -60 mV, and the pure inward receptor antibody (Fig 1B). In addition, of thirty neurons in the
peak current was determined by subtracting the background cur-
rent recorded in the absence of the ligand.13
Behavioral test.
Subjects: A total of twenty mice from wild-type and Nox2
null groups were obtained for experimentation. The mice were
housed 4 to 5 per cage in a temperature- and humidity-controlled
room on a 12 hour light-dark (light 06:00-18:00h) schedule. Mice
had free access to food and water throughout experimentation.
All studies were conducted during the light phase. The mice were
either 3 months or 12 months old when tested.
Y-maze measurements and testing procedure: The Y-maze ap-
ÀHOG  )LJ & WZHQW\WKUHH ZHUH LPPXQRUHDFWLYH WR WKH 1R[
antibody (Fig 1D). These results indicate that both NR1 receptors
and Nox2 are expressed in cortical neurons.
Activation of NMDA receptors results in ROS produc-
tion via NADPH oxidase in cortical neurons. To test the
notion that NMDA receptors are responsible for ROS produc-
tion in cortical neurons, the cultured neurons were exposed to
ƬP10'$DQGZHUHIRXQGWRKDYHDVLJQLÀFDQWLQFUHDVHLQ
DHE intensity (by 60±17%; n=7; p<0.01 vs. vehicle) (Fig 2A-E).
To further examine if ROS production is indeed mediated by
NMDA receptors, the effect of MK801, the NMDA receptor
paratus was composed of clear plastic; each arm was 30 cm long, antagonist, was tested.5 0.  Ƭ0 EORFNHG WKH 10'$
10 cm wide, and 18 cm high. A visit to an arm was recorded only induced increase in DHE intensity (p<0.01 vs. NMDA alone)
when the base of the mouse’s tail entered. Experimentation was )LJ'(6LPLODUO\WKH526VFDYHQJHU0Q7%$3Ƭ0DOVR
recorded onto a micro-computer using the Apple iMovie program blocked the NMDA-induced increase in DHE intensity (p<0.01
and saved into a LaCie hard disk for future analysis. In order to vs.FRQWURO)LJ'(0RUHLQWHUHVWLQJO\JSGVWDWƬ0D
minimize olfactory stimuli, the maze was sprayed with the glass VSHFLÀF 1R[ LQKLELWRU12 as well as apocynin (1 mM ), a non-
cleaner Windex (SC Johnson & Son, Racine, WI) and wiped with VSHFLÀF1$'3+R[LGDVHLQKLELWRUZHUHDOVRIRXQGWREORFNWKH

NEUROSCIENCE
cloth after each trial. The maze was set in a dimly illuminated NMDA-induced increase in DHE intensity (p<0.01 vs. control.)
and sound-attenuated room. Numerous visual cues were placed (Fig 2D-E). These results indicate that the activation of NMDA
behind the transparent plastic of the Y-maze walls and were kept receptors leads to ROS production via Nox2-containing NADPH
constant through all experiments and during the testing period.
One hour before the beginning of the experiment, the mice were
brought into a quiet room adjacent to the experimental room and
identical in light and noise conditions. All experiments were con-
ducted in the same apparatus and facilities.
Response to novelty and spatial memory: Experimentation con-
sisted of two trials separated by a 30-minute intertrial interval.
,QWKHÀUVWWULDORQHDUPRI WKH<PD]HZDVFORVHGZLWKDJXLO-
lotine door. Mice were placed in one of the two other arms, both
animals’ heads oriented away from the center of the maze. They
were then allowed free access to visit the two accessible arms for
5 minutes. After each trial was completed, the mouse was placed
into an isolated cage to prevent stress-induced aggressive behav-
ior. The position of the novel arm was at the left of the starting
arm for half of the animals and at the right for the other half (in
random order). The number and duration of visits to each arm
were recorded for every trial.17, 18
Statistical analysis. Data were expressed as mean ± SEM.
Two group comparisons were evaluated by the two-tailed, paired ),*85(  Immuncreativities of cultured cortical neurons of wild
type mice for NMDA receptor 1 (NR1) (A & B) and Nox2 (gp91)
or unpaired Student’s t test. P values less than 0.05 were con-
subunit of NADPH oxidase (C & D). The bar indicates 20 Ƭm.
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8 64
 Manuscripts
FIGURE 2. NMDA evokes ROS production in cortical neurons. (A-C)
Representative images of 30 ƬM NMDA-induced ROS production
LQFXOWXUHGFRUWLFDOQHXURQVRIZLOGW\SHPLFHHPEU\RV%ULJKW¾HOG
and DHE images in controls are shown in panels (A) and (B), re-
spectively. The DHE image at 15 minutes after application of 30
ƬM NMDA is shown in panel (C). The bar indicates 20 Ƭm. (D-E)
NMDA-induced free radical production in cultured cortical neurons
of mice embryos is dependent on Nox2-containing NADPH oxi-
ROS production between wild type and Nox2 null cortical neu-
rons and between two different ages, acutely dissociated corti-
cal neurons were analyzed.15 As shown in Fig. 4A-C, an exposure
RI Ƭ010'$OHGWRDVXEVWDQWLDOLQFUHDVHLQ'+(LQWHQVLW\
(p<0.01 vs. vehicle) in the wild-type cortical neurons of 3-month-
old and 12-month-old groups. However, in the cortical neurons
LVRODWHGIURPPRQWKROG1R[QXOOPLFHƬ010'$VLJQLI-
icantly decreased DHE intensity (p<0.01 vs. vehicle) (Fig 4A-C).
Cortical neurons from 12-month-old Nox2 null mice displayed
little changes from the baseline (p>0.05 vs. vehicle) (Fig 4B-C).
These results strongly suggest that (1) Nox2 NADPH oxidase
plays a central role in ROS production in adult mice neurons and
(2) without Nox2, NMDA could elicit a reduction in ROS levels
in 3 month-old cortical neurons.
Motor activity depends upon expression of Nox2. In
order to determine the contribution of Nox2 to cortex-associated
function in mammals, the total number of visits made by wild-
W\SHDQG1R[QXOOPLFHZDVFRPSDUHGGXULQJWKHÀUVWWULDOD
minute sample), as shown in Fig. 5A.17 Overall activity differed
VLJQLÀFDQWO\EHWZHHQPRQWKROGZLOGW\SHDQG1R[QXOOPLFH
(Fig. 5A, p<0.05, n=6). The total number of visits to the novel
arm during the second trial, after a 30-minute intertrial period,
DOVRGHPRQVWUDWHVDVLJQLÀFDQWGLIIHUHQFHEHWZHHQWKHORFRPRWRU
activity of wild-type and Nox2 null mice (Fig. 5B, p<0.01, n=6) in
both the 3-month- and the 12-month-old groups. A correlation
between locomotion and expression of Nox2 is clearly indicated.
GDVH'7KHWLPHUHVSRQVHFXUYHRI'+(¿XRUHVFHQFHLQWHQVLW\
Nox2-derived ROS production has no effect on mem-
in wild type mice cortical neurons following application of 30 ƬM ory. Previous work has indicated that overexpression of ROS
NMDA (?), NMDA plus the NMDA receptor antagonist MK801(ɶ
), NMDA plus ROS scavenger MnTBAP (ɻ). NMDA plus NADPH
may be responsible for cognitive dysfunction in aging.19, 20 To de-
oxidase Nox2 inhibitors gp91-ds tat (ɶ) and apocynin (Apo)(?). (E) termine whether the Nox2-containing NADPH oxidase-derived
ROS affect memory, the responses of mice to novelty (after an
NEUROSCIENCE

7KHKLVWRJUDSKGHPRQVWUDWHVWKHUDWLRFKDQJHVLQ'+(¿XRUHVFHQW
intensity in the presence of NMDA, NMDA plus MK801, MnTBAP, initial trial performance) were analyzed. For the second trial after
gp91-ds tat or apocynin. Control base levels are established before the 30-minute intertrial time, both the total amount of time spent
application of 30 ?M NMDA. **, p<0.01 vs. controls; ††, p<0.01 vs. in the novel arm and the ratio of novel to total arm visits are
NMDA alone.
shown in Fig 5C-D.171RVLJQLÀFDQWGLIIHUHQFHRI QRYHOW\H[SOR-
ration was noted between wild-type and Nox2 null mice of either
oxidase in cultured mice cortical neurons.
age group. These results indicate that the presence of Nox2 does
7KH1$'3+R[LGDVHLQKLELWRUDSRF\QLQEORFNV10'$
not inhibit cognitive functions but may play a key role in regular
mediated ionic currents. To determine the roles of NADPH
motor functions through its ROS production.
oxidase-derived ROS in NMDA-mediated functions, a whole-cell
patch-clamp was performed on the cultured cortical neurons to
determine if NMDA-evoked ion channel currents are affected by DISCUSSION
apocynin, the NADPH oxidase inhibitor. As shown in Fig. 3, apo-
F\QLQP0VLJQLÀFDQWO\LQKLELWHGWKHDPSOLWXGHRI WKH10'$
induced inward ion current in these neurons (-85.2±8.3 nA of
controls vs. -29.2±11.1 nA of apocynin-treatment; n=8, p<0.01).
These data indicate that NMDA-increased ROS also play an im-
portant role in maintaining the excitability of cortical neurons.
$JHGHSHQGHQW HIIHFWV RI 1R[GHULYHG 526 SURGXF-
tion. Since Nox2 null embryonic neurons could not survive from
the culture under experimental conditions, no data are available
for Nox2 null embryonic cultured cortical neurons. In order to a)
FRQÀUPWKHUHTXLUHPHQWRI 1R[IRUWKH10'$LQGXFHG526
production and b) compare the differences in NMDA-triggered
65 w w w . T H U R J . o r g T H U R J
The present studies have demonstrated that (1) the activation
of NMDA receptors in cortical neurons results in ROS produc-
tion, due largely to the activation of Nox2 NADPH oxidase; (2)
such NMDA-triggered, Nox2-dependent ROS production is age-
dependent, namely that in younger mice, a larger proportion of
ROS is contributed by Nox2 NADPH oxidase; and (3) Nox2 NA-
DPH oxidase-derived ROS are required for both neuronal excit-
ability in vitro and motor activity in vivo.
7KH ÀQGLQJV IURP WKLV VWXG\ SURYLGH VHYHUDO XQLTXH LQVLJKWV
into a pathway underlying ROS-linked changes from molecular
interactions to their behavioral consequence. (1) Using the ROS-
V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

tor activity. Together, these results provide cohesive new evidence

suggesting that excitation of NMDA receptors within the cortex
triggers NADPH oxidase to generate ROS as signaling molecules
for motor activity.
Relatively few previous studies have examined locomotor activ-
ity in relation to the cortex. Adriani et al. have studied the effects
of NMDA-receptor blockage on stimulating locomotor activity,
while Broberger et al H[SORUHG WKH HIIHFWV RI  FRUWLFDO LQÁXHQFH
on the motor-controlling basal ganglia.3, 23 Additionally, ROS have
EHHQLGHQWLÀHGDVVLJQDOLQJIDFWRUVXVHGE\$73ZLWKLQWKHQHX-
romuscular junctions of frogs.24 However, a direct correlation be-
tween locomotor activity and NMDA-receptor signaling within
FIGURE 3. The NADPH oxidase inhibitor apocynin suppressed the cortex has not been investigated until now, likely due to the
NMDA-evoked inward ion current in the cultured cortical neurons of
negative effects of ROS in aging and disease.
wild type mice embryos. (A) Representative traces 30 ƬM NMDA-
The technique employed in this study, the Y-maze test, is a two-
induced ion currents from the cultured cortical neuron using the
ZKROHFHOO SDWFK FRQ¾JXUDWLRQ RQ XSSHU SDQHO ,Q WKH SUHVHQFH trial memory task that has been previously validated in rats and
of NADPH oxidase inhibitor apocynin (1 mM), the amplitude of the mice. Here, it was used to demonstrate the roles of NADPH
NMDA-induced current was decreased (lower panel). (B) The his- oxidase in the memory and locomotor behavior of mice.17, 18 The
tograph demonstrates the changes in the amplitude of 30 ƬM NM- data acquired from this task are highly comparable to the regu-
DA-induced currents in the cortical neurons in controls (n=8) and in lar behavior of mice, since traditional methods to induce mice
the presence of apocynin (1mM) (n=8). **, P<0.01. into behavior, such as food deprivation, fear of water, or electric
foot-shock, were unnecessary. The test does not even involve pre-
sensitive dye DHE, it was determined that activation of NMDA learning or pre-training, and thus a genuine measure of memory
receptors elicits ROS production via Nox2 NADPH oxidase. and locomotion can be recorded with every performance. Most
DHE can be oxidized by ROS, but it is especially prone to cleav- importantly, the motivational component of the Y-maze test lies
DJHE\VXSHUR[LGH7KH526GHWHFWLRQPHWKRGZDVDIÀUPHGE\ in the natural tendency of mice to explore novel areas. In ad-
the results of this study; inhibition of DHE imaging intensity is dition, measurements of activity are quick, accurate, and fairly
induced by the ROS scavenger MnTBAP. (2) The data also indi- reproducible. Several animals can go through the trial simultane-
cate that the possibility of ROS-independent formation of DHE, ously, and the presence of an observer within the testing space is
due to auto-oxidation, is negligible. (3) The results from Nox2 null not needed.

NEUROSCIENCE
PLFH LQ FRPELQDWLRQ ZLWK VSHFLÀF SKDUPDFRORJLFDO DJHQWV IRU At the molecular level, ROS production by NADPH oxidase
NADPH oxidase such as gp91ds-tat and apocynin, also strongly relies on the assembly of cytoplasmic regulatory subunits and key
suggest that Nox2 is a key contributor of NMDA receptor-me- membrane-bound subunits to form the functional enzyme com-
diated ROS production in mice of different age groups. (4) The plex.21 Complete assembly of NADPH oxidase is required for full
patch-clamping study provided functional evidence that NADPH activity, although mutations in the gp91 membrane-bound unit
oxidase-derived ROS may play a crucial role in maintaining the DUHVXIÀFLHQWWRGLVDEOHWKH1$'3+R[LGDVHFRPSOH[FRPSOHWHO\
excitability of cortical neurons. (5) Without expression of Nox2, preventing oxidation and ROS production.9 Mutations in the gp91
there were parallel declines in the basal level of ROS and locomo- unit are most common in causing Chronic Granulomatous Dis-

FIGURE 4. Comparison of the NMDA-induced free radical production in dissociated neurons from 3-and12-month old mice. (A) The
WLPHUHVSRQVHFXUYHRI'+(¿XRUHVFHQWLQWHQVLW\LQPRQWKROGZLOGW\SHDQG1R[QXOOFRUWLFDOQHXURQVWRț010'$%7KH
WLPHUHVSRQVHFXUYHRI'+(¿XRUHVFHQWLQWHQVLW\LQPRQWKROGZLOGW\SHDQG1R[QXOOFRUWLFDOQHXURQVWRț010'$&7KH
histographs demonstrate that in addition to NMDA-induced potentiaion of ROS production in wild type neurons of both ages, NMDA
DOVRVLJQL¾FDQWO\HOLFLWHGUHGXFWLRQLQ526SURGXFWLRQLQPRQWKROG1R[QXOOQHXURQV S
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8 66
 Manuscripts

tablishing the oxidative stress hypothesis of aging as a direct link

between the two.20 It may be prudent to address with equal weight
the importance of NADPH-oxidase mediated ROS production
in normal movement, considering the consequences of inhibition
of normal motor functions.

CONCLUSION

The cerebral cortex is one of the major control centers of lo-

comotor activity in higher organisms. This study has successfully
demonstrated that NMDA receptors and NADPH oxidase are
essential for the increased levels of ROS in cortical neurons. In
particular, the membrane-bound gp91 (Nox2) subunit is a critical
FIGURE 5. Motor activity in 3-month-old and 12-month-old wild component of the NADPH oxidase complex, as NMDA-induced
type and gp91 (Nox 2) null mice. (A) The number of arm visits in ROS production was not noted in cortical neurons whose gp91
¾UVW¾YHPLQXWHWULDO%7KHQXPEHURIWRWDOYLVLWVWRQRYHODUPLQ subunits were either disabled or genetically manipulated. In addi-
VHFRQG¾YHPLQXWHWULDO S SWZRWDLOHG7WHVW$UP tion, this study has demonstrated that the lack of NADPH oxi-
discrimination in three-month-old and twelve-month-old wild type GDVHZLWKLQYHUWHEUDWHVZKLOHQRWVLJQLÀFDQWO\LPSDFWLQJPHPRU\
and Nox2 null mice. (C) The total time spent in the novel arm dur-
functions, clearly affects biotic motor activity. Collectively, such
LQJWKHVHFRQG¾YHPLQXWHWULDO'7KHUDWLRRIYLVLWVLQWKHQRYHOWR
WRWDOYLVLWVRIHDFKDUPLQVHFRQG¾YHPLQXWHWULDO data suggest that NMDA receptors play a role in initiating NA-
DPH oxidase-mediated ROS production in the cerebral cortex.
ease, which disrupts immunity and cognition. Thus, blockage of These results contribute to the growing evidence that ROS de-
gp91phox by either peptides or genetic manipulation has produced rived from NADPH oxidase are necessary signaling molecules in
considerable evidence that the subunit is essential for NADPH cortical neurons, and they support the key role of NMDA recep-
activation by stimulation of the NMDA receptor. tors in inducing ROS production within neurons.
Furthermore, the NMDA receptor, as a type of glutamate re- These questions represent a novel and promising avenue of
ceptor, is well-established as a means to induce ROS production neuroscience research. The role of the cerebral cortex in motor
in other CNS neurons by excitatory and excitotoxic response.5, 22 activity has been lightly outlined, but its mechanism of instructing
Although means of ROS production by NMDA receptor-medi- PRYHPHQW LV DQ LQQRYDWLYH DQG UHODWLYHO\ XQH[SORUHG ÀHOG 7KH
ated mechanisms other than NADPH oxidase cannot be ruled SUHVHQFHRI 1$'3+R[LGDVHZLWKLQDUHDVRI WKHFRUWH[VSHFLÀ-
NEUROSCIENCE

out, the NMDA-induced reduction of DHE intensity in Nox2 cally responsible for motor activity, such as the prefrontal lobe,
knockout neurons suggests that the enzyme is an important con- could further reinforce the function of NADPH oxidase as a
tributor to NMDA receptor-mediated ROS production in cortical chief producer of ROS for motor activity signaling; such activity
neurons. could be indicated by immunohistochemical staining. Other types
Currently, ROS production is considered the leading culprit in RI H[SHULPHQWVVSHFLÀFWRPRWRUDFWLYLW\FRXOGEHFRQGXFWHGRQ
inducing neuronal apoptosis in age-related diseases, particularly gp91 null mice, to reinforce the abnormal decrease in movement
Alzheimer’s disease. NADPH oxidase has been suggested as the in the absence of NADPH oxidase.
PHDQVE\ZKLFKÀEULOODUƢDP\ORLGSURWHLQVVLJQDOQHXUDOGHDWK25-
27
Although these recent developments appear contradictory to
REFERENCES
the present study, the results presented here aim to elucidate what
little is known about the mechanisms of ROS within the brain. 1. Bear MF, Connors B, Paradiso M, 2007. Neuroscience: Exploring the Brain,
Although overexpression of ROS has been proposed to be cru- Lippincott Williams & Wilkins.
2. Principles of Neural Science edited by Eric R. Kandel, James H. Schwartz,
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w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
68
 Manuscripts

Probing non-linear rheology with

free oscillations
NORMAN Y. YAO1, RYAN J. LARSEN2, AND DAVID A. WEITZ1,2
1
Department of Physics and 2School of Engineering and Applied Sciences,
Harvard University, Cambridge, MA 01238
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____________________________________________________
INTRODUCTION but incorporating higher harmonics into quantitative calculations
of non-linear elasticity and dissipation remains a challenge. A
Rheological measurements are exceptionally important in recent method of analyzing LAOS has focused on a geometrical
characterizing a material’s properties. Measurements are typically interpretation, whereby the non-linear stress is broken into elastic
performed in the linear regime, where a material’s response and viscous components, leading to a generalized storage and loss
maintains a direct linear relationship to the input stress or strain. modulus.2 This method utilizes Lissajous plots of elastic stress vs.
However, many materials also exhibit a non-linear response that strain and viscous stress vs. strain-rate to determine the non-linear
is both relevant and important. For example, many biopolymer elasticity and dissipation of the material. While interpretations of
networks exhibit a pronounced non-linear rheological response.9 LAOS data are a promising method to characterize non-linearity,
The importance of this non-linearity has inspired in vitro studies there exist other methods as well. One such method is the
of a variety of biological gels ranging from actin to collagen. Many differential measurement, which provides insight into non-linear
of these in vitro gels show remarkable strain-stiffening behavior. properties by superimposing a small oscillatory stress on a steady
Accurately characterizing the response in this non-linear stiffening pre-stress. In this method, an oscillatory stress is applied above a
regime is an important challenge. There currently exist several VKHDUVWUHVVƳ0), resulting in a total stress of the form ƳW Ƴ0 +
methods to characterize a material’s non-linear properties. The _ƤƳ0_VLQƹW. The resulting strain from the small oscillatory stress
most straightforward method is simply to extend the framework then takes the form ƤƣW  _Ƥƣ0_ VLQƹW I As long as _ƤƳ0|
PHYSICS

of the linear viscoelastic moduli (*Ȩ *ȩ) into the non-linear  Ƴ0 the measured strain is a pure sinusoid and thus directly
regime. This method interprets large amplitude oscillatory shear proportional to the applied oscillatory stress.6 Another method
data (LAOS) as though they were taken in the linear regime and involves the use of free oscillations, which result from an interplay
ÀWV WKH PDWHULDO·V UHVSRQVH WR D VLQJOH WULJRQRPHWULF IXQFWLRQ between the rheometer tool’s inertia and the sample’s elasticity.
Although the validity of *Ȩ *ȩ in the non-linear regime is Upon the application of a sudden shear stress, the sample’s strain
questionable, this method provides a simple way to capture response overshoots its equilibrium value, only to be pulled back
the onset of non-linearity. In this non-linear regime, an explicit by the sample’s elasticity. Although there has been discussion
sinusoidal driving force results in a non-sinusoidal response that is regarding the use of free oscillations in the non-linear regime,
not accurately governed by a single trigonometric function. This this method has yet to be used extensively or compared to other
means that the linear viscoelastic moduli are no longer adequate current methods for probing non-linearity.1
for interpretations of LAOS data. Large amplitude oscillatory A simple and accurate way to characterize the elasticity and
shear data have also been interpreted in other ways to quantify dissipation of a material at all stresses is vital to understanding a
material non-linearity. material’s responses in the non-linear regime. A comparison of these
A popular method for analyzing LAOS data is the Fourier- various methods is essential for determining internal consistency
Transform method, which utilizes the relative intensities of among the measurements and will further provide insight into the
higher harmonics as a measure of non-linearity.7,13 This method most effective way to characterize non-linear material properties.
provides an accurate assessment of the onset of non-linearity, ,Q WKLV SDSHU ZH SURYLGH WKH ÀUVW V\VWHPDWLF FRPSDULVRQ
69 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

between free oscillations and various other non-linear rheological - 4.0 Hz), *Ȩ ~20Pa, and *ȩ a3D 1RQOLQHDULW\ LQ WKH ÀEULQ
methods and describe the use of free strain oscillations to QHWZRUN·VUHVSRQVHRFFXUVDERYHDFULWLFDOVWUDLQƣc ~ 0.1, which
accurately capture the non-linear viscoelastic properties of an corresponds to an applied stress amplitude of approximately 1Pa.
in vitro biological gel. We examined four methods, including an
extension of the linear viscoelastic framework (*Ȩ*ȩ), a geometric RESULTS
interpretation of LAOS, the differential measurement, and free
oscillations. We utilized each method to quantify the elasticity We began by measuring *Ȩ *ȩ in the non-linear regime of
and dissipation of the sample at various points within the non- ÀEULQE\VZHHSLQJWKURXJKVWUHVVDPSOLWXGHVUDQJLQJIURP3D
linear regime. Upon comparison of the various measurements to 20Pa at a frequency of 0.1Hz. As shown in Fig. 1, both *Ȩand
of elasticity, we found *Ȩ to be substantially lower than the other *ȩ remained unchanged in the linear regime while they exhibited
measurements of non-linear elasticity. Excluding *Ȩ we found dramatic increases in the non-linear regime. Repeated tests
excellent consistency among the methods of measuring non-linear showed that the stress sweeps were reversible and that the same
elasticity. Accurately measuring the sub-dominant component of moduli were obtained each time. Although the simplest probe
DPDWHULDO·VUHVSRQVHLQRXUFDVHWKHGLVVLSDWLRQLVGLIÀFXOWZLWK of non-linear properties, the linear viscoelastic moduli assume
any test involving an oscillatory measurement. For example, we certain conditions that are not met in the non-linear regime. For
found that the differential loss modulus was extremely noisy and example, measurements of *Ȩ *ȩ assume that the input stress
was even negative at certain points. However, utilizing free strain and the measured strain are directly proportional, meaning that
oscillations provided for a measurement of non-linear dissipation the measured strain is a pure sinusoid. This assumption is valid in
that was much less noisy than the differential loss modulus. The the linear regime but is not valid in the non-linear regim.12
dissipation as calculated from free strain oscillations was also The second method tested was the geometric interpretation of
quantitatively consistent with our expectations and the assumption LAOS data. The assumptions inherent to the linear viscoelastic
that the material was not being altered by our measurements. PRGXOLDUHDYRLGHGZLWKDVWUHVVVWUDLQFXUYH$/LVVDMRXVÀJXUH
was obtained directly from the raw data of a single oscillation at
0.1Hz by plotting the applied stress as a function of the measured
METHODS
strain. The raw data was collected at a rate of 1000 points per
We utilized the biologically relevant and well-characterized second using a separate utility provided by TA Instruments.
PDWHULDO ÀEULQ ZKLFK DFWV DV WKH LQWHJUDO FRPSRQHQW RI  EORRG 7KH /LVVDMRXV ÀJXUHVDUH LQGHSHQGHQW RI  IUHTXHQF\7KHVKDSH
clots. Fibrin networks are an excellent model system with which of the Lissajous plot is able to provide information about the
to study the physics of biopolymer networks and are also an mechanical properties of the material, with a linear response being
important part of homeostatic processes. Most importantly here, an ellipsoid. Any deviation from a pure ellipsoid corresponds to a
the elasticity of these networks is highly non-linear. Upon the deviation from linearity. Additionally, the energy dissipated by the
addition of the enzyme thrombin (0.5U/ml), the glycoprotein material can be obtained by integrating the area contained within
ÀEULQRJHQ PJPO ZDV SRO\PHUL]HG WR IRUP in vitro ÀEULQ WKH/LVVDMRXVÀJXUH,QIDFWWKHDUHDHQFORVHGE\WKH¶¶ORRS··FDQEH
networks.8,11 This solution was gently mixed and loaded within interpreted as viscous damping, whereas the slope of the primary
10 seconds onto the home-made steel bottom plate of a stress- axis of the ellipse indicates the elastic modulus. )RUWKHÀEULQJHO
2

controlled rheometer (AR-G2, TA Instruments). the Lissajous curve enclosed very little area because of the elastic
We characterized the sample’s polymerization, which lasted nature of the network. Non-linearity was evident in the Lissajous

PHYSICS
approximately one hour, by measuring the linear viscoelastic ÀJXUH DV WKH ORRS GHYLDWHG GUDPDWLFDOO\ IURP D SXUH HOOLSVH DV
moduli. During polymerization, we found that the linear viscoelastic VKRZQLQ)LJD7RÀQGWKHHODVWLFPRGXOXVDWDJLYHQVWUHVV
moduli increased rapidly before reaching a plateau value. Upon full we plotted the elastic stress vs. strain curve in Fig. 2(b). The elastic
polymerization of the network, *Ȩ*ȩ both remained steady; we VWUHVVDWDVSHFLÀFVWUDLQZDVJLYHQE\WKHDYHUDJHRI WKHWZRVWUHVV
then measured the frequency dependence of the elastic and loss values on the Lissajous plot.2
Thus, using the elastic stress-strain
moduli. To ensure a linear response we maintained a maximum FXUYHWKHPRGXOXVDVDIXQFWLRQRI VWUHVVDPSOLWXGHƳ s
) is KsȨƳs)
applied stress below 0.01Pa and found that the linear moduli were >GƳGƣ@ Ƴs
. In order to determine a loss modulus at each stress,
frequency independent such that for all tested frequencies (0.001 we plotted viscous stress as a function of strain-rate, where the
YLVFRXVVWUHVVLVGHÀQHGWREHWKHDEVROXWHYDOXHRI WKHGLIIHUHQFH
FIGURE 1.The linear vis- between the elastic stress and the applied stress at a given strain.2
FRHODVWLF PRGXOL RI PJPO ¾- +RZHYHU EHFDXVH RI  WKH HODVWLF QDWXUH RI  ÀEULQ WKH YLVFRXV
brin obtained from a stress am- component of the stress was extremely small, causing the viscous
plitude sweep at a frequency of stress vs. strain-rate Lissajous to be noisy and indeterminate. In
0.1 Hz. addition, the measured strain was not a perfect sinusoid, which
is also requisite for accurate strain-rate plots. The importance
of accurately characterizing the sub-dominant component of a

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
70
 Manuscripts

PDWHULDO·VUHVSRQVHDWDOOVWUHVVHVUHTXLUHVDPHWKRGWKDWTXDQWLÀHV FIGURE 2
both elasticity and dissipation. FIGURE 3
We next examined the differential measurement, which provides
access to both viscoelastic moduli in the non-linear regime. To
calculate the differential elastic moduli we super-imposed a small
oscillatory stress ƤƳ0W _ƤƳ0_VLQƹW on a steady stress Ƴ0 or a pre-
stress. Thus, the applied stress as a function of time was ƳW Ƴ0
 _ƤƳ0_ VLQƹW. We measured the resultant oscillatory strain,
ƤƣW  _Ƥƣ0_ VLQƹW  I  :H FRQÀUPHG WKDW WKH UHVSRQVH ZDV
indeed linear and that the waveforms pure sinusoids for all Ƴ0
provided that ƤƳ0 Ƴ0/10 . The differential elastic modulus is given
FIGURE 2. Lissajous plot obtained from raw data of the instrument
by and the differential viscous modulus by XWLOLW\ $ 6WUDLQVWLIIHQLQJ EHKDYLRU RI ¾EULQ LV VKRZQ E\ WKH QRQ
ellipsoidal shape. (B) Elastic stress vs. strain, from which ( ) is
calculated.
.5 FIGURE 3. The differential moduli as a function of prestress at
0.1Hz.
The differential measurement provides a quantitative value for
both moduli at each prestress. The elasticity exhibits similar trends shown in Fig. 4. Using the KV model, we found that for a ringing
when compared to the elasticity obtained from the two previous frequency of ƹ and an exponential decay constant of Ƣ, where
tests, whereas .ȩ is extremely noisy, even becoming negative at the envelope of decay is given by eƢW, the elastic modulus is given
some prestresses, as shown in Fig. 3. Indeed, applying a prestress
ZLOOFDXVHDYLVFRHODVWLFPDWHULDOWR´ÁRZµDQGWKXVSURELQJWKH
loss modulus with an imposed oscillation would require the abil-
LW\WRDFFXUDWHO\VHSDUDWHHIIHFWVRI UHOD[DWLRQIURPWKRVHRI ÁRZ
7KLV VHSDUDWLRQ LV RIWHQ GLIÀFXOW DQG LQDFFXUDWH FDXVLQJ ODUJH
variations in the reported differential loss modulus.
The fourth method we considered was free oscillations; these
avoid the problems caused by the oscillatory measurements present
in all the other methods. If the steady shear stress is ramped up
slowly enough, the process would be quasi-static, and the strain
would be in equilibrium at all times. When the steady stress reaches
LWVÀQDOYDOXHWKHVWUDLQZLOODOVRUHDFKLWVÀQDOHTXLOLEULXPYDOXH
However, the sudden application of the steady shear stress causes
WKHPDWHULDO·VVWUDLQUHVSRQVHWRRYHUVKRRWLWVÀQDOHTXLOLEULXPYDOXH
only to be pulled back by the sample’s elasticity. This overshooting
is a consequence of the tool inertia and occurs as the rotational
energy of the bearing is transferred to strain energy. This type
of free strain oscillation would then result in an under-damped
PHYSICS
sinusoid, eventually reaching the equilibrium strain value, as shown
in Fig. 4. To better understand the free strain oscillations of these
viscoelastic networks, we modeled the networks as a set of purely
elastic springs and purely viscous dampers. We used a Kelvin-Voigt
(KV) model, which is represented as a purely viscous damper and a
purely elastic spring connected in parallel. The governing equations
for the model are: ƣTotal  ƣs  ƣd and ƳTotal  Ƴs  Ƴd , where the
s and the d subscripts denote the strain/stress of the spring and
damper, respectively. Thus, each component of the system feels
the same strain while the total stress of the system is a sum of each
of the stresses. This model captures free oscillations well: a sudden
DSSOLFDWLRQRI DFRQVWDQWVWUHVVƳc to a Kelvin-Voigt material results
in a deformation approaching the deformation for the pure elastic
material Ƴc /KcȨ with the difference decaying exponentially.3 We
subtracted the equilibrium strain and modeled the peaks using an
H[SRQHQWLDO UHJUHVVLRQ WKHUHE\ ÀQGLQJ WKH HQYHORSH RI  GHFD\ DV
71 w w w . T H U R J . o r g T H U R J V o l
by KcȨƳc  Dƹ2 Ƣ2 and the loss modulus by KcȩƳc  DƹƢ 
where , with I the moment of inertia of the tool, FƳ the
proportionality factor between stress and torque such that Ƴ )Ƴƴ
, and Fƣ the proportionality factor between strain and angular
displacement such that ƣ )ƣƨ .1,14 Although the simplest way to
obtain these equations is with a KV model, similar equations for KcȨ
and Kcȩ can also be obtained by calculating approximations from a
general relaxation function.4,10
DISCUSSION
To ascertain the optimum technique, we compared the elasticity
measured with each method. In the linear regime, all measures, *Ȩ
.Ȩ.sȨ and KcȨagreed closely with one another. In the non-linear
regime,.Ȩ.sȨ.cȨalso agreed closely with one another. By contrast
however, the extended linear elastic modulus *Ȩ was much lower
than the other measures of elasticity, as shown in Fig 5; moreover, *Ȩ
deviated further from the others as the non-linearity increased. This
is expected, as increasing non-linearity would result in additional
departure from the linear viscoelastic assumption that the response
strain is directly proportional to the input stress. The internal
consistency found within measurements of non-linear elasticity
does not extend to measurements of dissipation. In the non-linear
regime, .ȩ was dominated by noise, as shown in Fig 6; moreover,
although *ȩ and Kcȩ and showed similar increasing trends, their
magnitudes varied greatly. By contrast, in the linear regime, all
measurements of dissipation were consistent, such that *ȩ , .ȩ,
and Kcȩ agreed closely with one another. Combining measurements
of elasticity and dissipation, only the linear viscoelastic moduli and
the moduli obtained from free oscillations provided noise-free,
consistent values for both an elastic and loss modulus. For each
of these methods, the ratio of elasticity to dissipation remained
constant such that at all stresses *Ȩa*ȩ and KcȨa.cȩas shown
I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7

FIGURE 4. )UHHRVFLOODWLRQVLQWKHQRQOLQHDUUHJLPHREWDLQHGDWDVKHDUVWUHVVRI3D7KHGDPSLQJLV¾WWRDQH[SRQHQWLDOGHFD\UHGOLQH

FIGURE 5$FRPSDULVRQRIWKHVWRUDJHPRGXOLREWDLQHGIURPGLIIHUHQWPHWKRGVSORWWHGDVDIXQFWLRQRIPD[LPXPRVFLOODWRU\VWUHVVDP-
plitude for *ƌ of İ for KsƌRIİ0 for .ƌDQGRIİc for Kcƌ .
FIGURE 6.$FRPSDULVRQRIWKHYDULRXVORVVPRGXOLREWDLQHGIURPGLIIHUHQWPHWKRGVSORWWHGDVDIXQFWLRQRIPD[LPXPRVFLOODWRU\VWUHVV
amplitude for *ƍRIİ0 for .ƍDQGRIİc for Kcƍ
FIGURE 7.7KHH[WHQGHGOLQHDUYLVFRHODVWLFPRGXOLDQGWKHPRGXOLREWDLQHGIURPIUHHRVFLOODWLRQVSORWWHGDJDLQVWVWUHVVGHSLFWVLPLODU
trends. Namely, the ratios *ƌ*ƍ and Kcƌ.cƍ are constant for all stresses.
in Fig. 7. Indeed, as long as the material properties were unchanging, the geometric interpretation and the differential measurement.
we would expect that an increase in the storage modulus would Measurements of the non-linear dissipation by free oscillations
EHUHÁHFWHGLQWKHORVVPRGXOXVDVZHOO7KLVIDFWRULVFRQVLVWHQW however, were able to provide consistent and sensible results for
ZLWKWKHUDWLRIRXQGLQWKHOLQHDUUHJLPHRI WKHÀEULQJHOZKHUH*Ȩ the sub-dominant response of our network. While conventional
~20Pa and *ȩ ~2Pa. This suggests that both *Ȩ and Kcȩ correctly methods of determining the dissipation at each stress lack either
characterize the qualitative trends of the dissipation as a function consistency or accuracy, free strain oscillations provide both.
of stress. However, quantitatively, both linear viscoelastic moduli
suffer from the assumption of a single frequency response, which is ACKNOWLEDGEMENTS
no longer valid in the non-linear regime. Much of the information We thank Gareth McKinley and Randy Ewoldt for their useful
stored in the higher harmonics of the non-linear response is discussions and insights. This work was supported by the NSF
lost for both *Ȩ and *ȩ since higher harmonic amplitudes are (DMR-0602684 and CTS-0505929), and the Harvard MRSEC
disregarded. Our comparisons show that the elastic modulus (DMR-0213805).
obtained from non-linear free strain oscillations is consistent with
those obtained from a geometric interpretation of LAOS data and REFERENCES
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3. 'HPLUD\ + ´9LVFRHODVWLF PRGHO IRU DUWHULDOZDOO PDWHULDOVµ ,QWHUQDWLRQDO
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test and thus can be captured quickly. The simplicity of picking out 7. Kallus, S., N. Willenbacher, et al, “Characterization of polymer dispersions by
)RXULHUWUDQVIRUPUKHRORJ\µ5KHRORJLFD$FWD
the ringing frequency and the decay constant make this method an 8. /LX:/0-DZHUWKHWDO´)LEULQÀEHUVKDYHH[WUDRUGLQDU\H[WHQVLELOLW\DQG
elegant way to characterize the viscoelastic properties as a function HODVWLFLW\µ6FLHQFH
of applied stress, in the non-linear regime. 9. 6WRUP&--3DVWRUHHWDO´1RQOLQHDUHODVWLFLW\LQELRORJLFDOJHOVµ1DWXUH
435(7039), 191-194 (2005).
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CONCLUSION Rheologica Acta 6(2), 10 (1967).
11. :HLVHO-:)LEULQRJHQDQGÀEULQ´)LEURXV3URWHLQV&RLOHG&RLOV&ROODJHQ
We found internal consistency among a variety of methods DQG(ODVWRPHUVµ
12. :LOKHOP 0 ´)RXULHUWUDQVIRUP UKHRORJ\µ 0DFURPROHFXODU 0DWHULDOV DQG
regarding the characterization of non-linear elasticity. Utilizing Engineering 287(2), 83-105 (2002).
a geometric interpretation of LAOS data, a differential 13. :LOKHOP0'0DULQJHWDO´)RXULHUWUDQVIRUPUKHRORJ\µ5KHRORJLFD$FWD
measurement, and free strain oscillations provided an internally 37(4), 399-405 (1998).
14. Zolzer, U. and H. F. Eicke, “Free oscillatory shear measurements - an interest-
consistent set of tests to characterize non-linear elasticity. The LQJDSSOLFDWLRQRI FRQVWDQWVWUHVVUKHRPHWHUVLQWKHFUHHSPRGHµ5KHRORJLFD
sub-dominant response, in our case the dissipation, is more Acta 32(1), 104-107 (1993).
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Pelvic Fin Motion During

Steady Swimming and Maneuvers
WHITNEY KRESS1, EMILY STANDEN1, AND GEORGE LAUDER1

Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue,

Cambridge, Massachusetts 02138, USA1
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REFERENCES

1. 6WDQGHQ(0DQG*9/DXGHU´+\GURG\QDPLFIXQFWLRQRI GRUVDODQGDQDO
ÀQVLQEURRNWURXW6DOYHOLQXVIRQWLQDOLVµ7KH-RXUQDORI ([SHULPHQWDO%LRO-
RJ\
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WKHDGLSRVHÀQLQÀVKHVµ&DQDGLDQ-RXUQDORI =RRORJ\
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EHKDYLRUDOUHSHUWRLUHDQGK\GURG\QDPLFIRUFHVµ7KH-RXUQDORI ([SHULPHQ-
WDO%LRORJ\
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ÀVKHV7HOHVRWHL2VWUDFLLGDHDVD0RGHO6\VWHPIRU)LVKHV6ZLPPLQJZLWK
0DQ\ )LQV .LQHPDWLFVµ 7KH -RXUQDO RI  ([SHULPHQWDO %LRORJ\  
PHYSIOLOGY


5. 6WDQGHQ(0DQG*9/DXGHU´'RUVDODQGDQDOÀQIXQFWLRQLQEOXHJLOOVXQ-
ÀVKLepomis macrochirusWKUHHGLPHQVLRQDONLQHPDWLFVGXULQJSURSXOVLRQDQG
0DQHXYHULQJµ7KH-RXUQDORI ([SHULPHQWDO%LRORJ\
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VWHDG\SHFWRUDOÀQVZLPPLQJLQWKHVXUISHUFKHVµ7KH-RXUQDORI ([SHULPHQ-
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79 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

Biological, socioeconomic, and political

aspects of the Nassau grouper (Epinephelus striatus)
ÀVKHU\LQWKH7XUNVDQG&DLFRV,VODQGV
ANH-THU E. VO1*, ANGELA DIKOU2, AND STEVEN P. NEWMAN2

Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue, Cambridge, Mas-
sachusetts 02138, USA,1 and The School for Field Studies Center for Marine Resource Studies, 1 West Street,
South Caicos, Turks and Caicos Islands, British West Indies,2
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8QLRQIRUWKH&RQVHUYDWLRQRI 1DWXUH+RZHYHU7XUNVDQG&DLFRV,VODQGV7&,SURWHFWLYHUHJXODWLRQV
IRUWKH1DVVDXJURXSHUGRQRWFXUUHQWO\H[LVW7KLVVWXG\H[DPLQHGLWKHORFDWLRQVÀVKHGLLWKH
VL]HVRI 1DVVDXJURXSHUVODQGHGLLLWKHXWLOLW\RI ODQGHGFDWFKDQGLYWKHPRWLYHVRI VWDNHKROGHUV
UHJDUGLQJ WKH 7&, 1DVVDX JURXSHU 1DVVDX JURXSHUV FDXJKW IURP EDQN GURSRIIV ZHUH ODUJHVW LQ
PHDQVWDQGDUGOHQJWK6/“FP6'Q IURPRSSRUWXQLVWLFVSHDUÀVKLQJE\OREVWHU
GLYHUVVPDOOHVWLQPHDQ6/“FP6'Q DQGIURPOREVWHUSRWE\FDWFKLQWHUPHGLDWHLQ
PHDQ6/“FP6'Q 7KH7&,JRYHUQPHQWYDOXHG1DVVDXJURXSHUVIRUVRFLDODQG
HFRQRPLFXWLOLW\UHVWDXUDQWVGXHWRORFDODQGWRXULVWGHPDQGÀVKHUPHQIRULQFRPHIURP1DVVDX
JURXSHU VDOHV DQG ERWK ÀVKHUPHQ DQG ORFDO GRFNVLGH EX\HUV GXH WR WDVWH 7KLUW\ WZR SHUFHQW RI 
1DVVDX JURXSHUV FDXJKW DV E\FDWFK LQ OREVWHU SRWV DQG  RSSRUWXQLVWLFDOO\ VSHDUÀVKHG ZHUH
UHSURGXFWLYHO\ LPPDWXUH 7KHUHIRUH LQFUHDVLQJ ÀVKLQJ LQWHQVLW\ WR PHHW ULVLQJ WRXULVW GHPDQG
PD\ XQGHUPLQH WKH UHSURGXFWLYH SRWHQWLDO RI  WKH QDWXUDO VWRFN ZLWKRXW JRYHUQPHQW UHJXODWLRQ
____________________________________________________
INTRODUCTION PDWXUHLQGLYLGXDOVDUHDOVRVXVFHSWLEOHWRKHDY\ÀVKLQJSUHVVXUH
1DVVDX JURXSHU ZDV RQFH WKH PRVW FRPPRQO\ ODQGHG ÀVK
The Nassau grouper (Epinephelus striatus, Bloch) is a large, species in the western Atlantic and Caribbean. However, out
long-lived serranid found within coral reef communities in the of 60 to 80 known Nassau grouper spawning aggregations, an
western Atlantic and Caribbean. Late larvae and juveniles can be estimated third of these aggregations have either disappeared or
found among algal covered microstructures while older juveniles been overexploited, while the remaining ones are on the decline
shift to patch reefs.1 Adults positively associate with high relief ZLWKRQO\DIHZWKRXVDQGÀVKSHUDJJUHJDWLRQ11,16 This marked
substrates ranging in depths from the shoreline to at least 100 decrease in spawning aggregation sizes and numbers has been
m2,3,4 and demonstrate crepuscular activity rhythms.5,6 They are documented throughout the Caribbean as a result of the use of
non-specialized, bottom predators, reported to reach a total length KDQGOLQHVORQJOLQHVÀVKWUDSVVSHDUJXQVDQGJLOOQHWV12,15,16,17,18
of 120 cm,7 weigh up to 25 kg,8,9 and live up to 29 years.10 Further, over the past 30 years, the total population has
1DVVDX JURXSHUV DUH YXOQHUDEOH WR ÀVKLQJ PRUWDOLW\ EHIRUH experienced an estimated 60 % decline, and suitable habitat
reproducing. Males and females typically mature between 40-45 appears to be diminishing.18 Consequently, the Nassau grouper
cm standard-length (SL) and 4-8 years of age.11 The majority KDVEHHQFODVVLÀHGDVDQHQGDQJHUHGVSHFLHVE\WKH,QWHUQDWLRQDO
of Nassau groupers landed in Cuba, and at least 70% landed in 8QLRQIRUWKH&RQVHUYDWLRQRI 1DWXUH,8&1VLQFHDQGLV
the U.S. Caribbean, are reproductively immature due to shallow, currently a candidate for the U.S. Endangered Species List.18
LQVKRUHÀVKHGDUHDVDQGWUDSPHVKVL]HVXVHG11 However, as mature /HJDO PHDVXUHV VXFK DV FORVHG ÀVKLQJ VHDVRQV FORVHG ÀVKLQJ
SUSTAINABILITY

adults form spawning aggregations at consistent locations,9,12,13,14,15 areas, quotas, minimum weight restrictions, gear restrictions, or
SCIENCES

_______________________________ marine protected areas (MPAs) have been implemented in many

* Corresponding author. Mailing Address: Department of countries to help manage Nassau grouper stocks. However,
11,15,18,20,21

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Organismic and Evolutionary Biology, 22 Divinity Avenue,
of adequate enforcement, while Nassau grouper mobility and
Cambridge, Massachusetts 02138. Email: avo@fas.harvard.edu
migrations often undermine the goal of MPAs.4,22 ,Q DGGLWLRQ
w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
80
 Manuscripts

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pressures such as subsistence technology, little physical or social the attainment of all stakeholder objectives.
LQIUDVWUXFWXUH ORZ SRSXODWLRQ VL]HV XQGLYHUVLÀHG HFRQRPLHV
high unemployment rates, and low income that facilitate
overexploitation.23
STUDY SITE
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WKH 7XUNV DQG &DLFRV ,VODQGV 7&, WKH OLPLWHG GRFXPHQWDWLRQ
RI 7&,ÀQÀVKSURGXFWLRQDQGXVH24 and the lack of protective
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are commonly caught as bycatch or opportunistically in the lobster
ÀVKHU\24,25 Nassau groupers have historically been preferred
IRU WKHLU VL]H DQG TXDOLW\ RI  ÁHVK24 although they also afford
economic value to the dive tourism industry, with divers exhibiting
a higher willingness to pay to see more or larger individuals.26 An
DVVHVVPHQWRI 1DVVDX*URXSHUVWRFNVLVODFNLQJIRU7&,EXWDV
ÀQÀVKLQJSUHVVXUHKDVEHHQPLQLPDOWKHVWRFNVDUHDVVXPHGWR
be high. However, a tourism boom is imminent on South Caicos,
and demand for conch and lobster will increase while landings
will be restricted by biological and management constraints.
&RQVHTXHQWO\ GHPDQG IRU ÀQÀVK LV H[SHFWHG WR LQFUHDVH ZLWK
ÀVKHUPHQ HPSOR\LQJ DOWHUQDWLYH ÀVKLQJ VWUDWHJLHV27 through
UHGLUHFWLQJ WKHLU HIIRUWV WRZDUGV LQFUHDVHG ÀQÀVKLQJ LQWHQVLW\
As a consequence, present populations of Nassau groupers are
expected to decline.
Previous studies have not considered to what extent different
VRFLRHFRQRPLF DQG SROLWLFDO IDFWRUV KDYH LQÁXHQFHG ÀVKLQJ
SUHVVXUH DQG ÀVKLQJ PHWKRGRORJ\ VSHFLÀFDOO\ IRU WKH 1DVVDX
grouper. Given the current precarious status of this species, it is
important to understand all factors that may contribute to a decline
LQWKH7&,SRSXODWLRQVLI OHIWXQUHJXODWHG7KLVVWXG\LQYHVWLJDWHV
(i) the locations and habitats exploited for Nassau grouper via
end of the Bahamian Archipelago, are comprised of the Caicos,
Turks, and Mouchoir Banks (Figure 1). The Caicos Bank is a
shallow, oolitic limestone platform encompassing an area of 6,140
km2 and comprised of 64% sand, 18% mixed coral and algae, 7%
coral reefs, and 11% other habitats.28 Turks Bank, encompassing
an area of 324 km2, is comprised of 43% sand, 29% mixed coral
and algae, 26% coral reefs, and 3% other habitats, while Mouchoir
Bank, encompassing 1,109 km2, is predominantly coral and sand.28
Water depths range from 1-5 m throughout the banks, 20-30 m at
the top of the drop-offs, and over 1800 m beyond the drop-offs.
The economy of the Turks and Caicos is primarily based
RQ WRXULVP DQG VPDOO VFDOH ÀVKHULHV 7RXULVP LV D JURZLQJ
LQGXVWU\ WKDW FRQWULEXWHG URXJKO\ D WKLUG RI  WKH 7&, *'3 LQ
2005.29 Queen conch (Strombus gigas, Linné) and spiny lobster
(Panulirus argus, Latreille) form the basis of a US$169.2 million
H[SRUWRULHQWHG ÀVKLQJ LQGXVWU\ ZKLFK HPSOR\V QHDUO\  RI 
WKH7&,ODERUIRUFH30$OWKRXJKÀVKHULHVRSHUDWHIURPWKHLVODQGV
of Providenciales, Grand Turk, and South Caicos, South Caicos
VHUYHV DV WKH KRPH EDVH IRU WKH PDMRULW\ RI  WKH 7&, DUWLVDQDO
ÀVKLQJÁHHW&RPPHUFLDOÀVKHUPHQEULQJWKHLUGDLO\FDWFKWRWKH
docks located on the southwest side of South Caicos (Figure 1).
METHODS
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grouper landings, and (iv) the motives of various stakeholders in
WKH 7&, FRPPXQLW\ 7KLV EDVHOLQH NQRZOHGJH ZLOO DLG ÀVKHULHV
managers to better regulate stocks of this endangered species
LQ WKH 7&, DQG LQ RWKHU FRPSDUDEOH FRPPXQLWLHV LQ KRSH RI 
(Table 1). Fishermen were asked to indicate the areas they tar-
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WDWV DQG UHDVRQV IRU VXFK ÀVKLQJ VWUDWHJ\ ZHUH UHFRUGHG
'RFN ODQGLQJV GDWD Size and weight of Nassau groupers
in South Caicos were obtained from individuals landed between
November 22 to December 6, 2006, via: (i) lobster divers’
RSSRUWXQLVWLFVSHDUÀVKLQJQ LLOREVWHUSRWE\FDWFKQ 
DQGLLLEDQNGURSRII RUZDOOÀVKLQJZLWKK\GUDXOLFUHHOVQ 
2QH ZDOO ÀVKLQJ ERDW RSHUDWHV IURP 6RXWK &DLFRV 7KLV ERDW
remains at sea for multiple weeks per trip, and only one catch was
brought to the dock (November 30, 2006) within the duration
of this study. Seventeen percent of Nassau groupers landed by
OREVWHUSRWVDQGDOO1DVVDXJURXSHUVODQGHGE\ZDOOÀVKLQJZHUH
gutted before weighing. Both standard length (SL) and total length
SUSTAINABILITY

(TL) measurements were acquired in order to compare results

SCIENCES

with previous studies.

),*85( Location of (a) South Caicos within the Turks & Caicos 'LVWULEXWLRQ RI ODQGHG ¾VK ,t was noted through ob-
Islands (Rudd, 2003) and (b) landings dock in southwest South VHUYDWLRQ DQG LQWHUYLHZ ZLWK ÀVKHUPHQ ZKHWKHU HDFK ODQG-
Caicos. The approximate 100 m depth contour is shown for Caicos HG JURXSHU ZDV NHSW E\ ÀVKHUPHQ RU VROG WR ORFDO FRP-
and Turks Banks. munity members, the processing plant, middle men for
81 w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

7$%/(  4XHVWLRQV SRVHG WR 6RXWK &DLFRV ¾VKHUPHQ ZLWK 0LFURVRIWŠ ([FHO  DFFRUGLQJ WR WKH ÀVKLQJ PHWKRG
FRQFHUQLQJ1DVVDXJURXSHU¾VKLQJVWUDWHJ\LQWKH7XUNVDQG employed. Differences in total length, standard length, and weight
Caicos Islands. RI  1DVVDX JURXSHUV ODQGHG YLD GLIIHUHQW ÀVKLQJ PHWKRGV ZHUH
WHVWHG XVLQJ WKH QRQSDUDPHWULF .UXVNDOO:DOOLV 5DQN 6XP ƈ
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+RZPDQ\WUDSVGLG\RXXVH"LI DSSOLFDEOH GRYHDQGÀVKHGDORQJWKHVKDOORZVRI 3KLOOLS·V5HHI6HDO&D\V
GLVWULEXWLRQ WR 7&, UHVWDXUDQWV RU RWKHU FRQVXPHU JURXSV and Bush Cay as well as in “the Lakes,” a region encompassed
6WDNHKROGHUVXUYH\6SHFLÀFVHFWRUVRI WKH7&,FRPPXQLW\ by Ambergris Cays, Six Hill Cays, and Long Cay (Figure 2). The
were interviewed to determine the motivating factors inherent southwest region of Middleton Cay comprised a favorable area
in their valuation of the Nassau grouper (Table 2). The study IRUWKHGHSOR\PHQWRI OREVWHUFDVLWDVDUWLÀFLDOVWUXFWXUHVWRZKLFK
SRSXODWLRQRI RIÀFHUVLQWKH7&,'HSDUWPHQWRI (QYLURQPHQW lobsters aggregate for shelter. However, these commercial divers
DQG &RDVWDO 5HVRXUFHV '(&5 LQFOXGHG ÀYH LQGLYLGXDOV were restricted to areas less than 18.3 m deep with good visibility
DFFHVVLEOHE\WHOHSKRQHDQGWZRDGGLWLRQDORIÀFHUVZHUHVDPSOHG and minor current.
LQ VHSDUDWH LQWHUYLHZV 7KH VWXG\ SRSXODWLRQ RI  7&, UHVWDXUDQW
owners or managers involved 68 restaurants that were accessible
by telephone, and all 68 restaurants were contacted to determine
whether they served Nassau grouper. Only thirteen out of 60
3URYLGHQFLDOHVUHVWDXUDQWVÀYHRXWRI VL[*UDQG7XUNUHVWDXUDQWV
and both South Caicos restaurants fully responded to all questions
in the survey. Out of approximately 140 commercially licensed
ÀVKHUPHQLQ6RXWK&DLFRVWKHVWXG\SRSXODWLRQLQFOXGHGWKRVH
ZKR ÀVKHG IRU 1DVVDX JURXSHUV DQG XWLOL]HG WKH GRFN 7KH
VDPSOHFRQVLVWHGRI DFRRSHUDWLYHVXEVHWRI WHQÀVKHUPHQZKR
either landed Nassau groupers or were available for a personal
interview. A study population of approximately ten local groups
of dockside buyers regularly purchased groupers, and nine were
sampled within the time period of the study.
'DWD DQDO\VLV /RFDWLRQV RI  GLIIHUHQW ÀVKLQJ PHWKRGV LQ
WKH 7&, ZHUH LQFRUSRUDWHG LQWR D PDS FUHDWHG XVLQJ *DUPLQ
7ZR ODUJH VFDOH OREVWHU SRW ÀVKLQJ ERDWV ! WUDSV DQG DW
least one small scale boat (<200 traps) mainly targeted White Cay,
Seal Cays, Bush Cay, and to a lesser extent, Fish Cays (Figure 2).
)LVKLQJYHVVHOZLWKWRWDOWUDSVDQGÀVKLQJYHVVHOZLWK
275 total traps, placed transects of 25 lobster pots at depths of
21.3-27.4 m in areas to the southwest of White Cay, to the southeast
of Bush Cay, and among the islands of Seal Cays. These habitats
are comprised of a large, low-gradient bank with seagrasses and
corals. However, the placement of these transects were frequently
DOWHUHG DV ÀVKHUPHQ FRQVXOWHG SHUVRQDO DQQXDO UHFRUGLQJV RI 
lobster activity and migrations. Transects were accordingly moved
with changing weather conditions, lunar phases, and seasonal
SURJUHVV WR PD[LPL]H WRWDO OREVWHU FDWFK ZKLOH WKH VL]H RI  ÀVK
E\FDWFKGLGQRWIDFWRULQWRÀVKLQJVWUDWHJ\
7KH RQO\ 6RXWK &DLFRV ZDOO ÀVKLQJ ERDW ÀVKLQJ YHVVHO 
concentrated its efforts predominantly at the southern end of
0DS6RXUFHŒ YHUVLRQ  9DULDWLRQV LQ ÀVK OHQJWK ZHLJKW
and catch destination within the market were graphically explored
7$%/(  &RPPXQLW\ VHFWRUV WDUJHWHG IRU VXUYH\ DQG WKH
TXHVWLRQVSRVHGWRGHWHUPLQHPRWLYHVIRUYDOXLQJWKH1DVVDX
grouper in the Turks and Caicos Islands.
w w w . T H U R J . o r g T H U R J
&DLFRV%DQN)LJXUH,WDOVRÀVKHGWKHZHVWHUQZDOORI &DLFRV
Bank, the southern wall of Turks Bank, and the Mouchoir Bank.
While the Mouchoir Bank yielded much larger Nassau groupers
and red snappers, the southern end of Caicos Bank yielded
the greatest numbers of both species, resulting in it being the
SUHIHUUHGÀVKLQJDUHD)LVKLQJYHVVHOW\SLFDOO\ÀVKHGDWGHSWKV
of 91-366 m with four hydraulic reels using eight hooks per reel.
'RFN ODQGLQJV GDWD Nassau groupers comprised 15.0%
of the total wet weight catch, while red snappers comprised the
UHVWRI WKHFDWFKODQGHGYLDZDOOÀVKLQJRQ1RYHPEHU
1DVVDX JURXSHUV FRPSULVHG  RI  WKH WRWDO QXPEHU RI  ÀVK
bycatch landed via lobster pots from November 22 to December
  :DOO ÀVKLQJ \LHOGHG WKH ODUJHVW 1DVVDX JURXSHUV ZLWK
PHDQOHQJWKVRI “FP6'7/Q “
SUSTAINABILITY
cm S.D. SL, mature) and mean weights of 10.29 ± 5.29 kg S.D.,
SCIENCES
Q /REVWHUSRWV\LHOGHGLQWHUPHGLDWHVL]HGLQGLYLGXDOVZLWK
PHDQOHQJWKVRI “FP6'7/Q “
cm S.D. SL, immature) and mean weights of 2.97 ± 2.01 kg S.D., n
ZKLOHFRPPHUFLDOGLYHUV·RSSRUWXQLVWLFVSHDUÀVKLQJODQGHG
the smallest Nassau groupers with mean lengths of 38.26 ± 7.05
V o l I I s s u e 1 S p r i n g 2 0 0 8
82
 Manuscripts
),*85($UHDV¾VKHGE\GLIIHUHQWPHWKRGVLQWKH7XUNVDQG&D-
LFRV ,VODQGV RSSRUWXQLVWLF VSHDU¾VKLQJ E\ FRPPHUFLDO GLYHUV DW
up to 10.0 m depths (downward diagonal stripes), lobster pots at
However, a number of factors limited the capability of the
'(&5 WR HVWDEOLVK VSHFLÀF SURWHFWLYH PHDVXUHV IRU WKH 1DVVDX
JURXSHU 6FLHQWLÀF GDWD DQG SXEOLF DZDUHQHVV UHJDUGLQJ WKH
VWDWXVRI WKH1DVVDXJURXSHULQWKH7&,ZHUHODFNLQJ3RDFKLQJ
by foreign vessels complicated regulation planning. Staff and
ÀQDQFLDOUHVRXUFHVIRUWKH'(&5ZHUHOLPLWHGDQGRWKHUSUHVVLQJ
governmental issues such as the enforcement of marine protected
areas, the sustainable development of tourism, and the control of
illegal immigrants existed.
Restaurants.—Of the 68 restaurants, 63.2% served Nassau
grouper, and of the restaurants serving Nassau grouper, 83.7%
were located on Providenciales, 11.6% on Grand Turk, and 4.7%
on South Caicos. Among the 19 restaurants that responded to
the survey, 57.9% obtained their Nassau grouper supply locally
PGHSWKVXSZDUGGLDJRQDOVWULSHVDQGZDOO¾VKLQJE\ although the supply was not dependable. The average local supply
K\GUDXOLFUHHOVDWPGHSWKVKRUL]RQWDOVWULSHV price of Nassau grouper to these 19 restaurants was US$18.32
per kilogram of cleaned meat while the average price per dish was
FP6'7/Q “FP6'6/LPPDWXUHDQGPHDQ
US$20.07 (Table 3).
ZHLJKWVRI “NJ6'Q )LJXUH
Although the most common reason why restaurants valued
7KHUH ZDV D VWDWLVWLFDOO\ VLJQLÀFDQW GLIIHUHQFH LQ WRWDO OHQJWK
Nassau grouper appeared to be tourist demand, it was mentioned
ƈ SDQGVWDQGDUGOHQJWKƈ S
WZLFHWKDW1DVVDXJURXSHUZDVQRWWKHSUHIHUUHGVHUYHGÀVKRI 
  RI  ODQGHG 1DVVDX JURXSHUV DPRQJ ÀVKLQJ PHWKRGV
the chef or the restaurant owner because of inconsistent supply
)XUWKHU WKHUH ZDV D VWDWLVWLFDOO\ VLJQLÀFDQW GLIIHUHQFH LQ WKH
DQGORZSURÀWPDUJLQ$OOEXWRQHUHVWDXUDQWWKDWREWDLQHGDORFDO
weight of un-gutted Nassau groupers landed by lobster pots and
supply of Nassau grouper stated that the supply was undependable.
VSHDUÀVKHGE\RSSRUWXQLVWLFFRPPHUFLDOGLYHUVƈ S
Despite this discrepancy, the restaurants kept Nassau grouper on
DQGLQWKHZHLJKWRI JXWWHG1DVVDXJURXSHUVODQGHGE\
the menu due to tourist demand.
OREVWHUSRWVDQGE\ZDOOÀVKLQJƈ S 
)LVKHUPHQ³/REVWHUSRWÀVKHUPHQFRPPRQO\YDOXHG1DVVDX
'LVWULEXWLRQRIODQGHG¾VK Opportunistic commercial divers
JURXSHU EHFDXVH WKH SURÀW WKDW WKH\ HDUQHG IURP WKH VDOH RI 
VSHDUÀVKHG1DVVDXJURXSHUVHLWKHUIRUSHUVRQDOFRQVXPSWLRQRU
their bycatch comprised supplementary income (Table 4). On
DV IDYRUV IRU IULHQGV ZKR KDG UHTXHVWHG WKH ÀVK ZKHUHDV ZDOO
average US$600 from Nassau grouper bycatch sales were earned
ÀVKHUPHQZHUHKLUHGE\DORFDOSURFHVVLQJSODQWDQGWKHUHIRUH
per trip, which was frequently enough to cover the cost of diesel
the entirety of their catch was sold directly to restaurants on
and supplies (approximately US$335). Other valuations included
Providenciales (Figure 4). The distribution of Nassau groupers
taste, obligation to sell groupers to the local processing plants,
IURP OREVWHU SRW ÀVKHUPHQ ZDV PRVW GLYHUVH ZLWK UHFLSLHQWV
REOLJDWLRQ WR VHOO ÀVK WR ORFDO FRPPXQLW\ PHPEHUV GHVLUH IRU
LQFOXGLQJ WKH ÀVKHUPHQ WKHPVHOYHV IULHQGV RI  ÀVKHUPHQ ORFDO
personal consumption, and desire to give groupers to friends and
community members, middle men selling to restaurants, and local
family members that valued the taste of Nassau grouper.
processing plants.
Local Buyers.—Of the local buyers, 55.6% valued the taste
6WDNHKROGHU VXUYH\ The most important reasons for why
of the Nassau grouper most importantly while 44.4% did not
GLIIHUHQWVHFWRUVRI WKH7&,FRPPXQLW\YDOXHG1DVVDXJURXSHU
rarely overlapped between sectors (Table 3). Tourist and local
demand were most important for restaurants while taste was the
RQO\FRPPRQDOLW\DPRQJÀVKHUPHQDQGORFDOEX\HUV
DECR.—The DECR valued the Nassau grouper for economic
DQGVRFLDOEHQHÀWZKLFKIHHGWKHORFDOGHPDQG(FRQRPLFYDOXH
was placed on the availability of Nassau grouper for the tourism
LQGXVWU\ DV WKH QDWXUDO UHVRXUFHV FRPSULVH D VLJQLÀFDQW VRXUFH
RI  DWWUDFWLRQ DQG LQFRPH IRU WKH 7&, )XUWKHU ÀQÀVKHU\ ZDV
valued as a means to diversify the lobster- and conch-dominated
SUSTAINABILITY
ÀVKHULHV7KH'(&5FRQVLGHUHGWKHFXUUHQW7&,1DVVDXJURXSHU
SCIENCES
stock to be an underutilized resource that could be exploited given
high demand. Therefore, during the years 2003-2004, the DECR
IRUPDOO\ GHFLGHG WR EHJLQ HQFRXUDJLQJ WKH ODQGLQJ RI  ÀQÀVK
WR PD[LPL]H VRFLDO EHQHÀW IRU WKH ÀVKHUPHQ DQG WKH ÀVKHULHV
industry.
83 w w w . T H U R J . o r g T H U R J V o l
YDOXH1DVVDXJURXSHUDQGSUHIHUUHGRWKHUÀVKVSHFLHV7DEOH
Buyers who did not value the Nassau grouper commonly cited
WKHSUHVHQFHRI ZRUPVLQWKHÁHVK
DISCUSSION
7&, 1DVVDX JURXSHU VWDWXV The species targeted by
ÀVKHUPHQ LQ WKH 7XUNV DQG &DLFRV GHWHUPLQHG WKHLU ÀVKLQJ
VWUDWHJ\ DQG ORFDWLRQ /REVWHU SRW ÀVKHUPHQ DQG FRPPHUFLDO
divers principally targeted lobsters while Nassau groupers
comprised an opportunistic catch. Consequently, ideal lobster
habitats were pursued, and any overlap between Nassau grouper
and lobster habitats was not intentionally sought. As the areas
LPPHGLDWHO\VXUURXQGLQJWKH7&,KDGEHHQFRQVLGHUHGRYHUÀVKHG
for lobster, “the Lakes,” Seal Cays, and Bush Cay were chosen
I I s s u e 1 S p r i n g 2 0 0 8
 Manuscripts

0% 0%
FIGURE
120
3 FIGURE 4 0% 0%

100
(a)
Mean Total Length (cm))

80
fis hermen co
60 ),*85(  Variation (mean ± S.D.) in total 40% fis hermen's

40 length, standard length, and weight of local cons um

proces s ing p
20
1DVVDX JURXSHUV ODQGHG YLD GLIIHUHQW ¾VKLQJ middle men
methods in the Turks and Caicos Islands 60% res taurants
0
100 Hydraulic R eels W all L obs ter P ot B y-C atch L obs ter Divers ' GXULQJ 1RYHPEHU DQG 'HFHPEHU  $OO
90
F is hing Opportunis tic C atch
1DVVDX JURXSHUV ODQGHG YLD ZDOO ¾VKLQJ (b)
0%
Mean Standard Length (cm) '

F is hing Method
80
DQG   RI 1DVVDX JURXSHUV ODQGHG YLD
70 5%
60 lobster pots were gutted before weighing.
50
7% 12%
40 fis hermen
30
),*85(  'HVWLQDWLRQ DQG XWLOLW\ RI 1DVVDX fis hermen
20 JURXSHU FDWFKHV ODQGHG YLD D OREVWHU GLYHUVµ local cons
10
RSSRUWXQLVWLF VSHDU ¾VKLQJ E OREVWHU SRW proces s in
0 39% middle me
18
Hydraulic R eels W all L obs ter P ot B y-C atch L obs ter Divers ' E\FDWFK DQG F ZDOO ¾VKLQJ ZLWK K\GUDXOLF
res taurant
16
F is hing O pportunis tic C atch
reels in the Turks and Caicos Islands during 37%
F is hing Method
14 1RYHPEHU DQG 'HFHPEHU  ¾VKHUPHQ
Mean Weight (kg) '

12
FRQVXPSWLRQ ŵ ¾VKHUPHQµV JLIW WR IULHQGV (c)
10
0%
8 (ŵ), local consumption (ŵ), processing plant fis herm
fis herm
6 Ɛ UHVWDXUDQWV YHUWLFDO VWULSHV PLGGOH PHQ
4 local co
2
VHOOLQJWRUHVWDXUDQWVKRUL]RQWDOVWULSHV proces s
0 middle
Hydraulic R eels W all L obs ter P ot B y-C atch L obs ter Divers '
res taur
F is hing Opportunis tic C atch
F is hing Method 100%

LQVWHDG :DOO ÀVKHUPHQ GLUHFWO\ WDUJHWHG 1DVVDX JURXSHU DQG PHDQ6/ZDV“FP6'Q ZKLOHGDWDIURPIDOO
WKHUHIRUHVSHFLÀFDOO\ÀVKHGDGXOWKDELWDWVWKDWZRXOG\LHOGPRUH 2004 indicated a mean TL of 57.75 ± 11.08 cm S.D. and a mean
Nassau groupers of smaller sizes over areas with fewer, larger 6/ RI    “  FP 6' Q   )LQDOO\ ZDOO ÀVKHUPHQ
1DVVDXJURXSHUVLQRUGHUWRPD[LPL]HSURÀW yielded the largest Nassau groupers, corresponding to their
:KLOH ZDOO ÀVKHUPHQ HPSOR\HG VSHFLDOL]HG ÀQÀVK JHDU WKH VSHFLDOL]HGÀVKLQJJHDUDQGVWUDWHJ\
use of hydraulic reels along drop-offs for Nassau grouper has The distribution of Nassau groupers landed by different
RQO\EHHQIRUPHUO\GRFXPHQWHGLQWKH/HVVHU$QWLOOLDQ,VODQGV32 PHWKRGV YDULHG DFFRUGLQJ WR OHYHOV RI  ÀVKHUPHQ·V REOLJDWLRQV
Previous studies have noted the harvest and overexploitation to stakeholders. Opportunistic commercial divers possessed
of Nassau groupers from spawning aggregations by hand lines, the freedom to land and utilize Nassau groupers subjectively
ORQJOLQHVÀVKWUDSVVSHDUJXQVRUJLOOQHWV12,15,16,17,18,19 However, ZKLOH ZDOO ÀVKHUPHQ ZHUH REOLJDWHG WR WXUQ RYHU DOO ODQGHG
ÀVKHUPHQLQFOXGHGLQWKLVVWXG\ZHUHXQDZDUHRI DQ\ORFDO7&, Nassau groupers to their employers for sale to local markets in
Nassau grouper spawning aggregations. 3URYLGHQFLDOHV /REVWHU SRW ÀVKHUPHQ ZHUH FRPSHOOHG WR VHOO D
The mean size of landed Nassau groupers differed among portion of their bycatch to the processing plants at the rate of
ÀVKLQJ PHWKRGV 2SSRUWXQLVWLF FRPPHUFLDO GLYHUV VSHDUÀVKHG 86SHUNLORJUDPRI ZKROHÀVKDVRSSRVHGWR86SHU
the smallest individuals due to factors including free diving kilogram of cleaned meat supplied to restaurants, in exchange
UHVWULFWLRQV WKH IHDU RI  DWWUDFWLQJ VKDUNV GLIÀFXOW\ RI  KDQGOLQJ for ice, bait, and supplies. However, they also sold to their loyal,
DODUJHFDWFKXQGHUZDWHUDQGWKHGHVLUHWRODQGDÀVKDGHTXDWHO\ local dockside customers with the potential to earn US$6.63-8.62
VL]HGIRUDVLQJOHPHDO&RQWUDVWLQJO\OREVWHUSRWÀVKHUPHQ\LHOGHG SHUNLORJUDPRI ZKROHÀVK)LQDOO\IULHQGVDQGIDPLO\PHPEHUV
intermediate sizes of Nassau groupers. Fishermen mentioned that dictated the devotion of a portion of landed Nassau grouper
both small and extremely large Nassau groupers were negatively bycatch to personal ends.
selected by lobster pots determined by the mesh and pot opening Main objectives regarding the Nassau grouper varied among
VL]HV*LYHQWKHWHQLQFKGLDPHWHUSRWRSHQLQJVPDOOHUÀVKKDYH stakeholders. However, Nassau groupers in the Turks and Caicos
a higher probability of escaping after entrance, and extremely were valued only to the extent of their economic and social utility.
ODUJHÀVKDUHXQDEOHWRÀWWKURXJKWKHRSHQLQJ)XUWKHUOREVWHU Their biological and ecological importance did not appear to
SRWÀVKHUPHQWKURZEDFN1DVVDXJURXSHUVXQGHUNJLQWKH factor in their valuation scheme.
SUSTAINABILITY
SCIENCES

KRSHVWKDWWKH\ZLOOJURZELJJHUDVVPDOOÀVKDUHXQGHVLUDEOHWR Adverse weather conditions prolonged the soak time of lobster

their buyers. A previous study measuring Nassau groupers landed pots from one day to four days for a sampled trip which, according
via lobster pot bycatch in South Caicos during fall 2004 yielded WRÀVKHUPHQPD\KDYHHQKDQFHGWKHSUREDELOLW\RI ÀVKHVFDSH
VLPLODUOHQJWKV6FKRROIRU)LHOG6WXGLHVXQSXEOLVKHGGDWD,QWKH and thereby affected the bycatch size and composition. However,
present study, the mean TL was 55.66 ± 12.84 cm S.D., and the daily catch measurements for the remaining duration of the study

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8
84
 Manuscripts

7$%/(1DVVDXJURXSHUVXSSO\WRUHVWDXUDQWVLQWKH7XUNVDQG&DLFRV,VODQGV

Restaurant Location Supply 6WHDG\6XSSO\" Supply Price Dish Price 0RVW,PSRUWDQW9DOXD- Targeted Consumers
Source (US$/kg) (US$) tion Reason
1 Providenciales imported yes 23.21 tourist and local demand tourists and locals
2 Providenciales imported yes 23.21 23.95 tourist demand mostly tourists
3 Providenciales local no 12.16-14.37 12.00-15.00 tourist and local demand mostly locals
4 Providenciales imported yes 17.68 22.00 tourist and local demand tourists and locals
5 Providenciales imported yes 11.05-13.26 25.00-30.00 tourist demand mostly tourists
6 Providenciales imported yes 11.05-13.26 25.00-30.00 tourist demand mostly tourists
7 Providenciales imported yes 11.05-13.26 25.00-30.00 tourist demand mostly tourists
8 Providenciales imported yes 46.41 34.00 IUHVKORFDOÀVK tourists
9 Providenciales local no IUHVKORFDOÀVK tourists and locals
10 Providenciales local and local-no, 19.99 easily accessible mostly tourists
imported imported-yes
11 Providenciales local and local-no, ” • IUHVKORFDOÀVK tourists and locals
imported imported-yes
12 Providenciales imported yes 22.10 10.00-20.00 tourist and local demand tourists and locals
13 Providenciales local and local-no, local: 19.89-24.31, 10.00-24.00 tourist and local demand local and tourists and
imported imported-yes imported: 13.26 ex-patriots
14 Providenciales local no local demand
15 Grand Turk local no 15.47-17.68 popular with everyone mostly locals
16 Grand Turk local no 13.26-14.37 12.00-18.00 IUHVKORFDOÀVK tourists and locals
17 Grand Turk local no 13.26 5.00-18.00 local supply tourists and locals
18 South Caicos local no 11.05-13.26 • tourist demand mostly tourists
19 South Caicos local no 5.53-6.63, ÀOOHWHGÀVKLVSRSXODU mostly locals
QRWÀOOHWHG

served to minimize this trip’s effect on the data. Furthermore,
although the small data set in this study limits the conclusions
WKDWPD\EHGUDZQWKHGDWDFRPSULVHVWKHÀUVWLQWKHVFLHQWLÀF
literature to complement landed Nassau grouper measurements
DQGFDWFKVL]HVZLWKLQIRUPDWLRQUHJDUGLQJÀVKHGKDELWDWORFDWLRQ
and quality as well as the underlying socioeconomic and political
circumstances driving the harvest of this endangered species.
,PSOLFDWLRQVSeal Cays and Bush Cay are located approximately
48 kilometers from South Caicos, and Turks Pass and Mouchoir
%DQNDUHORFDWHGDWOHDVWNLORPHWHUVDZD\7KHUHIRUHÀVKLQJDW
Seal Cays and Bush Cay requires an extended work day in order to
FRPSHQVDWHIRUWUDYHOWLPHDQGÀVKLQJDW0RXFKRLU%DQNUHTXLUHV
remaining at sea for weeks per trip in order to make the trip
SURÀWDEOH 0DQ\ ORFDO ÀVKHUPHQ DSSHDUHG XQZLOOLQJ WR FRPPLW
to the long work hours, and these unconventional work hours
will most likely continue to discourage local workers until the
GHPDQGIRUÀQÀVKEHFRPHVKLJKHQRXJKWRPDNHWKHSD\ORFDOO\
desirable. Contrastingly, there appeared to be no shortage of
foreign workers willing to commit to the work, but work permits
IRU IRUHLJQ ODERU KDYH SURYHG GLIÀFXOW WR REWDLQ 7KHVH IDFWRUV
SUSTAINABILITY
(range 24.5-63.5 cm). Consequently, 32% of the landed individuals
had not reproduced, and as the SL of Nassau groupers landed
YLDRSSRUWXQLVWLFVSHDUÀVKLQJE\FRPPHUFLDOGLYHUVZDV“
6.02 cm S.D. (range 26.0-40.5 cm), 80% of the landed individuals
were reproductively immature. Of the total landed immature
individuals, 82% were caught as lobster pot bycatch while 18%
ZHUH RSSRUWXQLVWLFDOO\ VSHDUÀVKHG $V WKH ÀVKHU\ SHUVLVWV DQG
expands, the continued removal of immature individuals from the
stock may impact stock preservation and growth. Additionally,
as females increase in size, their egg production potential
increases exponentially.33 Therefore, the gametes produced by
WKHODUJHVWLQGLYLGXDOVODQGHGYLDZDOOÀVKLQJPD\EHFUXFLDOIRU
WKHSHUSHWXDWLRQDQGUHFUXLWPHQWÀVKLQJRI WKHVWRFN$VÀVKLQJ
LQWHQVLW\UHVSRQGVWRLQFUHDVHGÀQÀVKGHPDQG1DVVDXJURXSHU
landings may adversely affect the reproductive potential of the
stock without regulations in place to protect Nassau grouper
critical life stages. Alternatively, if Nassau groupers continue
WR EH ODQGHG IDU IURP 7&, QHDUVKRUH ZDWHUV WRXULVWVGLYLQJ LQ
the immediate vicinity maintain the chance of seeing Nassau
JURXSHUVDQGWKHRSSRUWXQLW\IRUWKHGLYHLQGXVWU\WRSURÀWIURP

ZLOOFRQWLQXHWRVKDSHWKHJURZWKRI WKH7&,ÀQÀVKLQGXVWU\DV the existence of Nassau groupers will be conveniently reserved

SCIENCES

ORFDODQGWRXULVWGHPDQGIRUÀQÀVKLQFUHDVHV as long as habitats and larval recruitment remain un-impacted

$QXPEHURI IXWXUHELRORJLFDOUDPLÀFDWLRQVRI WKH7&,1DVVDX by increasing levels of development and population density.
JURXSHUÀVKHU\PD\EHDQWLFLSDWHG1DVVDXJURXSHUVUHSURGXFWLYHO\ Future management efforts must increase public education
mature at approximately 40-45cm SL.11,QWKLVVWXG\WKH6/RI  WRZDUGVWKHVWDWXVRI WKH1DVVDXJURXSHULQWKH7&,*LYHQWKDW
85 Nassau groupers landed via lobster pots was 43.85 ± 10.59 cm S.D.
w w w . T H U R J . o r g T H U R J V
the community predominantly values the social and economic
o l I I s s u e 1 S p r i n g 2 0 0 8

7$%/(7KHPRVWLPSRUWDQWUHDVRQWKDWYDULRXVVHFWRUVRI
WKHFRPPXQLW\YDOXH1DVVDXJURXSHULQWKH7XUNVDQG&DLFRV JURXSHUKDELWDWV$OOODQGHG1DVVDXJURXSHUVEURXJKWSURÀWVWR
,VODQGV '(&5 GHQRWHV WKH 'HSDUWPHQW RI (QYLURQPHQWDO ÀVKHUPHQVDWLVIDFWLRQRI WDVWHWRORFDOEX\HUVDQGWRXULVWVDQG
and Coastal Resources of the Turks and Caicos Islands. HFRQRPLFLQFUHDVHVIRUWKHZKROHRI 7&,7KH1DVVDXJURXSHU
utility of Nassau groupers, public awareness towards the
ecological importance of Nassau groupers must be improved
before any desired Nassau grouper regulations are established
and expected to be effective. Analysis of lobster pot bycatch
composition throughout the duration of this study suggested that
haemulids and serranids, predominantly Nassau groupers, were
PRVWDEXQGDQWLQWKHDUHDVWDUJHWHGDURXQGWKH7&,6FKRROIRU
Field Studies, unpublished data). Given that many tourists valued
IUHVKORFDOÀVKLQWKHUHVWDXUDQWVDFKDQJHLQPDUNHWLQJVWUDWHJ\
WRHQFRPSDVVERWKKDHPXOLGVDQGVHUUDQLGVPD\DOOHYLDWHÀVKLQJ
pressure and demand for the Nassau grouper.
The utilization of local knowledge from various sectors of the
FRPPXQLW\FRPSULVHVDVLJQLÀFDQWWRROIRUZHLJKLQJVWDNHKROGHU
objectives and informing management decisions. The large-
VFDOH DSSOLFDWLRQ RI  LQWHUYLHZLQJ ÀVKHUPHQ ZLWK PDSV DQG
subsequently incorporating the data into geographic information
Manuscripts
DQGUHYHDOHGWKDWRQO\ZDOOÀVKHUPHQVSHFLÀFDOO\WDUJHWHG1DVVDX
appeared to be most valued socially and economically.
+RZHYHU GHYHORSLQJ D VXVWDLQDEOH 1DVVDX JURXSHU ÀVKHU\ WR
PHHWWKHREMHFWLYHVRI DOOVWDNHKROGHUVLQWKH7&,UHTXLUHVDGGLWLRQDO
UHVHDUFK%HIRUHWKHH[SHFWHGLQFUHDVHLQÀVKLQJLQWHQVLW\WRPHHW
increasing demand, more data must be collected to broaden the
EDVHOLQHIRUWKH1DVVDXJURXSHUÀVKHU\$GGLWLRQDOO\WKHFRPSOHWH
status of the Nassau grouper stock is currently unknown, as the
dock landings data do not fully portray a depiction of the number,
VL]H DQG KHDOWK RI  WKH H[LVWLQJ 7&, SRSXODWLRQV 7KHUHIRUH D
GHWDLOHGÀVKHULHVLQGHSHQGHQWSRSXODWLRQDVVHVVPHQWLVHVVHQWLDO
WRIXOO\XQGHUVWDQGLQJWKHLPSDFWRI WKH1DVVDXJURXSHUÀVKHU\
on the stock. Finally, developing effective ways of encouraging
EX\HUV WR YDOXH GLIIHUHQW \HW DEXQGDQW ORFDO ÀQÀVK VSHFLHVPD\
prove to be a useful management tool.
*LYHQ WKH ÀQDQFLDO DQG KXPDQ UHVRXUFH OLPLWDWLRQV FXUUHQWO\
impeding such research from being accomplished, and given
high and continually increasing Nassau grouper demand in the
7&,WKHSUHFDXWLRQDU\SULQFLSOHLVUHFRPPHQGHGXQWLOUHVHDUFK
needs can be met. Minimum Nassau grouper size limits, which
ZRXOG PRVW DIIHFW OREVWHU SRW ÀVKHUPHQ DQG RSSRUWXQLVWLF
lobster divers, should be established to guarantee at least one
reproductive event per individual before it may be landed. Further,
maximum Nassau grouper size limits, which would most affect
ZDOO ÀVKHUPHQ VKRXOG DOVR H[LVW LQ RUGHU WR SURWHFW WKH PRVW
sexually productive individuals from being landed. As the Nassau
grouper is an endangered species of international concern, basic
protective regulations are recommended for its conservation.
Public education is also suggested to encourage understanding and
cooperation. Marketing strategies may incorporate haemulids or
lutjanids in addition to serranids to relieve demand for the Nassau
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methods employed in this study may contribute to the development
of a comprehensive understanding of the Nassau grouper’s status
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ZLWKWKH7&,FRPPXQLW\WRPDQDJHDVXVWDLQDEOH1DVVDXJURXSHU
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CONCLUSION
grouper, and the future establishment of periodic monitoring of
the stock should be considered to permit adaptive management.
%H\RQGWKHVSHFLÀFLW\RI WKH7&,DQGWKH1DVVDXJURXSHUWKH
research methods of this study may be applied to comparable
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VWDWXVRI DPDULQHVSHFLHV,I VLPLODUÀVKLQJLQWHQVLW\ELRORJLFDO
aspects, and political and socioeconomic values are encountered,
the management recommendations suggested in this paper may
apply.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr. J. Claydon, E. Magarian,
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portion of the landings were reproductively immature, while it &RDVWDO5HVRXUFHVDQGWKHNH\ÀQDQFLDODQGÀHOGORJLVWLFDOVXSSRUW
is hypothesized that the largest landed individuals may be crucial provided by the Center for Marine Resource Studies, the School
to the perpetuation of the stock. The localization of Nassau IRU)LHOG6WXGLHV6RXWK&DLFRV,VODQG7XUNVDQG&DLFRV,VODQGV
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List of Threatened Species. Available: www.iucnredlist.org. (7 November 2006).

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