THURJ Vol. 3 Issue 1

9RO,VVXH6SULQJ
#(#(!50)&/.#)(5#(5
'#5).5)&),-55*85gf
Ļ5"/'(5-#-5 ),5
&#'.5"(!55*85gm
'/&.#(!5'/..#)(-5
),5,-#-.(5.)5 5
*85kj
$POHSBUVMBUJƲOTȁƺ
.FFSB"USFZB
GPǚXJOƢJOł
#Ĝ˞.BOVTDSJQU
.FFSB"USFZBHSFXVQJO"OO"SCPS .JDIJHBOCFGPSFIFBEJOHUPUIFFBTU
DPBTUGPSDPMMFHFBOEUIFXFTUDPBTUGPSHSBEVBUFTDIPPM"U)BSWBSE
.FFSBEFEJDBUFENPSFUIBOUISFFZFBST BOEBMMPGIFSVOEFSHSBEVBUFTVN
NFST UPDIFNJDBMCJPMPHZSFTFBSDIJO1SPGFTTPS%BWJE-JVTMBCPSBUPSZ
.FFSBJTFYUSFNFMZHSBUFGVMUPIFSHSBEVBUFTUVEFOUNFOUPS ,FWJO&TWFMU
GPSIJTJOWBMVBCMFHVJEBODFEVSJOHIFSXPSLUPDPOGFSSFTJTUBODFUP)*7JO
GFDUJPOCZEFWFMPQJOHNFUIPETGPSUIFTBGF FO[ZNBUJDFYDJTJPOPGUIFHFOF
FODPEJOHBO)*7DPSFDFQUPS
0VUTJEFPGUIFMBC .FFSBDPNCJOFEIFSFOUIVTJBTNGPSFRVFTUSJBOTQPSUT
BOEėFMEIPDLFZCZKPJOJOHBOEDPNQFUJOHXJUIUIF)BSWBSE1PMP
5FBNãBUSFBTVSFEFYQFSJFODFTIFXPVMEOPUIBWFCFFOBCMFUPFYQMPSF
PVUTJEFPGUIFJOUFSDPMMFHJBUFMFBHVF4IFBMTPQMBZFEĚVUFJOUIF)BSWBSE
1PQT0SDIFTUSB"TB,JSLMBOE)PVTFSFQSFTFOUBUJWFPGUIF3FTPVSDF&đ
DJFODZ1SPHSBN 3&1
TIFTPVHIUUPJOTQJSFTPDJBMDIBOHFUPXBSETNPSF
FOWJSPONFOUBMSFTQPOTJCJMJUZĉSPVHIUIFGSJFOEMZSPMFPG3&1 .FFSBEJT
DVTTFEUIFJNQPSUBODFPGTVTUBJOBCJMJUZXJUIIFSDMBTTNBUFT BOEIFMQFE
UIFNUPEFWFMPQFWFSZEBZIBCJUTUPTBWFFOFSHZBOEXBTUFGFXFSSFTPVSDFT
*O.FFSBTTFOJPSZFBS IFSIPOPSTUIFTJTXPOIFSBĉPNBT5)PPQFT
QSJ[FGPSPVUTUBOEJOHTDIPMBSMZSFTFBSDI"SPVOEUIFTBNFUJNF UIF/B
UJPOBM4DJFODF'PVOEBUJPOBXBSEFE.FFSBBQSFTUJHJPVT(SBEVBUF3FTFBSDI
'FMMPXTIJQGPSUIFQVSTVJUPGHSBEVBUFTUVEJFT*O+VOF .FFSBHSBEV
BUFEGSPN)BSWBSE$PMMFHF $VN-BVEFJO'JFMEXJUIBO"#EFHSFFJO
$IFNJDBMBOE1IZTJDBM#JPMPHZ
"ěFSFYQMPSJOH*OEJB &HZQU +PSEBO BOE5VSLFZGPMMPXJOHHSBEVBUJPO
BOEHFĨJOHIFSNPUPSDZDMFMJDFOTF .FFSBNPWFEUPUIF4BO'SBODJTDP#BZ
"SFBUPQVSTVFB1I%JO$IFNJTUSZBUUIF6OJWFSTJUZPG$BMJGPSOJB #FSLF
MFZ"TUVEFOUJOUIF$IFNJDBM#JPMPHZ(SBEVBUF1SPHSBN TIFJTJOIFS
ėSTUZFBSPGHSBEVBUFTUVEJFT.FFSBTQBTTJPOTBSFTDJFODFBOEUIFFOWJSPO
NFOU#FDBVTFTIFCFMJFWFTHMPCBMDMJNBUFDIBOHFJTUIFHSFBUFTUDIBMMFOHF
PGPVSUJNF JOHSBEVBUFTDIPPM.FFSBBJNTUPDPNCJOFIFSJOUFSFTUTCZEF
WFMPQJOHTVTUBJOBCMFFOFSHZTPMVUJPOTGPSUIFXPSMEUISPVHIBQQMJFE DIFN
JDBMCJPMPHZSFTFBSDI
!"#$%&'(')**$&'+','-./012'34+4 LETTERS
April, 2010
Dear Harvard Community:
As the desire among undergraduates to gain formative and substantive experiences in research contin-
ues to grow and flourish at Harvard, so does the great work of the students who put together The Harvard
Undergraduate Research Journal to reveal some of the thought-provoking, inspiring endeavors among
our young scholars across a wide range of disciplines.
Dean Evelynn Hammonds, early in her appointment, described her wish that every undergraduate
have an opportunity to pursue a compelling research experience. Indeed, with programs such as PRISE
and the opening of the new Office for Undergraduate Research Initiatives, Harvard College is poised to
further support our students who are engaging Harvard faculty and other senior researchers around the
world with imaginative and intellectually stimulating scientific projects.
The articles that appear in this journal each term endure as a cumulative testimony to some of the im-
pressive efforts in research undertaken by our undergraduates. Best wishes and thanks to the THURJ staff
not only for reporting to us some of these stories, but for contributing to the further development of a
meaningful community among undergraduate scientists as well.
Yours truly,
Gregory A. Llacer
Director Harvard College Office for Undergraduate Research Initiatives
Director Harvard College Program for Research in Science and Engineering (PRISE)
555678$/96"/2 !
LETTERS !"#$%&'(')**$&'+','-./012'34+4
The Harvard Undergraduate Research Journal
April, 2010
Dear Harvard Community,
It is our great pleasure to introduce the fifth issue of The Harvard Undergraduate Research Journal
(THURJ). For five semesters, we have been showcasing and encouraging the brilliant and diverse array
of scientific pursuits present on campus, and this newest issue represents our continued commitment
toward the integral role of the undergraduate research experience as part of a student’s scientific educa-
tion. Our relevant and insightful feature articles reflect this great diversity of scientific undertakings on
campus, with topics spanning from evolution in mice to global climate change. We are proud to present
research articles spanning the social and basic sciences, from economics to neurobiology to astrophysics.
Our prize-winning article from Meera Atreya puts forth a novel method of conferring HIV-1 resistance
through in vivo enzyme injection, an exceptional example of the inspired and ingenious research we pub-
lish with pride.
As evident from the extraordinary work in the coming pages, the dedication and passion for scientific
endeavors on campus is incredible, and THURJ is truly honored to promote and share this enthusiasm
for science with the rest of the Harvard community. We have taken on an active role in hosting speak-
ers such as Noam Chomsky and Francis MacDonald to inform and inspire the undergraduate body, and
we continue to promote events like the Research Panel in order to facilitate real student engagement in
research. As our journal begins its third year, we hope to continue these new efforts and discover new
directions so that we may further enrich the scientific undertakings on campus.
Our publication could not have been possible without the dedicated work of our staffers. We would like
to extend our gratitude to our Peer Review Board and the Harvard faculty, graduate students, and associ-
ates who reviewed our manuscripts and ensured their high caliber. Moreover, our staffers on the Content,
Design, Business, and Social and Public Relations Board have been extraordinary in their creativity and
dedication, without which we would not have this fantastic issue. Perhaps most importantly, THURJ is
grateful for the continued support of Dean Evelynn Hammonds, HMS Dean Jeffrey Flier, Professor Ste-
ven Freedman, Provost Steven Hyman, FAS Dean Michael Smith, FAS Dean for the Physical Sciences Jer-
emy Bloxham, Professor Richard Losick, and Harvard College. We give special thanks to our co-founders,
John Zhou and Shoshana Tell, and wish them the best as they graduate from the College. And of course, a
final thanks goes to you, the reader; we hope that this issue will delight your intellectual curiosity. Enjoy!
Sincerely,

Grace Cho John Mei
Co-Editor-in-Chief Co-Editor-in-Chief
" :8&';</=</>'?1>&/2/<>$<7&'@&*&</A8'B"$/1<#
!"#$%&'(')**$&'+','-./012'34+4 CONTENTS
Contents
Features
8 Science and Service: the story of one
man’s quest to explore science with
children in Liberia
Jeffrey Atwood ‘13
10 Organismic and Evolutionary Biology

Professor Hoesktra on uncovering
evolution in field mice (front cover)
Stacy Rush ‘11
12 A review of Richard Dawkins’s latest

book: the Greatest Show on Earth
Caroline Huang ‘13
14 Developments in reprogramming cell

degeneration
Jung Soo Lee ‘12
17 Harvard professors develop new tools

to monitor and predict climate change
Patrick Snodgrass ‘13
20 At the heart of an effort to merge

industry and academia is the Harvard
Clinical Research Institute
Yi Cai ‘12
!!!"#$%&'"(&)
!"#$%&'(')**$&'+','-./012'34+4 CONTENTS
Managing Editor of Content Executive Board Design Chair

Jen Gong ‘12 Co-Editors-in-Chief Amanda Lu ‘13
Managing Editor of Peer Review and John Mei ‘12 and Grace Cho ‘12 Manager for Social and Public
Submissions Business Manager Relations
Monica Liu ‘12 Varun Bansal ‘13 Janet Song ‘13
Boards Faculty Advisory Board

Alán Aspuru-Guzik, Ph.D David Jeruzalmi, Ph.D
Business Assistant Professor of Chemistry and Chemical Associate Professor of Molecular and Cellular
Ruoyu Zhang, ‘13 Biology Biology
Kevin Fan ‘13 Paul Bamberg, Ph.D Efthimios Kaxiras, Ph.D
Senior Lecturer on Mathematics Gordon McKay Professor of Applied Physics
Content
Michael Brenner, Ph.D and Professor of Physics
Alissa D’Gama ‘11 - Associate Managing
Glover Professor of Applied Mathematics and George Lauder, Ph.D
Editor Applied Physics Professor of Biology and Alexander Agassiz
Sophie Wharton ‘11 - Associate Managing Myron Essex, D.V.M., Ph.D Professor of Zoology
Editor Mary Woodard Lasker Professor of Health Richard Losick, Ph.D
Patrick Snodgrass ‘13 Sciences in the Faculty of Public Health Maria Moors Cabot Professor of Biology
Jung Soo (Tom) Lee ‘12 Brian Farrell, Ph.D L. Mahadevan, Ph.D
Stacy Rush ‘11 Professor of Biology Lola England Professor of Applied
Caroline Huang ‘13 Jeffrey Flier, M.D. Mathematics
Jeffrey Atwood ‘13 Dean, Harvard Medical School, and George C. David Mooney, Ph.D
Yi Cai ‘11 Reisman Professor of Medicine Gordon McKay Professor of Bioengineering
Nicole Francis, Ph.D Hongkun Park, Ph.D
Peer Review and Submissions Associate Professor of Molecular and Cellular Professor of Chemistry and of Physics
William Sun ‘13 - Associate Editor Biology Steven Pinker, Ph.D
Helen Yang ‘11 - Associate Editor Steven Freedman, M.D., Ph.D Johnstone Family Professor of Psychology
Jessica Zeng ‘12 - Associate Editor Associate Professor of Medicine Tobias Ritter, Ph.D
Patrick Snodgrass ‘13 - Associate Editor Robin Greenwood, Ph.D Assistant Professor of Chemistry and Chemical
Abby Schiff ‘11 - Associate Editor Associate Professor of Business Administration Biology
Allen Shih ‘13 - Copy Editor Guido Guidotti, Ph.D Eugene Shakhnovich, Ph.D
Nathan Kim ‘13 - Copy Editor Higgins Professor of Biochemistry Professor of Chemistry and Chemical Biology
Isabella Wechsler ‘13 David Haig, Ph.D Irwin Shapiro, Ph.D
George Putnam Professor of Organismic and Timken University Professor
Xuezhi Dong ‘12
Evolutionary Biology Zhigang Suo, Ph.D
Peter Zhang ‘13
Marc Hauser, Ph.D Allen E. and Marilyn M. Puckett Professor of
Jeanine Sinanan-Singh ‘13 Professor of Psychology Mechanics and Materials
Keli Liu ‘13 Dudley Herschbach, Ph.D David Weitz, Ph.D
Johanna Lee ‘13 Frank B. Baird Jr. Professor of Science Mallinckrodt Professor of Physics and of
Katherine Xue ‘13 John Hutchinson, Ph.D Applied Physics
Akachimere Uzosike ‘13 Abbott and James Lawrence Professor of
Doris Chen ‘13 Engineering and Gordon McKay Professor of
Edward Kogan ‘12 Applied Mechanics
Jacob Cedarmaum ‘12
Jacob Weatherly ‘12
John Liu ‘11 Design
Andrew Chen ‘12
Faculty Reviewers
Ritchell van Dams ‘11 - Associate Chair
Johnny Hu ‘11 Jung Soo (Tom) Lee ‘12 David Jeruzalmi, Ph.D
Meng Xiao He ‘11 Allen Shih ‘13 Associate Professor of Molecular and Cellular
Sway Chen ‘12 Shimwoo Lee ‘13 Biology
Ye Zhao ‘13 Casey Alcantar ‘13 Harvard MCB Department
Nicholas Tan ‘12 Peter Zhang ‘13 Arthur P. Dempster, Ph.D
Eva Gillis-Buck ‘12 Research Professor of Theoretical Statistics
Chioma Madubata ‘11
Vanisha Yarbrough ‘10 Harvard Statistics Department
Darius Li ‘12 Bob Westervelt, Ph.D
Justine Cheng ‘13
Social and Public Relations Mallinckrodt Professor of Applied Physics and of
Preya Shah ‘13 - Associate Manager Physics
Alex Mays ‘12
Caroline Huang ‘13 Guido Guidotti, Ph.D
Fiona Wood ‘13
Rohini Shivamoggi ‘13 Higgins Professor of Biochemistry
Jeanine Sinanan-Singh ‘13 Shaye J.D. Cohen, Ph.D
Jung Soo (Tom) Lee ‘12 Nathan Littauer Professor of Hebrew Literature
Shannon Purcell ‘12 and Philosophy
!!!"#$%&'"(&)
CONTENTS !"#$%&'(')**$&'+','-./012'34+4
Contact About Us
General: contact@thurj.org
The Harvard Undergraduate Research Journal (THURJ) showcases
Advertising: advertising@thurj.org
peer-reviewed undergraduate student research from all science and
Subscribing: subscriptions@thurj.org
quantitative social science disciplines. As a biannual publication,
Submissions: submissions@thurj.org THURJ familiarizes students with the process of manuscript
Website: http://www.thurj.org submission and evaluation. Moreover, it provides a comprehensive
forum for scientific discourse on the cutting-edge research that impacts
our world today.
Copyright 2010 The Harvard At its core, THURJ allows students to gain insight into the peer
Undergraduate Research Journal.
review process, which is central to modern scientific inquiry. All
THURJ manuscripts are rigorously reviewed by the Peer Review Board
No material appearing in this publication (consisting of Harvard undergraduates), and the top manuscripts
may be reproduced without written that they select are further reviewed by Harvard graduate students,
permission of the publisher, with the
exception of the rights of photographs post-doctoral fellows, and professors. This process not only stimulates
which may only be granted by the faculty-student collaboration and provides students with valuable
photographer. The opinions expressed feedback on their research, but also promotes collaboration between
in this magazine are those of the
contributors and are not necessarily
the College and Harvard’s many graduate and professional schools. In
shared by the editors. All editorial rights addition to publishing original student research papers, THURJ is also
are reserved. an important medium for keeping the Harvard community updated on
science research-related news and developments.
!"#$%&'(&')$*+)#','&)-&.#$/#0#&'1"$23-'+&4
Features
FEATURE Volume 3 Issue 1 | Spring 2010
6ʎȲʑQȪɏʋQɍ6ʑʢʧLȪɏ!
!"#$%&&#"'()*+',#-%.
Photos courtesy of Adam Cohen
By Jeffrey Atwood ‘13, THURJ Staff unaware of all the graduate students, but there are just
as many graduate students as undergraduate students,
Since his youth, Adam Cohen’s passion has been for and there is a whole other part of the university going
scientific inquiry. Growing up in Manhattan, the cur- on independent of the classes.”
rent Assistant Professor of Physical Chemistry at Har- Cohen’s chemistry interest came from an unlikely path.
vard remembers picking up pieces of electronics from As a senior in high school, he built a scanning tunneling
the garbage, taking them home, and trying to fix them. microscope as an entry to a science contest. This led him
In seventh grade, he enrolled in a graduate level course to work in a lab in MIT, helping to develop new scanners
in electronics, which led him to build his own lab in his to detect land mines, which in turn sparked an interest in
house, complete with all the tools to build complex cir- physical chemistry. In Whitesides’s lab, he continued to
cuits. Despite his intense interest in science, Cohen does research sensor technology. Concentrating in both chem-
not only focus on his research. Electronics led to physics, istry and physics, he described his research as “applying
which he studied as an undergraduate at Harvard. There physical insights to chemical problems.” After earning
he participated in several extracurricular activities: he P.h.D’s from both Oxford and Stanford, he returned to
travelled abroad in Ecuador as part of Let’s Go, a travel Harvard as an assistant professor of chemistry. Even
writing program, and taught science to young children though he has an extensive resumé, Cohen still finds time
with the ExperiMentors, a PBHA group which still exists for his non-research pursuits. Graduate student Yiqiao
today on campus. He also worked in George Whitesides’s Tang stated that one the reasons he was attracted to the
chemistry lab for all four years as an undergraduate. lab was Cohen’s devotion to his students and researchers.
Coming back to Harvard as a professor showed Cohen Despite the change from building electronics at home,
a different side of the university: “As an undergrad I was to working in a lab at Harvard, the same desire to go
8 The Harvard Undergraduate Research Journal

Volume 3 Issue 1 | Spring 2010 FEATURE
a personal connection, and as a professor at Harvard, he

invited him to speak, and this gave him the idea to travel
to Liberia and see what he could do to help.
In Liberia, the team met with civic and religious leaders
and examined the nation’s educational facilities, from its
Photos courtesy of Adam Cohen
elementary schools to the sole medical school in the coun-

try. They even gave sermons at local churches explaining
the benefits of science education. One of their main find-
ings was that much of the aid to Liberia is wasted. The
equipment sent can’t be used, either because the citizens
don’t know how to use it, or because they don’t have the
infrastructure. Together, the group focused on ways to
improve scientific literacy without the equipment and
teaching aids that universities in the United States take
Professor Cohen teaches a class to Liberian students. for granted. Cohen’s group showed the students at the
local medical university how to do a DNA extraction on
beyond the bounds of human knowledge motivates his a tomato, which Cohen called, “probably the first DNA
research. “To make a prediction, do an experiment, and purification ever done in Liberia.” They also tried to
see that prediction born out in reality is amazing,” Cohen teach the people about basic issues that were relevant to
explains. His research covers a wide variety of topics, the local community, such as the germ theory of disease
from the theoretical to the practical. For example, one and malnutrition. He plans to make a return trip in the
of his current projects is to gain a basic understanding summer to lead a workshop at the University of Liberia
of mucus and how bacteria can in the hopes that these people will
travel through it, a subject that “Perhaps his most important eventually teach others and reedu-
the scientific world knows sur- contribution to his research- cate a nation that lost its youngest
prisingly little about. Much of generation of scientists, doctors,
his research, as he puts it, is to ers is to teach them to be and teachers to civil war.
“develop tools that other scien- unafraid to be curious, and With so many demands, Cohen
tists can use in order to make still manages to make time to take
discoveries. We build new ways
pursue their ideas.” care of his lab members and stu-
of visualizing molecules, new ways of controlling their dents. He throws parties and cooks for his researchers at
positions, and new ways of controlling their internal states his house. Perhaps his most important contribution to his
and reactivity.” Despite being a relatively young profes- researchers is to teach them to be unafraid to be curious,
sor, he has already received several prestigious awards, and pursue their ideas. Graduate student Yiqiao Tang
including the Camille and Henry Dreyfus Award for New summed up his lesson as, “Premature is not such a bad
Faculty, and the DARPA Young Faculty Award. thing; it’s always good to talk to people, and to hear their
One of Professor Cohen’s most unique projects, how- comments about this idea.” As a scientist with a strong
ever, is not research-based. Over the summer of 2009, connection to students, Cohen has one word of advice
Professor Cohen and other scientists traveled to Liberia for young future scientists: “Work in a lab.” However,
to try and improve the quality of science education in that he follows up with a word of caution. “There’s an issue
country. Cohen’s connection with Liberia is personal. The that people sometimes run into: that doing well in sci-
adviser for his high school science club was from Liberia, ence classes here has very little to do with being a good
and many times, Cohen would see him collecting school scientist…The key to being a good scientist is asking the
supplies to send to his family in Liberia. The two formed right questions.”
www.thurj.org 9
!"#!"#$#
$%&#!$%
Evolving in the
Hoekstra lab
Photos courtesy of Hopi Hoekstra

By Stacy Rush ‘11, THURJ Staff
H opi Hoekstra likes mice. A professor in Harvard’s

Organismic and Evolutionary Biology depart-
ment, Hoekstra is cracking the fundamental relationship
between genes, traits and the effect of the environment
on survival and reproduction. She originally worked with Above: Hopi Hoekstra observing a field mouse.
grizzly bears but quickly realized that to answer any large
questions she needed large sample sizes—which neces- compared to mice on the Santa Rosa Island (Hoekstra
sitated the use of small organisms. Mice breed prolifically, et. al., 2006). The color difference was driven by selec-
are easily maintained in a lab setting—and, as Hoekstra tion for camouflage, as many predators are visual hunt-
adds, “they do happen to be very cute.” ers. Hoekstra’s team pegged the same Mc1R gene as a
While doing her postdoctorate, Hoekstra spent her contributor to the adaptive pattern. A single nucleotide
time trapping rock pocket mice in sunny Arizona. She change weakens the receptor so that it does not respond to
found that mice that lived on lava flows—dark colored signaling as well, creating a light-colored coat phenotype.
rock—tended to have dark coats, while mice living in a That the coat color of the mice matches their surround-
lighter environment tended to have light coats. In other ings is only evident through field observations, which
words, coat color “closely [matched] the environment is why Hoekstra stresses the importance of a hands-on
in which [the mice] inhabit.” On the black lava “light- approach. “You just walk out on these brilliant white
colored mice were big targets for predation because they sand beaches where you see there’s very little vegetative
are these bright golden beacons running around on this cover and it just makes sense that mice that are lighter in
black lava.” color are going to be more camouflaged,” she says. This
But Hoekstra’s small subjects held big lessons in store. research led National Geographic to hail Hoekstra as a
Back in the lab, scientists tracked down a genetic source new Darwin just in time for Darwin’s 200th birthday,
for the phenotype. Hoekstra’s team pegged the melano- which was this past year.
cortin-1 receptor gene (Mc1R) as a key ingredient for the Hoekstra has since branched out to study other mice
difference. A mutation allowed mice to camouflage with characteristics, including tail length variation, repro-
their environments—an impressive example of evolution ductive traits, and behaviors. According to Jesse Weber,
in action. a graduate student in her lab, “every diverse thing you
Hoekstra did not stop there. She found the same phe- can think of for adaptation in rodents…is fair game in
nomenon in Floridian populations of beach mice. On the lab.”
Florida’s Gulf coast, mice displayed a dark coat color Weber is currently studying the evolution and genetics
Photos courtesy of Hopi Hoekstra

Above: Camoflauge, a light mouse matching its environment.
of instinctive burrowing in two species of Peromyscus, or ‘extended phenotype’—it’s not actually held within our
deer mice. Peromyscus polionotus builds complex burrows skin, yet it’s controlled by genes,” explains Hoekstra. For
while Peromyscus maniculatus builds simple burrows. further research, the lab also has what Hoekstra calls a
The simple burrow consists of just one entrance while “mouse farm” - similar in concept to an ant farm. “You
the complex burrow also contains an escape exit that is can videotape [the mice] burrowing in a two-dimensional
dug up to just below the surface. This allows the mice structure and they will build these complex burrows,”
to make a hasty getaway in times of danger. When P. Hoekstra says.
maniculatus and P. polionotus are crossed and then re- Reflecting on the challenges of her work, Hoekstra
crossed, the hybrid progeny mice will build variations of says that it is “very integrative. For any given project to
the complex and simple burrows. To enable observation get the complete picture of adaptation, it requires essen-
of just this sort of behavior, the lab houses giant four-by- tially working across disciplines. We do ecological work
five feet sandboxes which Hoekstra calls “phenodomes” ….work at the organismal level … work at the devel-
because “that’s where we measure phenotype.” The mice opmental level down to the genetics and the molecular
are put in the phenodomes for two days and allowed to biology…. It’s hard in some sense to do all of it and all of
build a burrow. They are then removed and researchers it well.” However, she adds, the combination of all these
make a cast of their burrow using a kind of hardening aspects of biology “makes our work fun.” As she said in
foam. The cast is then studied just like any other mor- National Geographic, “Opening a trap with a mouse is a
phological trait. “This is what Richard Dawkins calls an little like Christmas morning.”
www.thurj.org 11
By Caroline Huang ‘13, THURJ Staff doubt, beyond sane, informed, intelligent doubt, beyond
doubt evolution is a fact.” In his preface, Dawkins notes
“Do you believe in evolution?” that in his previous books he has assumed the theory of
People either accept evolution as a fact or they don’t evolution without providing the facts that confirm it,
believe in it—rarely do you meet a person without an but catalyzed by the glaring reality of these 40 percent
opinion. When I posed this question to my roommate I of Americans the “history-deniers,” he sets out to prove
was attempting to be diplomatic, but I regretted it almost the theory of evolution in The Greatest Show on Earth.
immediately afterwards. Was the question too prying? As Dawkins explains in his second chapter, evolution
Should I have attempted to segue smoothly into the sub- is often misunderstood in popular culture as the idea that
ject, with some backup topic ready to return to, instead humans evolved from other apes. Instead of claiming that
of opening our conversation with that particular inquiry? we evolved from modern chimpanzees or orangutans,
It was odd to talk and think about a scientific theory the theory of evolution holds that we shared a common
as if it was a taboo ancestor with them that
subject; normally was neither ape nor
only social issues “Beyond reasonable doubt, human, but would even-
merit this aura of tually, over millions of
untouchability. The beyond serious doubt, beyond years and a staggering
theory of evolution
may be unique in its
sane, informed, intelligent doubt, number of generations,
evolve into the differ-
capacity to stir up beyond doubt evolution is a fact.” ent primates that we see
conflict and discom- today. He also disperses
fort. I would not feel -Richard Dawkins another common mis-
at all uneasy posing conception by explaining
!""#$%&'%()*$+,&)-+.(/(012('+3)--)*$
a similar question to my roommate about quantum theory that evolution itself involves an entire species—not just
(which is generally much less well-known and under- one organism. The mechanisms of evolution only work
stood), and likely she would not have paused for one tell- because of the variation in each species that allows nature
ing second before replying with an air of certainty: “no.” to select for certain traits in a population.
November 24, 2009—just shy of three months after After explaining the basics of evolution, Dawkins
Richard Dawkins published his book The Greatest Show meticulously discusses the different agents of evolution
on Earth: The Evidence for Evolution—marked the 150th and the evidence left behind by those paths that we can
anniversary of the publication of Charles Darwin’s semi- examine. He even goes as far as to explain the methods
nal work of evolutionary biology, On the Origin of Species. that we can use to date the fossils and other remains
Charles Darwin’s own 200th birthday was in February of and how they can be cross-checked with each other. He
the same year. The flap of Dawkins’ newest book informs doesn’t state facts; he explains how the processes involved
us that a 2008 Gallup poll found that over 40 percent in evolution and studying evolution work.
of Americans deny evolution. Ostensibly they are his It is evident from his writing that Dawkins is an expert
audience for this book, for he spends most of the first on the subject of evolution and is well-practiced against
chapter discussing the viewpoints of his “40-percenters” battling the non-believers. To counter the Creationist
and stating that the message of the book is to convince idea that the world is less than 10,000 years old, he draws
the reader that “beyond reasonable doubt, beyond serious upon evidence from radioactive dating. In the face of the
argument that animals are too perfectly evolved to have the end of his book in his chapter concerning theodicy.
been the product of an unconscious design-by-nature, Theodicy is the attempt to reconcile the goodness of
he shows the ridiculously long detour of the recurrent God with the presence of evil in the world. In evolution-
laryngeal nerve in a giraffe’s neck—some extra 15 feet— ary terms, one might expect a beneficent creator to reduce
that serves as a reminder of its gilled ancestors. Gaps in the amount of suffering in the world, but that is not the
the fossil record? We are lucky to have a fossil record case. The natural state of the world is one of famine, of
at all, and given the miniscule chances of fossilization pain and suffering, and of distress—all this clearly demon-
our collection is actually quite large and compelling. To strated in most predator-prey relationships. The specific
refute the idea that evolution from a simple, singled-celled example that Dawkins chooses to highlight was one that
organism, even if it were possible, would have taken lon- disturbed Darwin as well—the behavior of ichneumon
ger than the approximately four billion years since life wasps. The females meticulously paralyze and lay eggs in
first appeared on earth, Dawkins quotes J.B.S. Haldane, live caterpillars. Their eggs hatch into larvae that proceed
one of the most important figures in neo-Darwinism: to consume the caterpillar inside out in a manner which
“You did it yourself in nine months.” maximizes suffering by starting with the least important
The book is accessible to anyone with an open mind innards before consuming the essential ones—such as
and an interest in the subject. Dawkins knows exactly the heart—to keep the caterpillars alive as long as they
how long to linger on a par- can. This insures that the meat
ticular subject and how far in “Perhaps, more than is fresh for the offspring. Do
depth his audience will care to caterpillars feel pain? Dawkins
go into a certain experiment. anything else, this book hopes not.
Plus, the accompanying pic- Even theodicy, however, is
tures and supplementary color is a manifestation of the not a compelling argument
pages make for an even more
enlightening and enjoyable
idea that science and re- for some. The existence of evil
in the world is justified easily
reading experience.
Now the question remains:
ligion simply don’t mix. “ enough as a result of Adam and
Eve’s abuse of free will that led
if you are a Creationist why would you read this book? to their expulsion from Eden. Perhaps, more than any-
I am reminded of a time when my other roommate, thing else, this book is a manifestation of the idea that sci-
a vegan, attended a debate on vegetarianism. Before the ence and religion simply don’t mix. By this, I don’t mean
conversation began, the representative from PETA asked to say that one can’t be both a scientist and religious, but
those who were present, “How many of you are vegan or that faith-based evidence should not be used to support
vegetarian?” More than 90 percent of the audience raised or denounce scientific theory, just as scientific evidence
their hands. Is Dawkins merely preaching to the choir? is not used to sustain faith. The existence of a place like
Despite professing to address his 40-percenters, I can- Eden and the motivations and actions of a divine Creator
not say that his book is written for a Creationist audience. are all part of the supernatural, and science does not deal
His tone often borders on belligerent and his comparison with the supernatural.
of the evolution-denier to the Holocaust-denier could After reading The Greatest Show on Earth, there is no
only serve to alienate the very people he seems to want to doubt that the overwhelming evidence for evolution can
convince. However, he writes as if trying to engage in the be found in sources as varied as every organism on the
!""#$%&'%()*$+,&)-+.(/(012('+3)--)*$
discussion with the opponent, stating and then refuting planet. Yet in the face of religion does any of this matter? It
each of the standard arguments against evolution brought is clearly not lack of evidence that has left so many people
up by Creationists. unconvinced; maybe what Dawkins has shown is that it is
The evidence that Dawkins manages to fit into a book impossible to write a book about evolution for Creation-
of easily readable length is vast—from fossil records to ists. But this is no fault of the author whose argument is
scientific experiments, from selective breeding by humans seamless; rather it is a testament to the irreconcilability
to sexual reproduction in fish—but evolution-deniers of science and religion in this debate.
may be most convinced by the arguments he presents near
www.thurj.org 13
!"#"$%&'()*+"),-.,/).')
) 0"("'"$1*&#")0&%"1%"%2)
the future directions of !"#!$%!&''()% technologies
By Jung Soo Lee ‘12, THURJ Staff
Illustration from Wikimedia Commons
T
hink of Prometheus, the Greek Titan eternally These kinds of questions have led to the field of regen-
condemned by Zeus for stealing fire from him erative biology, which has gained a great deal of attention
and sharing it with the mortals. For his crime, he for its controversial yet groundbreaking findings in stem
was punished by being chained to a rock while an eagle cell research. One of the areas of stem cell research that
pecked away at his liver every day, only to have it grow has attracted growing interest from the greater research
back for months and years of continuous torture. community and public is nuclear reprogramming.
While the story of this cruel and unusual form of pun- Dr. Konrad Hochedlinger, an assistant professor of
ishment has captivated the minds of many, it also presents Harvard Medical School and a principal investigator at
an interesting and yet fundamental question of regenera- the Massachusetts General Hospital, leads a stem cell
tive biology. How is it that as little as 25% of a liver can lab that is doing some of the most groundbreaking and
regenerate to form a fully sized liver? What is it about cutting edge research on reprogramming.
the liver that allows it to fix itself in such a drastic way? Nuclear reprogramming is actually just what it sounds
like. Reprogramming in general refers to the concept Although reprogramming may seem like the perfect
of changing the wiring, or the global gene expression, solution for curing various degenerative diseases, there
of a cell in order to change its identity. In the case of are still many obstacles to overcome in the original repro-
stem cell research, reprogramming refers to the genetic gramming method described by Yamanaka and Takahashi
manipulation of a differentiated cell – a skin fibroblast, a before they can be used to treat diseases in humans.
cardiomyocyte, or even a β cell of the pancreas – to turn First of all, the efficiency of reprogramming remains
it into a cell that resembles an embryonic stem (ES) cell. very low and the procedure is quite time-consuming.
The product of successful reprogramming is referred to as In order for the procedure to be useful for medical pur-
an induced pluripotent (iPS) cell. In 2006, Yamanaka and poses, it must yield considerable numbers of iPS cells
Takahashi of Japan first reported the derivation of iPS cells for researchers to manipulate in the laboratory. As Dr.
from mouse fibroblast cells in their seminal paper. Just Hochedlinger describes, a possible strategy for improving
a year later, various groups had succeeded in generating efficiency is to “pick different cell types from ones used
human iPS cells. Since then, today [skin cells, fibroblasts].
reprogramming has become “. . . the efficiency of repro- We’ve shown in the blood system
one of the major, if not the that if you start with immature
main, focus of today’s stem gramming remains very cells, cells are more efficiently
cell research. converted to iPS cells.” This
To the average person, low and the procedure is approach makes intuitive sense,
this almost seems like a
technique straight out of a
quite time-consuming” since less differentiated and
“younger” cell types are closer in
sci-fi movie. By reversing identity to embryonic stem cells.
the time on a differentiated cell, researchers have been These “immature” cells would have less distance to travel
able to generate iPS cells that exhibit two important char- before they are reprogrammed into iPS cells.
acteristics of an embryonic stem cell: self-renewal, or Another way to prove efficiency is to “inactivate
the ability to indefinitely make more copies of itself, and pathways for inducing senescence in cells,” as Dr.
pluripotency, or the capacity to differentiate into more Hochedlinger explains. Senescence refers to the point at
defined cell types. which cells have aged so much that they stop dividing.
For Dr. Hochedlinger, this phenomenal differentiation
potential of embryonic stem cells and iPS cells drew him
to stem cell research. He found it “fascinating that you
can capture in a petri dish undifferentiated cells that can
be coaxed into all cell types of the body.”
It is this much talked about concept of pluripotency
and iPS cells that has gained the interest of many medical
researchers who believe that the technique may allow the
Illustration from Wikimedia Commons

development of novel regenerative therapies and treat-
ments for degenerative diseases of the human body.
Take, for instance, Parkinson’s disease, in which degen-
eration of midbrain dopaminergic neurons leads to severe
loss of motor skills along with many other debilitating
symptoms. In the near future, scientists may be able to
take the patient’s skin cells from a simple biopsy and
reprogram them into iPS cells. Those iPS cells can then
be directed to differentiate into dopaminergic neurons
which can then be transplanted back into the patient.
Not only would this kind of therapy replace cells that are
A diagram illustrating the goal of using iPS cell technology
lost during disease, but it would also bypass the problem
to treat various regenerative diseases. A tissue sample can be
of immune rejection that always complicates transplant harvested from a patient, reprogrammed into iPS cells, and
procedures because the replacement dopaminergic neu- differentiated into a desired cell type for re-transplantation
rons would be generated from the patient’s own skin cells. back into the patient.
www.thurj.org 15
Because the cell types used in reprogramming are often study them] at different time points when they’re not
older, adult cells and the process takes many days to com- yet iPS cells.” The Hochedlinger lab is also studying the
plete, allowing cells to reach an immortal state to allow identity of iPS cells. “We’re trying to understand if embry-
them to avoid aging and senescence prior to reprogram- onic stem cells are equivalent to iPS cells,” explains Dr.
ming may improve the efficiency of the process. Hochedlinger. “We’ve developed a system in mice, so we
Even if some of these barriers to use of iPS cells in can compare genetically identical ES and iPS cells. We’re
therapies are overcome, it may be awhile before they can using genome wide technologies to really look at every
help treat numerous degenerative diseases. It is still not nucleotide in the genome to figure out what the difference
known “how to make a variety of true cell types from is between the two cell types.”
pluripotent cells,” says Dr. Hochedlinger. “We know how With the many stem cell labs working to further refine
to make cardiomyocytes and neurons, but that’s about the process of nuclear reprogramming, medical therapies
it. Another problem is how to engraft cells. Once you’ve using reprogrammed cells may not be out of the question.
made neurons, you have to know how to graft them The original studies of Yamanaka and Takahashi that led
to make them functional.” Yet to the derivation of the first iPS
another issue lies in the nature cells have led other researchers
of iPS cells specifically. “We don’t“ ‘We don’t fully under- to develop different reprogram-
fully understand if iPS cells are
exactly equivalent to ES cells—if
stand if iPS cells are ex- ming methods. For example,
direct lineage reprogramming
they do everything ES cells do.” actly equivalent to ES involves the reprogramming of
Despite the seemingly end- one adult cell type directly into
less number of issues with cells – if they do every- another cell type without passing
reprogramming that need to be through an iPS cell stage.
addressed, researchers are cur- thing ES cells do.’ ” Although at this point, it is dif-
rently trying to solve all of these ficult to assess how realistic the
problems in order to perfect the promising technique. expectations of using reprogramming technology for
Dr. Hochedlinger’s lab, for example, seeks to study disease treatments are, iPS cells are already making great
not the end result of iPS cells, but the actual process of contributions to researchers’ understanding of diseases.
reprogramming. “Reprogramming takes about 10 days Deriving iPS cells from diseased cell types give research-
to 2 weeks in culture. We have no good understanding of ers the ability to study diseases in a petri dish as they
what’s going on in those 2 weeks,” says Dr. Hochedlinger. develop and unfold. By using iPS cells as human disease
“We’ve isolated cell surface markers that are downregu- models, researchers are coming closer to developing new
lated and upregulated in cells that eventually become treatments for many incurable disorders.
iPS cells. These markers allow us to pull out cells [to

New Frontier in
Climate Change
by Patrick Snodgrass ‘13, THURJ Staff
A lthough not completely accepted

by the public, the idea that
humans can adversely affect
Anderson was instrumental in the
passage of the Montreal Protocol,
a document banning the use of
,OOXVWUDWLRQE\&DVH\$OFDQWDUಫ7+85-67Dᚎ
global climate is fast becoming chlorofluorocarbons (the main

a commonplace notion. Yet the agents causing depletion of the
subject of atmospheric chemis- ozone layer). He understands
try research addressing climate that the public remains largely
change did not even exist just unconvinced that humans have
thirty years ago. directly caused climate change
Since the 1970s, the tools such as global warming. How-
available to researchers in the ever, he stresses that the major-
field have evolved from rudimen- ity of the scientific community
tary weather balloons to complex, supports the view that climate
infrared satellite instruments. At the change is primarily caused by
same time, Harvard has emerged as one human factors.
of the leading worldwide research centers in
atmospheric chemistry and climate change.
Housed in Harvard’s Department of Earth and Plan-
“He understands that
etary Sciences, atmospheric chemists are investigating the public remains largely
everything from how to model the effect of pollution on
global warming to how to develop optical instruments to unconvinced that humans
create an accurate climate record. Their research provides
insight into how policy-makers can help mitigate the have directly caused climate
effect of human activity on the environment.
change such as global
Lasers and Satellites warming.”
In addition to the continued work on global warming, However, it is necessary to generate data to demon-
research is now transitioning toward understanding the strate the connection between human activity and climate
broader effects that changes in atmospheric composition to influence public policy. This is the motivation for many
have on the climate. James Anderson, the Philip S. Weld of Anderson’s current research endeavors.
Professor of Atmospheric Chemistry, recognizes that “Setting in place a very high accuracy record of how
research in the field requires more robust as well as more the entire coupled climate structure was of paramount
accurate climate data. He has witnessed the development importance,” says Anderson. “And so, in 1996, we pro-
of the field and made many of the measurements that posed to build a highly accurate small satellite that would
established the existence of the Antarctic ozone hole. be in the infrared spectrum.”
www.thurj.org 17
Although he faced some setbacks along the way, the discrepancy between the measured and simulated
Anderson successfully built this instrument. The device data, and ultimately to improve the model’s ability to
will hopefully create a long-term global record of climate, forecast atmospheric composition. This demonstrates
while achieving a new level of accuracy. why the instruments developed by the Anderson Group
This device measures the spectra of infrared radiation are so important to this field.
emitted by the earth. The data can be used to generate Chemical transport models like GEOS-Chem are cru-
global temperature distributions, atmospheric composi- cial to an understanding of climate systems since they can
tion, and radiative forcing, all of which are necessary to reveal how chemicals released into the environment affect
create a precise picture of global climate change. climate. By pairing these models with climate change
This instrument is meant to help create a climate moni- models, scientists discovered that the release of green-
toring system, key to the development of the field. “This house gasses like CO2 into the atmosphere may be a major
country doesn’t have a climate observing system,” says cause of global warming.
Anderson. “It only has pieces of what you would call a Eric Leibensperger, a graduate student in the lab group,
very rickety system, and it’s simply not capable of provid- recently demonstrated how these chemical transport
ing hard scientific evidence for public policy.” models can be used to understand changes in climate.
The infrared satellite instrument will hopefully pro- Leibensperger simulated the changes over time in the
vide the data necessary to better understand the earth’s atmospheric concentration of aerosols (particles of air
dynamic climate structure. pollution). He then used a global climate model to trans-
late these changes into their effects on global tempera-
Modeling of Atmospheric Chemicals and Cli- tures. In the process, Leibensperger showed that “if you
mate remove the U.S. sources of aerosols, you actually warm
the United States.” He also verified that this warming
Collecting data with atmospheric instruments is not effect would be felt only locally above the US.
an end in itself. Rather, atmospheric chemists use climate
and chemical models to interpret the raw data supplied An Interconnected Climate System
by researchers like Professor Anderson with the intent of
understanding the interaction between the atmosphere Despite improvements in modeling technology and
and climate. data collection, climate and chemical models still struggle
The Harvard Atmospheric Chemistry Modeling to replicate the complex climate structure. Professor Ste-
Group, led by Professor Daniel ven Wofsy, who studies sources and
Jacob and Dr. Jennifer Logan, is sinks of CO2 and other chemicals in
at the helm of atmospheric mod- “Chemical transport the atmosphere, is well acquainted
eling with the novel GEOS-Chem
chemical transport model. The
models like GEOS- with the challenges of climate models.
“What I think is the most impor-
GEOS-Chem model calculates
three-dimensional atmospheric
Chem are crucial to tant scientific issue that slops over
to the policy and social realms is
composition using estimates of an understanding of that the [climate] system is highly
emissions of various chemicals interactive and nonlinear and that
into the atmosphere. It simulates climate systems” the one thing we really know about
the flow of these chemicals when these systems is that they are hard to
they are blown by winds and when they react with other predict,” says Wofsy. “In fact, they are generally thought
chemicals. of as being unpredictable.”
Central to this model is the data collected by satellites. The reason that these complex models often break
“Satellites, for a modeler like myself,” says Professor Jacob, down is that the atmosphere is intricately connected to
“are very exciting because satellite observations mean the environment in ways we do not yet understand. The
nothing without a good model.” atmospheric changes caused by chemical emissions do
Discrepancies between the measured satellite data not stop with changes in global temperatures.
and the data simulated with the GEOS-Chem model are Professor Anderson explains this phenomenon: “I like
revealed when the two sets of data are compared. The to make the analogy that a 1°C increase in global mean
GEOS-Chem model is subsequently modified to reduce temperature, global warming, is to the climate as a 2%
default rate on a mortgage is to the financial structure. It What Now?

triggered a sequence of events in the climate and finan-
cial structures that led to collapse, but the feedbacks in Although there are already noticeable consequences
the financial system are what really caused the system due to climate change, its future effects promise to be
to implode.” far worse than any observed to date. “Something bad is
The new frontier of climate change research is shifting going to happen,” says Wofsy. “What, I don’t know, but I
toward understanding these climate feedbacks. Although do know that it is going to be sudden.”
the Anderson Group is continuing to work on their infra- Both Wofsy and Anderson agree that establishing pub-
red satellite instrument, the group is now also exploring lic policy sooner rather than later is necessary to curb the
these feedbacks. effect of human activity on climate. “The role of govern-
Two main focuses of the Anderson Group’s new ment here is unprecedented,” says Anderson. “For eco-
research are the Arctic ice caps and the glaciers on nomic competitiveness, for national security, we have to
Greenland. The melting and collapse of these systems move very quickly to renewable energy and fortunately,
have the potential to exacerbate climate change and global we have it.” The United States has the resources to power
warming. two thirds of its energy with
To better forecast the future
of the Greenland glaciers, the “The United States has the solar and wind power. It just
requires time to develop.
Anderson Group is developing
instruments to map the topog-
resources to power two However, with climate
research in an early stage
raphy and three-dimensional -thirds of its energy with of development, it seems
structure of the glaciers. In unlikely that politicians
addition, the group is develop- solar and wind power.” would support changes in
ing underwater instruments to environmental public policy
gather information about the Arctic ice caps. based on current findings. Establishing unequivocal evi-
The endeavors of Professor Wofsy and the Anderson dence is a huge feat, let alone drafting legislation with a
Group are providing the field with much-needed infor- price tag that taxpayers would approve.
mation on the interaction between the climate and envi- Yet Anderson is optimistic about the future: “I have no
ronment. This information is critical to the development doubt that we can do it. But the government has got to
of the field since it is necessary to develop more accu- step in and develop the economic infrastructure.”
rate models and to understand the mechanisms through But Anderson is not the only one with this positive atti-
which human activity affects the environment. tude. Rather, it pervades Harvard’s atmospheric chemistry
and climate change research groups. These researchers are
determined to not only understand the complex climate
system but also to fight for its preservation.
www.thurj.org 19
The Synergistic Combination of
&
at the HARVARD CLINICAL

RESEARCH INSTITUTE
By Yi Cai ‘11, THURJ Staff
C ollaborative efforts between the Harvard Medi-

cal School, Beth Israel Deaconess Medical Center
(BIDMC), and Partners HealthCare founded the non-
in 2000, CDAC became a part of the institute formed to
facilitate collaboration between BIDMC, Massachusetts
General Hospital, and Brigham and Women’s Hospital.
profit institution, the Harvard Clinical Research Institute
(HCRI). In this joint venture headquartered near the
Boston University campus, industry meets academia to
The Mission of the Institute
advance clinical research in multiple areas, from medical Currently, HCRI continues to provide full-service clini-
device trials to quality of life assessments. cal trials and is regarded as one of the premier institutions
of its field. HCRI is affiliated with Harvard and consists
of professionals involved with the institute at a formal or
The Origins informal level. However, it also has characteristics that
HCRI was founded with much of the same collabora- set it apart from other clinical research organizations.
tive zeal and academic focus that the institute continues For one, Executive Director of Clinical Investigations
to emphasize today. At the Harvard Medical School about Dr. Donald Cutlip says that what makes HCRI unique
a decade ago, Dr. Eugene Braunwald, Dr. Joseph Martin, is the “availability of typical commercial services within
Dr. Victor Dzau, and Dr. Ray Dolin shared the vision of a a world-class academic environment.” That is, academic
union between academia and industry to create a clinical physicians and scientists are involved throughout each
research organization that would contribute to the field stage of a trial, and the institute is supervised by a Board
in a productive and responsible way. Their brainchild of Directors composed of academic professionals. Fur-
came to life with the launch of HCRI in 2000. thermore, HCRI upholds a strict conflict of interest policy.
The institute also involved entities from BIDMC and As Dr. Cutlip notes, “this adds a level of independent
Partners Healthcare. For instance, the then Cardiovascu- oversight that is not seen in most commercial clinical
lar Data Analysis Center (CDAC) at BIDMC became an research organizations or certainly when individual
important part of HCRI. Dr. Jeffrey Popma, a founder of industry sponsors oversee their own trials.”
CDAC, recalls the inspiration behind the beginning of the HCRI’s emphasis on academics does not go unno-
center. At the time, he says, “the databases to house the ticed. Dr. Cutlip accredits HCRI’s “academic reputa-
data were not very sophisticated.” So, CDAC was founded tion and ability to work with industry without the usual
“to assist in the generation of reliable data and to facilitate conflicts” as crucial in their winning a proposal for an
an interface with industry.” In the mid-1990s, CDAC car- important research project called Dual Anti-Platelet
ried out many clinical trials that would come to transform Therapy (DAPT). The project, which just started this
the field of coronary stenting. After the founding of HCRI past fall, aims to design and manage a large public health
trial for the study of appropriate clot prevention therapy HCRI still seems to be steadily making progress toward
for patients who receive coronary stents (small tubes its original mission each day.
placed in coronary arteries that are blocked in order One new goal for the future of HCRI, according to
to treat coronary heart disease). This study was man- Dr. Cutlip, is to “reach out as an academic leader to
dated by the Food and Drug Administration (FDA), is help improve clinical research methods, which includes
sponsored by four large stent manufacturers and four development of industry models for adjudication of
large drug companies, and is projected to involve over clinical events within clinical trials, an area in which we
20,000 patients. Indeed, “it is a high-profile study and the have become recognized as a leader.” HCRI has recently
proposal was won during a very competitive process.” founded the Academic Research Consortium, a collabora-
tion of other academic research organizations, industry,
and the FDA that aims to standardize clinical trial designs
Facing the Future and endpoint definitions for clinical trials of coronary
HCRI faces many challenges moving forward, espe- “HCRI continues to provide
cially in the current economic climate. Inevitably, inno-
vation in the field has slowed as a result of clinical trials full-service clinical trials
moving along longer timelines than in the years before.
Consequently, the economic side of clinical research and is regarded as one of
has become increasingly significant. Dr. Matthew Reyn-
olds, Director of HCRI’s Economics and Quality of Life
the premier institutions of
Research Center, says that it is now more important for
“developers of new technologies, such as our sponsors,
its field.”
to demonstrate not only the clinical value, but also the stents and heart valves. In its collaborations with the gov-
economic value of their techniques.” ernment, HCRI has had a powerful impact on the produc-
However, HCRI has been pleasantly surprised by their tion of biomedical products and on their implementation.
performance in the dire economic climate. “Last year, HCRI also hopes to play a role within Academic Research
HCRI overcame economic challenges by sound business Consortium to help improve the conflict disclosure and
practices and careful economic planning,” says Spencer management process. Dr. Cutlip says that clarification
Goldsmith, President of HCRI. Dr. Reynolds also credits of these policies within Harvard institutions has been a
the federal stimulus package with creating many new major development, and extending such developments
opportunities for comparative effectiveness research. Cur- to the rest of the field is an important issue.
rently, HCRI is undertaking two NIH-funded compara- HCRI has clearly made great contributions to the
tive effectiveness projects and in the process of designing field of clinical research, due in part to its academically-
more. Dr. Cutlip explains that some prospective studies oriented mindset. As Dr. Popma reflects, “without the
plan to “compare non-surgical heart valve replacement collaboration between academics and industry, I am not
with open heart surgery in patients who are at higher sure that the advances that have happened over the past
risk for open heart surgery. This is a very novel therapy decade could have been achieved.” As HCRI celebrates
that is in its infancy but could have major impact on its decade anniversary, it has not only many accomplish-
future treatments.” Thus, despite economic challenges, ments to commemorate, but also many more to expect.
www.thurj.org 21
!"#$%&'"())*"!+!"#$%&
One night in the spring of 2007, in the fluorescent white confines of

Cabot Library, two freshmen labored on their problem set for Physical
Sciences 1. Talk of p-orbitals and reaction rates turned to talk of under-
graduate research. Other top schools had research journals, reasoned
John Zhou ‘10 and Shoshana Tell ‘10, so why doesn’t Harvard have one?
From this discussion arose what would become The Harvard Under-
graduate Research Journal, Harvard’s only undergraduate journal that
peer reviews and publishes original undergraduate research.
John and Shoshana spent an entire semester laying out the ground-
work for the journal before beginning to recruit members and accept
submissions; they looked for potential executive board members,
talked with professors and deans, and established financial support for
a journal whose first issue would be door dropped to every undergrad-
uate on campus and distributed among graduate students and faculty.
This issue, the first of the third volume, marks the last issue that John
and Shoshana will see as undergraduates, before they pursue grander
endeavors outside of the College. Despite their emeritus status, the
two co-founders have inevitably lent a helping hand to their successors
as best they could; for THURJ, their graduation will be bittersweet.
For those of us who have been lucky enough to work with John and
Shoshana personally, we will remember their academic brilliance, sense
of institutional development, political acumen, energy, and persever-
ance. And for those of us to whom John Zhou and Shoshana Tell are
just legendary names, we unequivocally see their legacy from cover to
cover. Today, THURJ retains the flow of writing, reviewing, and editing
that it has had since its inception. We retain the regular staff meetings,
speaker events, and celebration of our passion for original research.
We continue their mission to provide undergraduates not only with a
forum for scientific discovery but also with exposure to the process by
which high quality, peer reviewed science is produced.
To the co-founders of The Harvard Undergraduate Research Journal,

we wish you both the best and that you continue to use your creative
THURJ Co-Founders talents in extraordinary pursuits.
John Zhou ‘10 (top), Shoshana Tell ‘10 (bottom)
Sincerely,
!"#$%&'(($)($!"#$*'+,'+-$./-#+0+'-1'&#$2#3#'+4"$5)1+/'6
www.thurj.org 23
Fall 2008
Volume 1, Issue 2
“The impacts of stricter high school graduation requirements
on youth crime” by Alice Chen won the manuscript prize for Fall
2008 issue. Her work examined the association between state
high school exit examinations and course graduation require-
ments and youth crime.
Fall 2007
John Zhou and Shoshana Tell co-
founded The Harvard Undergrad-
!"#$%&'"())*"!+
uate Research Journal and spent
!"#$%
a semester garnering support
from departments and faculty.
+,$)-.,"+,&"/&!$0
2007 2008 2009
Spring 2008 Spring 2009

Volume 1, Issue 1 Volume 2, Issue 1
For our inaugural issue, we spotlighted “Biological, The best manuscript for Spring 2009 issue went to
socioeconomic, and political aspects of the Nassau “Elasticity in ionically cross-linked neurofilament
grouper (Epinephelus striatus) fishery in the Turks and networks” by Norman Yao. Neurofilament networks
Caicos Islands” by Anh-Thu E. Vo. Vo’s study determined protect the neuron from external physical stress. Yao’s
that the increasing fishing intensity will eventually work examined problems associated with neurofila-
compromise the reproductive potential of the natural ments, ranging from polymer elasticity to nonequilib-
stock without a government regulation to curtail it. rium statistical mechanics.

Fall 2009 February 2010

Volume 2, Issue 2 A Talk on Linguistics with Noam
Our prize winning manuscript was “Still images of motion Chomsky
picture: Using static crystal structures to understand the THURJ was honored to host Noam Chom-
behavior of a DNA glycosylase” by Kimberly Oo. DNA glyco- sky of MIT to discuss some of his views on
sylase is a DNA repair enzyme that removes damaged bases. linguistics. As a part of THURJ’s continued
The article revealed the structure of a catalytic intermediate effort to outreach into social sciences as
during the excision of the damaged base via crystallization well as the basic sciences and engineering,
data. Dr. Chomsky was
a great speaker
who enlightened
a packed house
about theories like
generative gram-
mar.
2010
September 2009 October 2009 Spring 2010

Cambrian Explosion Halloween Talk with Dr. An-
THURJ began the year with a joint
Volume 3, Issue 1
drew Berry
event with various science groups at On Halloween, Dr. Andrew Berry
the Queen’s Head Pub. We hosted sci- came to speak about a fascinating
ence bands like Thousand Bands and study of evolution in beetles.
Gertler’s Law and invited Dr. Andrew
Berry to host for the night. The event
provided a fun place for freshmen and
returning members alike to mingle and
socialize.
April 2010
November 2009 A Talk on Snowball Earth
Theory with Francis MacDon-
A Talk on Healthcare Reform
by Dr. Arnold Relman ald
Professor Francis MacDonald from the
Dr. Relman, Professor of Medicine and
Department of Earth and Planetary Sci-
Social Medicine at Harvard Medical
ences came to speak about the Snow-
School came and discussed alternative
ball Earth Theory, which hypothesizes
ways of thinking about the healthcare
that the earth was entirely covered in
reform. With the recent election of
ice at one point. He discussed some
President Obama, healthcare was a
of the perspectives on this theory and
heated topic on campus and many
implications of such a theory.
interested students attended.
www.thurj.org 25
Volume 3 Issue 1 | Spring 2010 RESEARCH
Social stress and scapegoatism:

An economic model
David Joosten ‘11
Economics
Harvard College
Introduction This paper analyzes scapegoatism from the perspective of the

individual members of a social network, their social utilities, and
“You didn’t want to come. The average man don’t like the decisions that result. Using basic psychological descriptions of
trouble and danger. You don’t like trouble and danger. the effects of a scapegoat event7 on the members of a social group,
But if only half a man – like Buck Harkness, there – this paper formalizes an economic model to describe this dynamic
shouts ‘Lynch him! Lynch him!’ you’re afraid to back social process. Furthermore, the model’s results lead to several
down – afraid you’ll be found out to be what you are – general conclusions about the factors that increase the likelihood
cowards – and so you raise a yell, and hang yourselves of scapegoat events.
onto that half-a-man’s coat-tail, and come raging up Scapegoat events largely occur because a particular stress dam-
here, swearing what big things you’re going to do. The ages a group’s solidarity or the strength of its social connections. To
pitifulest thing out is a mob; that’s what an army is – a alleviate some of the social repercussions of this stress, the group
mob; they don’t fight with courage that’s born in them, members can choose to blame an individual within the group,
but with courage that’s borrowed from their mass.” thereby sacrificing that individual for the good of the remaining
– Colonel Sherburn, The Adventures of Huckleberry group members’ social connections. In other words, scapegoat-
Finn by Mark Twain1 ing relieves the social impact of the stress, or the social stress, and
enables the group as a whole to regain solidarity.8
The term scapegoat originated from a Jewish practice described The decision to pursue scapegoatism or not is an individual deci-
in Leviticus XVI in which a townspeople would confess their trans- sion on the part of each group member that depends on the relative
gressions over a sacrificial goat on Yom Kippur and subsequently values of various factors. Generally, the social utility each member
cast the goat out into the dessert to die, thereby purifying the town could regain by scapegoating must be compared to the social util-
of its sins.2 Today, the term is applied freely to a variety of situations ity lost if a particular member were excluded from the group due
in which a collective fault or social stress is blamed on a particu- to the occurrence of a scapegoat event. Since different members
lar minority.3 Modern historians have used the term to describe of the group are more or less valuable to the group as a whole, the
political developments, from protectionist tax policies to the Jew- comparison between the relative social utilities of scapegoating or
ish Holocaust itself.4 In economics, the term has been applied to not may be different for each group member. Therefore, the decision
a variety of situations, such as to describe those affected by labor each group member makes depends on his particular circumstances
policies or the focus of investors’ attention in the currency mar- within the group and the resulting group decision is dependent on
kets.5 Psychologists, particular social psychologists, have conducted group composition as well as the social stress inhibiting solidarity.
experiments and described the dynamic features of scapegoatism.6 However, although group members’ individual decisions may
Although the original, ritual meaning of scapegoat is no longer be different, they are subject to many of the same factors and so are
practiced today, the term has come to describe a dynamic social significantly correlated. The social stress discounts all of the links
process witnessed in a variety of fields. between the members in a group universally, so that a larger social
stress will compel every member to increase the opportunity cost
of not scapegoating in his decision on whether to scapegoat or not.
1 Mark Twain. The Adventures of Huckleberry Finn, (New York: Harper and
Brother Publishers, 1912), 203. Additionally, the value of the weakest member of the group, the
2 Calum Carmichael, “The Origin of the Scapegoat Ritual,” Vetus Testamen- would-be target of a scapegoat event, factors into every member’s
tum, Vol. 50, Fasc. 2 (Apr., 2000), pp. 167-182. decision through the cost incurred if he chooses to scapegoat.
3 “Labour: The Scapegoat,” Economic and Political Weekly, Vol. 9, No. 26
(June 29, 1974), p. 995. Overall, the group as a whole is more likely to scapegoat its
4 Claude R. Foster, Jr, “Historical Antecedents: Why the Holocaust,” Annals weakest member when the social stress inhibiting all group con-
of the American Academy of Political and Social Science, Vol. 450, (July, 1980), nections is high relative to the value of the weakest member. This
pp. 1-19.
Edson W. Spencer, “Japan: Stimulus or Scapegoat?” Foreign Affairs, Vol. 62, result helps explain the prevalence of scapegoat events in the fol-
No. 1 (Fall, 1983), pp. 123-137. lowing situations:
5 “Labour: The Scapegoat,” Economic and Political Weekly, Vol. 9, No. 26 t When a group is large and the weakest member’s value
(June 29, 1974), p. 995.
Philippe Bacchetta and Eric van Wincoop, “A Scapegoat Model of Exchange- comprises only a small fraction of the group members’
Rate Fluctuations,” The American Economic Review, Vol. 94, No. 2 (May, 2004),
pp. 114-118. 7 The concentrated casting of blame for a collective social stress on a particu-
6 Louis A. Zurcher and Kenneth L. Wilson, “Role Satisfaction, Situational lar individual
Assesment, and Scapegoating,” Social Psychology Quarterly, Vol. 44, No. 3 (Sep., 8 Kenneth Westhues, “At the Mercy of the Mob,” OHS Canada, Vol. 18, No. 8
1981), pp. 264-271. (Dec., 2002), pp. 30-36.
www.thurj.org 27
RESEARCH Volume 3 Issue 1 | Spring 2010
values Scapegoat Model

t When a group experiences a high level of variation in
social stress
t When group member values are right-skewed Network structure
t When group leaders support a scapegoat event The social group is comprised of k members, or nodes, that
Previous studies of historical scapegoat events confirm these are connected to all other members of the group. The complete
driving factors. In the most extreme cases of historical scapegoat inter-connection of the group serves as the most socially cohesive
Economics
events, all of the above factors are present at the time of the scape- configuration of the group’s social network, and so it acts as an effec-
goat event. Whether one considers the post-WWI Red Scare, the tive baseline from which to draw conclusions about the prevalence of
scapegoat Charles Mitchell at the time of the 1929 Stock Market scapegoatism. Furthermore, a completely inter-connected network
Crash, or the rise of anti-Semitism during the Third Reich in Ger- accurately describes many real-world social groups such as those
many, a great social stress coupled with a belief that the minority that are found in corporations, on athletic teams, and in families.
held weak social values is present in all of them.9
Although there has been a great focus on scapegoatism in his- Connection structure
torical literature and in the field of social psychology (particularly As a result of the complete inter-connection of the group, there
in organizational behavior), economic literature has not explored is the following composition of network connections:
t ∑ i=1 i total connections (of which k–1 connect to the
k−1
the topic in-depth.10 Bandiera, Barankay, and Rasul (2007) discuss
the effects of manager favoritism and subsequently the incentive would-be scapegoat)
systems that can remove favoritism in the workplace.11 However, Each member of the group (excluding the would-be scapegoat)
their paper does not focus on the opposite of favoritism – scape- faces the following:
goatism, in which a member in the workplace is isolated from his t k–2 non-scapegoat connections
peers and deliberately excluded socially. Additionally, Chwe (2000) t 1 scapegoat connection
focuses on the integration of social network theory and game theory
by exploring social coordination and the requirements necessary Social values
for coordination.12 In essence, his paper helps fuel the concepts of Each of the k group members is assigned a social value (denoted
link variation and social values found in this paper, and specifically by Vi) drawn from a random, normal distribution centered so that
the barriers to achieving a scapegoat event. While Chwe discusses 5% of the social values are negative. This social value is exogenous to
these dynamics generally, this paper focuses on the specific char- the model and is meant to describe the value of each member of the
acteristics of coordination as they relate solely to the occurrence group to the group as a whole. One could imagine a particular team
of scapegoat events. working on a project requiring physical strength and certain team
The rest of the paper is structured as follows: the first section members being inherently stronger than others. In this model, the
sets up the model of the social group, including a few stochastic stronger team members are denoted as having higher social values
variables and underlying assumptions. The second section analyzes than the rest of their team. Furthermore, one could imagine that
the results of various simulations. The last section analyzes some some team members are so weak that they actually detract from
examples from two papers on organizational behavior within the the group’s efforts, which would result in a negative social value.
framework of model. The conclusion discusses the implications of Note that these social values are different from the link values that
these results and potential areas of further research in the area of are discussed subsequently.
scapegoatism.
Links and social stress
The social stress (denoted by δ) is the discount factor applied
universally to all links within the social network of the group. All
links are assumed to have a value of 1 that is subsequently multi-
plied by (1–δ) to arrive at the current value of the social link. The
assumption that links have a base value of 1 is made to simplify
9 Claude R. Foster, Jr, “Historical Antecedents: Why the Holocaust,” Annals the model – the social values discussed above have been chosen
of the American Academy of Political and Social Science, Vol. 450, (July, 1980),
pp. 1-19. instead to represent the diversity of group members. One could
David R. Colburn, “Governor Alfred E. Smith and the Red Scare, 1919-20,” also potentially vary the link values in this model to differentiate
Political Science Quarterly, Vol. 88, No. 3 (Sep., 1973), pp. 423-444. – Political group member’s individual links with one another in addition to
Scientist Colburn characterizes the rise of Gov. Alfred E. Smith and the social
tensions that brought about the post-WWI Red Scare in the United States. their social values, but the conclusions of this paper are unaffected
Thomas F. Huertas and Joan L. Silverman, “Charles E. Mitchell: Scapegoat by assuming their base value of 1.
of the Crash?” The Business History Review, Vol. 60, No. 1 (Spring, 1986), pp. 81- The social stress is perhaps the key ingredient into the model, and
103. – Historians Thomas Huertas and Joan Silverman discuss Charles Mitchell’s
role in the 1929 Stock Market Crash and his subsequent role as scapegoat during so it deserves particular attention. The social stress is considered
the Depression. exogenous to the model – it represents the combined effect of all
10 Such as in: Zurcher and Wilson, “Role Satisfaction, Situational Assesment, external factors on the social network and the links therein. This
and Scapegoating,” Social Psychology Quarterly, Vol. 44, No. 3 (Sep., 1981), pp.
264-271. model targets the following characteristics for the social stress:
11 Oriana Bandiera, Iwan Barankay, and Imran Rasul, “Social Connections t It is centered around zero so that it has a 50% probability
and Incentives in the Workplace: Evidence from Personnel Data,” (revisions re- of inhibiting link values
quested, Econometrica).
12 Michael Suk-Young Chwe, “Communication and Coordination in Social t It is derived from a distribution which makes extreme
Networks,” Review of Economic Studies, Vol. 67, (2000), pp. 1-16. outcomes unlikely
t It is bounded Simulation Results and Analysis

t It cannot increase the link values beyond their base value
of 1
Overall, to achieve these characteristics, the social stress is drawn Group size
at random from a normal distribution, its cumulative probability is To gauge the effects of varying group size, this paper presents
calculated, and then centered at zero (by subtracting 0.5 from it). the results of a simulation with a group size of 3 and compares it to
Additionally, any negative values are set to zero to ensure that link a simulation with a group size of 10 each run for 1,000 trials. The
Economics
values are never greater than 1.13 These modifications ensure that all sample proportion of the frequency of scapegoat events for each
the targeted characteristics listed above are satisfied in the model. simulation is shown in the following table:
Voting ^
Group Size p n
When a social stress is imposed on the social network, each
group member must decide whether he will attempt to scape- 3 29.8% 1000
goat the stress or not. This is a personal decision that may vary 10 68.1% 1000
from member to member depending on group composition. In
comparing the opportunity to regain social utility by scapegoat- Running a statistical test to compare the proportion means yields
ing versus the cost of excluding the weakest group member, the a highly statistically significant result at the 5% significance level18
model applies the following condition14 for a particular member in which the frequency of scapegoat events for the group of size 10
(member X) of the group choosing to vote for a scapegoat event: is greater than that for the group of size 3. The simulation results
confirm the reasoning that the greater the number of members in
δ [Σik=1 (Vi ) −VX −Vweakest ] > (1 −δ) Vweakest the group, the greater the opportunity cost of not scapegoating
After each member evaluates his situation and votes whether to is for each member relative to the value of the member with the
scapegoat or not, there is a scapegoat event if and only if all members weakest social value.
but one (the weakest member) vote in favor of scapegoating.15 In this If the situation from the perspective of member X in Figure 1
model, the weakest member will never vote in favor of scapegoat- and Figure 2 above is compared, the increase in opportunity cost
ing because he would never benefit from the solidarity a scapegoat for larger groups is made apparent. While member X in Figure 1
event would bring about, but would rather lose all social utility by must sacrifice 50% of his links to gain a proportion δ on 50% of his
his exclusion from the group. links, member X in Figure 2 must sacrifice only 25% of his links
to gain a proportion δ on 75% of his links. Although this need not
Trials16 always be the case19, member X in Figure 2 on expectation would
Each trial represents the introduction of a particular social stress be more likely to pursue a scapegoat event than member X in Fig-
on the social network of the group, each group member’s evalua- ure 1 because less of a sacrifice is required proportionally to the
tion of the comparison discussed previously and vote, and finally expected gain.
the aggregate result of the individual group members’ votes. Each
trial is independent of all other trials. The procedure of each trial Variance of social stress
is detailed as follows: In analyzing the effect of the social stress on the frequency of
1. For each member of the group (not including weakest): scapegoat events, this paper presents the results of a simulation
a. Calculate the social utility lost due to the social stress. in which the social stress is that which is described in the original
b. Calculate the social utility of the weakest member. model specifications and compares it to the results of a simulation
c. Vote in favor of scapegoating if L > W, against scape- in which the social stress’s variance is subsequently quadrupled,
goating otherwise. each run for 1,000 trials. The sample proportion of the frequency of
2. Tally the votes and perform a scapegoat event if the votes scapegoat events for each simulation is shown in the following table:
in favor of scapegoating sum to k - 1 (i.e. unanimous
decision of all members not including the vote of the
weakest member).
3. Repeat, drawing new social values and a new social
stress.17
13 This is a simplification made to avoid issues with members whose social

values are negative, which when multiplied by a link value greater than 1 (a nega-
tive social stress) would actually yield a more negative social utility, an illogical
result considering there is a negative social stress, or positive effect on the social
network.
14 Note that if the social stress is equal to 0.0, there is a scapegoat event iff
Vweakest < 0.0. In other words, when there is no social stress, only members that
detract from the group can be scapegoated. Figure 1. Figure 2.
15 One could use different voting systems, such as simple majority, but the
implications of voting by unanimous decision lead to conclusions supported by
models of leadership, discussed in the simulation results under “Leadership.” are essentially running the same simulation but with different random values.
16 For the actual PHP program used to produce the data, refer to Appendix B 18 See Appendix A for the full statistical test [editor’s note: published online].
[editor’s note: published online]. 19 If social values are negative, then member X in Figure 2 would be more
17 Note that we still have the original total number of members, so that we likely to want a scapegoat event.
www.thurj.org 29
yields a statistically significant result at the 5% significance level23

^ in which the frequency of scapegoat events for the right-skewed
Variance of Social Stress p n distribution of social values is greater than that for the original,
normal distribution. These results support the reasoning that as the
Original 43.2% 1000 distribution of social values is skewed to the right, the probability
4*(Original) 52.0% 1000 that Vweakest is low enough relative to the other members’ social values
to set off a scapegoat event increases24. In other words, the likelihood
Economics
Running a statistical test to compare the proportion means yields of having a member weak enough to scapegoat increases under a
a statistically significant result at the 5% significance level20 in which right-skewed distribution.
the frequency of scapegoat events for the adjusted variance is greater
than that for the original variance. The simulation results support Leadership
the idea that the more variant the social stress is, the greater the In determining the effect of leadership on the probability of
likelihood is of a scapegoat event. scapegoat events, this paper presents the results of a simulation
A greater social stress leads to a greater probability of a scape- in which the social values distribution is the same as that which is
goat event, since the social stress increases the opportunity cost of described in the original model description and compares it to a
not choosing to scapegoat and decreases the cost of choosing to simulation in which the kth group member’s social value is the sum
scapegoat21. As shown in Figure 3, since the Modified Social Stress of all other members’ social values. The results of running these
(social stress with greater variance) is greater than the Original simulations each for 1,000 trials is found below:
Social Stress for all values greater than zero, the probability of a
scapegoat event for the Modified Social Stress is greater than that ^
Model Type p n
for the Original Social Stress.
Original 48.6% 1000
Social value dispersion Leader 50.4% 1000
To understand the effects of varying the dispersion of social
values, this paper presents the results of a simulation in which the Running a statistical test to compare the proportion means
social value distribution is that which is described in Section I (nor- does not yield a statistically significant result at the 5% significance
mal distribution) and compares it to the results of a simulation in level25. However, a deeper analysis of the data reveals that the voting
which the social value distribution is right-skewed22, each run for dynamics of the two simulations are very distinct. A leader event is
1,000 trials. The sample proportion of the frequency of scapegoat defined as a trial in which there is not a scapegoat event due to the
events for each simulation is shown in the following table: single vote against scapegoating26 by the member with the highest
social value. The proportion of trials in which there is no scapegoat
event due to a leader event is shown in the following table:
^
Distribution of Social Values p n ^
Model Type p n
Normal 51.4% 1000
Right-Skewed 59.3% 1000 Original 2.02% 514
Leader 7.78% 496
Running a statistical test to compare the proportion means
Running a statistical test to compare the proportion means
yields a statistically significant result at the 5% significance level27
in which the frequency of leader events for the Leader Model is
greater than that for the Original Model. In fact, in all cases, the
strongest member of the group is the least dependent on the social
network for social utility, and is therefore always the least likely to
vote for a scapegoat event.
The effect of a leader in the social group28

Figure 4 above presents a possible social group in the Original
Model whose strongest member has a social value of 4. Figure 5
23 See Appendix B for the full statistical test.

24 Note that the proportion of members whose social values are negative has
been kept at 5%, so that the number of negative social values in the group does
Figure 3. play a role in the results presented.
25 See Appendix B for the full statistical test.
26 This does not include the vote of the weakest member, who always chooses
20 See Appendix B for the full statistical test. not to scapegoat. The justification for exactly why the weakest member chooses
21 The opportunity cost of not choosing to scapegoat is: not to scapegoat is found in Section I, “Voting.”
δ [Σik=1 (Vi ) −VX −Vweakest ] > (1 −δ) Vweakest , and therefore the probability of a scapegoat 27 See Appendix B for the full statistical test.
event is increasing as δ increases, assuming positive social values. 28 In the Figures, the numbers within the circles indicate the social value of
22 The right-skewed distribution is modeled by using the Χ 2 Distribution. each node.
a scapegoat event may decrease social value of the group as a whole,

each individual member gains in terms of social utility, and it is this
that drives a group to perform a scapegoat event even in situations
when the weakest member still has a positive social value.
Social role and scapegoatism

Zurcher and Wilson (1981) find that one’s ability to fulfill a
Economics
particular role in a group setting is highly correlated with group
satisfaction and individual performance through a Marine field
exercise done with Naval Reservists.31 Based on a previous field exer-
cise, subjects were told to choose whether they had more satisfaction
Figure 4. Figure 5. with a “Naval role,” a “civilian occupational role,” or anywhere in
between on a gradient scale. All subjects were then put in groups
presents the same social group in the Leader Model. In either Fig- to complete assignments that strongly favored one role and then
ure 4 or 5, the social utility the strongest member receives from his the other. Zurcher and Wilson find that those subjects who iden-
social network is the same. Therefore, the decision of the strongest tified with neither role rated the assignments the most negatively,
member is left unaffected by his own social value. However, the followed by those subjects who participated in an assignment con-
same is not true for the other members of the group. While the trary to their chosen role. This result can be explained through
members whose social values are 2, 2 and 3 in Figure 4 lose 4δ as the framework of the scapegoat model developed in this paper in
a result of the social stress imposed on the social network, those the following manner: Those who participated in an assignment
members in Figure 5 lose 8δ. These members from Figure 5 have a contrary to their chosen role gained from their social network the
higher opportunity cost for choosing not to scapegoat relative to most since they were able to rely on their fellow members whose
their counterparts in Figure 4, and subsequently are more likely to self-described role related to the assignment. Those members who
choose to scapegoat. As a result, it is the strongest member’s vote identified with neither role did not gain as much from their social
that is both the limiting factor and the deciding factor to achieving network since they were more like their fellow members whose
a scapegoat event, and this result is borne out in the data under the chosen field related to the assignment.
Leadership Model. In fact, the study finds that those who identified with neither the
“Naval role” nor the “civilian occupational role” were actually “likely
to scapegoat someone within the setting”32. Since these subjects
were neither differentiated from the “Naval role” group members
Examples nor the “civilian occupational role” group members, they stood to
gain the least from their social network, and therefore would have
had the lowest overall social utility. As a result, these subjects’ dis-
Mobbing satisfaction with the assignment reached the critical level required
According to Westhues (2003), the term mobbing is defined to choose a scapegoat event.
as a “collective campaign by co-workers to exclude, punish, and
humiliate a targeted worker.”29 Mobbing is a specific example of
the scapegoatism, and there are two characteristics of mobbing as
described by Westhues that relate to the conclusions of the model Conclusion
developed in this paper. Firstly, Westhues characterizes mobbing as
an act that is “initiated most often by a person in a position of power This paper develops a model for scapegoatism and examines the
or influence.”30 In most cases of documented mobbing, workplace factors that lead to higher probabilities of scapegoat events through
tensions are only released through the form of mobbing when it is controlled simulations. First, the paper finds that larger group sizes
legitimized by a manager. This feature of mobbing can be explained (or more specifically, group sizes in which the minority is small)
in the model by the critical importance of the strongest member lead to higher incidence rates of scapegoatism on expectation. This
in determining whether a scapegoat event is successful or not (as occurs as a result of the low marginal social utility the weakest
shown through the Leadership Model in the simulation results). group member provides the rest of the group members relative to
Since the strongest member of a group has the least to gain by their total social utility. Second, environments in which a social
scapegoating in terms of social utility, it is this member who acts as network experiences highly variant levels of social stress (δ) tend to
the barrier to a successful scapegoat event in borderline situations. have higher rates of scapegoatism. As the probability that a social
Secondly, Westhues describes scapegoating as “an effective if stress is greater than the critical point required for a scapegoat
temporary means of achieving group solidarity, when it cannot be event increases, the probability that a scapegoat event occurs also
achieved in a more constructive way.” In terms of the scapegoat increases. Third, certain types of social value distributions, namely
model, this gain in solidarity is captured by the opportunity cost of right-skewed distributions, are more likely to lead to higher rates
choosing not to scapegoat, or δ [Σik=1 (Vi ) −VX −Vweakest ].>Although
− of scapegoatism. This occurs as a result of the greater probability
that a given group has a member whose social value is low enough
29 Westhues... See also Noa Davenport, Ruth Schwartz, and Gail Elliott,
Mobbing: Emotional Abuse in the American Workplace (Ames, Iowa: Civil So-
ciety Publishing, 1999) 31 Zurcher and Wilson…
30 Westhues, paragraph 9. 32 Zurcher and Wilson, pg. 270.
www.thurj.org 31
References
to merit a scapegoat event. Lastly, leaders emerge as the deciding
vote in thwarting scapegoat events as a result of their low reliance Bacchetta, P. and Wincoop, E. (2004) “A Scapegoat Model of Exchange-Rate
on the social network relative to the rest of the group members. Fluctuations”, The American Economic Review 94: 2-114.
This conclusion results from the unanimous voting structure the Bandiera, O., Barankay, I., and Rasul, I. (2008) “Social Connections and Incen-
model utilizes as its framework for determining when scapegoat tives in the Workplace: Evidence from Personnel Data”, (revisions requested,
events occur and the independence a group leader’s vote has from Econometrica).
Economics
his own social value. Charmichael, C. (2000) “The Origin of the Scapegoat Ritual”, Vetus Testa-
These scapegoat factors have implications that reach a variety of mentum 50: 2-167.
fields: sociology, history, economics, and particularly organizational Chwe, M. S.-Y. (2000) “Communication and Coordination in Social Networks”,
behavior. The model recommends that to achieve solidarity, groups Review of Economic Studies 67: 1.
should be relatively small, should avoid having critically high levels Colburn, D.R. “Governor Alfred E. Smith and the Red Scare, 1919-20”, Political
of social stress imposed upon them, should have members whose Science Quarterly 88: 3-423.
social values are not skewed-right, and should have clearly defined Davenport, N., Schwartz, R., and Elliott, G. (1999) Mobbing: Emotional Abuse
group leaders. These group characteristics lead to the highest levels in the American Workplace, Ames, Iowa: Civil Society Publishing.
of social utility for each of the group members, and can help deter Foster, C.R. (1980) “Historical Antecedents: Why the Holocaust”, Annals of the
group dynamics from negatively impacting group objectives. American Academy of Political and Social Science 450: July-1.
Huertas, T.F. and Silverman, J.L. (1986) “Charles E. Mitchell: Scapegoat of the
Crash?” The Business History Review 60: 1-81.
Spencer, E.W. (1983) “Japan: Stimulus or Scapegoat?”, Foreign Affairs 62: 1-123.
Twain, M. (1912) The Adventures of Huckleberry Finn, New York: Harper and
Brother Publishers.
Unknown (1974) “Labour: The Scapegoat”, Economic and Political Weekly 9:
26-995.
Westhues, K. (2002) “At the Mercy of the Mob,” OHS Canada 18: 8-30.
Zurcher, L.A. And Wilson, K.L. (1981) “Role Satisfaction, Situational Assesment,
and Scapegoating”, Social Psychology Quarterly 44: 3-264.

Circulating hormone adrenomedullin and

its binding protein protect neural cells from
hypoxia-induced apoptosis
Stephanie M. Wang ‘13 and Weng-Lang Yang
The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA
Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. In addition to necrosis,
neural cells exposed to the condition of oxygen depletion during ischemia can also undergo apoptosis, which
significantly contributes to brain injury. Adrenomedullin (AM), a multifunctional hormone, in combination with
its enhancing binding protein, AMBP-1, has been shown to effectively reduce tissue damage under hemorrhage
and ischemia/reperfusion in animal models. To evaluate a beneficial effect of AM/AMBP-1 administration in
brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma
SH-SY5Y cells. After exposure to 1% O2 for 20 h, the neural cells were injury with a reduction of the cellular ATP
Neuroscience
levels and an increase of lactate dehydrogenase released in culture medium. Pre-administration of AM/AMBP-1
significantly reduced the hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of
TUNEL-positive cells and activation of caspase-3 in comparison to those cells exposed to hypoxia alone. AM/
AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia
exposure. Correspondingly, treatment of forskolin, a stimulator of cAMP production, also protected neural cells
from hypoxia-induced injury. Inhibition of PKA, a downstream target of cAMP, by KT5720 abolished the protec-
tive effect of AM/AMBP-1 on hypoxia-induced apoptosis. These results indicate that AM/AMBP-1 elevates cAMP
levels, followed by activating PKA activity, to protect neural cells from the injury caused by hypoxia. This study
suggests that AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.
acids, acts as a circulating hormone to induce various biological

Introduction activities in a paracrine or autocrine manner8. AM is multifunc-
tional in nature and can regulate the proliferation, differentiation,
Cerebral ischemia results from an insufficient supply of blood and migration of a number of different cell types as well as exert
and oxygen to the brain after injuries. It is the underlying cause its regulatory abilities on blood pressure, water, and electrolyte
of neuron death during stroke and brain trauma, two leading balance9. A specific AM binding protein (i.e., AMBP-1) has been
perpetrators of death in the United States [1]. According to the identified in mammalian blood [10] and further purified and char-
National Center of Injury Prevention and Control, about 1.4 million acterized by Pio et al. as human complement factor H [11]. AMBP-1
Americans sustain traumatic and ischemic brain injuries annually. can facilitate AM to confine in the interstitial space for the accession
More than 10% of those injured are either severely disabled or die, to AM receptors and modulate AM biological activity. Moreover,
and patients surviving from this disease require massive sending AMBP-1 can prolong half-life of AM in the circulation by protect-
on long-term rehabilitations such as speech, occupational, and ing it from protease degradation (reviewed by Zudaire et al.[12]).
physical therapies1. So far, very limited treatments are available for Administration of AM combined with AMBP-1 has been shown to
patients with stroke and brain trauma. Although tissue plasminogen have protective effects on tissue damage caused by low oxygen and
activator has been approved by the FDA, it is only beneficial for a blood supply conditions, such as gut ischemia-reperfusion [13] and
small number of patients [2]. Development of new therapeutics is hemorrhagic shock [14] in animal models. These studies prompt us
imperative in this field. to evaluate the effectiveness of AM/AMBP-1 on protecting brain
During the brain damage, two major processes that lead to cell damage under ischemic condition.
death: necrosis and apoptosis. Within the core of the ischemic Here, we used an in vitro cell model to study neuron injury
area, where blood flow is most severely restricted, necrotic cell caused by oxygen depletion. Human neuroblastoma SH-SY5Y cells
death is dominant and occurs within a few days after damage. In were treated with retinoic acid to differentiate into neuron-like cell
the periphery of the ischemic area, where collateral blood flow can type and exposed to hypoxic condition at 1% O2. We first identi-
buffer the full effects of the damage, which may start several days fied the injury and apoptosis of differentiated SH-SY5Y cells after
after transient ischemia, is mainly apoptosis [3,4]. In principle, the hypoxia exposure. We then examined the effect of AM in combined
apoptotic cascades during brain damage are reversible, which can with AMBP-1 on hypoxia-induced cell injury and apoptosis. We
be the major target of therapeutic interventions [5-7]. further elucidated the signaling pathway mediated by AM/AMBP-1
Adrenomedullin (AM), a potent vasoactive peptide was discov- in regulating hypoxia-induced apoptosis in differentiated SH-SY5Y
ered by Kitamura et al. [8]. This peptide, consisting of 52 amino cells.
www.thurj.org 33
of medium per well, followed by 5 min shaking. Subsequently, 50 µl

Materials and Methods of the substrate buffer solution (luciferase/lucerin) was added and
shaken for another 5 min. The reaction was developed in the dark
Cell culture and materials for 10 min and measured on a luminescence plate reader. Protein
The human neuroblastoma cell line SH-SY5Y was obtained from levels in the cell lysate were determined by a DC protein assay kit
the American Type Culture Collection (ATCC; Manassas, VA). Cells from Bio-Rad (Hercules, CA). ATP levels were expressed as a ratio
were cultured in Dulbecco’s Modified Eagle Medium/F12 Medium of ATP in light units/mg protein.
(1:1, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum,
100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L-glutamine. Lactate dehydrogenase (LDH) assay
SH-SY5Y cells were maintained at 37°C in a humidified incubator The release of LDH in the cell culture supernatant was measured
containing 95% air and 5% CO2. SH-SY5Y cells were treated with according to the protocol provided by the manufacturer (Pointe
retinoic acid (RA, 10 µM, Sigma, St. Louis, MO) for 5 days to dif- Scientific; Canton, MI). The higher LDH activity detected in the
ferentiate into neuron-like cell type [15]. Typical morphology of cell supernatant, the more numbers of dead and dying cells. The
the differentiated SH-SY5Y cells is shown in Figure 1B. Differenti- LDH activity was normalized to protein concentration.
ated SH-SY5Y cells were plated into 96-, 12-, or 24-well plates 72 h
before experiments. AM and AM receptor antagonist (AM 22-52) Terminal deoxynucleotidyl transferase dUTP nick end
were obtained from Phoenix Pharmaceuticals (Belmont, CA) and labeling (TUNEL) assay
AMBP-1 was obtained from Cortex Biochem (San Leandro, CA). SH-SY5Y cells were grown on a chamber slide. After 20 h in
Forskolin and protein kinase A (PKA) inhibitor, KT5720 were from hypoxia, cells were fixed with 4% paraformaldehyde for 1 h at
Tocris Bioscience (Ellisville, MO). Anti-cleaved caspase-3 polyclonal room temperature. Cells were washed with PBS (phosphate buffer
Neuroscience
rabbit antibody (Cat. No. 9661) was from Cell Signaling (Danvers, saline; pH 7.2) and permealized on ice for 2 min with 0.1% Tri-
MA) and has been applied to other study for Western blotting [16]. ton X-100/0.1% sodium citrate. After washing with PBS, cells were
Anti-actin antibody was from Sigma. incubated with a TUNEL reaction mixture from Roche Applied
Science (Indianapolis, IN) at 37°C for 1 h. The slides were sealed
Hypoxia treatment with Vectashield mounting medium containing propidium iodide
Hypoxia was produced using a sealed chamber containing 1% (PI) from Vector Labs (Burlingham, CA). The green fluorescent,
O2, 5% CO2, and 94% N2, and placed in an incubator at 37°C. Dif- TUNEL-positive cells were counted under a fluorescent microscope.
ferent agents were added to the differentiated SH-SY5Y cells 30 min Total cell number in the field was determined by PI staining.
before exposure to hypoxia. After 20 h incubation in the hypoxic
chamber, culture media and cells were collected for further analyses. Western blot analysis for cleaved caspase-3
SH-SY5Y cells were lysed in cell lysis buffer containing a protease
Adenosine triphosphate (ATP) detection assay inhibitor cocktail (Roche Applied Science; Indianapolis, IN). Total
ATP levels in the differentiated SH-SY5Y cells were determined protein (25 µg) from cell lysate was loaded on 4-12% Bis-Tris gels
by ATPlite kit from PerkinElmer (Boston, MA), according to the (Invitrogen) and subjected to electrophoresis using MES-SDS run-
manufacturer’s instructions. Briefly, after hypoxia, 50 µl of cell lysis ning buffer (Invitrogen). After electrophoresis, gels were transferred
solution was added into cells cultured in a 96-well plate with 100 µl to 0.2-µm nitrocellulose membranes (Invitrogen) and blocked with
Figure 1. Differentiation of human neuroblastoma cells. SH-SY5Y cells were (A) untreated or (B) differentiated through treatment with retinoic
acid (10 μM). The differentiated cells show typical neuron morphology, such as dendrites and neuronal processes.

5% nonfat dry milk in 10 mM Tris-HCl with 0.1% Tween-20, pH 7.5 Results

(TBST). The membranes were incubated with anti-cleaved caspase-3
polyclonal rabbit antibody or anti-actin antibody overnight at 4°C.
Afterwards, the membranes were washed with TBST and incubated Effect of AM/AMBP-1 on cellular damage under hypoxia
with HRP-linked anti-rabbit IgG (Southern Biotech, Birmingham, To assess the cellular damage of SH-SY5Y cells under hypoxia
AL) for 1 h at room temperature and detected using chemilumi- (1% O2), we measured the ATP levels in these cells. The correlation
nescence and autoradiography. Intensity of the band was analyzed between ATP levels and cell death has been well established in
by Bio-Rad GS-800 Calibrated Densitometer. neural cells [17]. Oxygen depletion for 20 h resulted in a significant
decrease of ATP levels in SH-SY5Y cells by 32.6% in comparison to
Measurement of 3’-5’-cyclic adenosine monophosphate normoxia (Figure 2). We then examined the effect of AM/AMBP-1
(cAMP) and PKA activity on hypoxia-induced cell injury. It has been reported that an admin-
An aliquot of 100 µl of cell lysate was used for each cAMP mea- istration of AM at 100 nM exert an optimal effect on inhibiting
surement. Intracellular cAMP content was measured using a cAMP cytokine release in macrophage stimulated with lipopolysaccharide
Biotrak enzyme-immunoassay system (GE Healthcare; Bucking- [18]. Furthermore, AM co-administered with AMBP-1 at ratio of
hamshire, UK), according to the manufacturer’s instructions. A 2:1 has better outcomes than AM treatment alone [18]. Therefore,
standard curve was performed to calculate the concentration of we selected two doses of AM/AMBP-1 (100/50 and 200/100 nM) to
cAMP in cell lysate. To determine PKA activity, cells were grown examine whether these molecules could protect SH-SY5Y cells from
on 24-well plates, exposed to hypoxia, lysed in cell lysis buffer and hypoxia-induced cell injury. As shown in Figure 2, AM alone and
applied to PKA activity assay kit from Stressgen (Ann Arbor, MI), AMBP-1 alone at 100 nM and 50 nM, respectively, didn’t change the
according to the manufacturer’s instructions. The cAMP levels and ATP levels of cells exposed to hypoxia. When AM was administered
Neuroscience
PKA activity were normalized to protein concentration. at 200 nM, there was a slight increase of ATP levels in hypoxia-
treated cells, but it didn’t reach a statistical significance. However,
Statistical analysis when AM co-administered with AMBP-1 at 100/50 nM, the ATP
All data were expressed as the means ± SE (standard error) of at levels of hypoxia-treated cells had an 83.4% increase in comparison
least four independent experiments and were compared by one-way
analyses of variance (ANOVA) and the Student-Newman Keuls’
test. Differences in values were considered significant if p<0.05.
Figure 2. Effect of AM, AMBP-1, and AM/AMBP-1 on cellular Figure 3. Effect of AM/AMBP-1 on LDH release after hypoxia.
ATP levels after hypoxia. Differentiated SH-SY5Y cells were incubated Differentiated SH-SY5Y cells were incubated in normoxia or hypoxia
in normoxia or hypoxia (1% O2) for 20 h, in the presence of AM alone, (1% O2) for 20 h, in the presence of vehicle or AM/AMBP-1 (100/50 nM).
AMBP-1 alone, or AM/AMBP-1 at the indicated concentration. ATP levels The release of LDH in the culture medium (A) and cellular ATP levels in
in total cell lysates were determined. Data are presented as means ± SE cell lysate (B) were measured. Data are presented as means ± SE (n=4-6).
(n=6). *p<0.05 vs. Normoxia (open bar); #p<0.05 vs. Hypoxia alone. *p<0.05 vs. Normoxia; #p<0.05 vs. Hypoxia+Vehicle.
www.thurj.org 35
Hypoxia+AM/AMBP-1
Neuroscience
Figure 4. Effect of AM/AMBP-1 on formation of apoptotic cells after hypoxia. Differentiated SH-SY5Y cells were incubated in normoxia or
hypoxia (1% O2) for 20 h, in the presence of vehicle or AM/AMBP-1 (100/50 nM). Apoptotic cells were identified by TUNEL assay with green fluores-
cence labeling. (A) A representative filed of cells under fluorescence microscope. (B) The apoptotic rate was determined by the number of TUNEL-pos-
itive cells divided by the total number of cells in 6 fields. Data are presented as means ± SE (n=6-8). (C) Total cell lysates were subjected to Western blot
analysis. Representative blots against cleaved caspase 3 and b-actin are shown. Blots were scanned and quantified with the densitometry. Band intensity
of cleaved caspase-3 was normalized to the corresponding band intensity of b-actin. Data are presented as means ± SE (n=4). *p<0.05 vs. Normoxia;
#p<0.05 vs. Hypoxia+Vehicle.
to hypoxia controls (Figure 2). Similar results were also observed detected in the normoxia controls, while its intensity in the cells
at 200/100 nM of AM/AMBP-1 (Figure 2). Thus, the AM/AMBP-1 exposed to hypoxia was increased by 68.4% in comparison to the
at 100/50 nM was applied to the following experiments. normoxia controls. In contrast, the level of cleaved caspase-3 in the
To confirm the protective effect of AM/AMBP-1 on hypoxia- cells exposed to hypoxia in the presence of AM/AMBP-1 became
induced injury, we analyzed the release of LDH in the supernatant, similar to that in the normoxia controls (Figure 4C). Taken together,
another marker for measuring cell injury and death. LDH activity these results indicate that AM/AMBMP-1 can protect SH-SY5Y
was increased by 41.4% under the condition of hypoxia in compari- cells from apoptosis induced by hypoxia.
son to the normoxia controls (Figure 3A). When adding 100/50 nM
of AM/AMBP-1, the LDH activity of the hypoxia-treated SH-SY5Y Effects of AM/AMBP-1 on cellular cAMP levels after hypoxia
cells reduced to the level similar to that of the normoxia controls It has been indicated that AM can transmit its signal through
(Figure 3A). Correspondingly, the ATP levels of AM/AMBP-1 the receptors to regulate cellular cAMP levels for executing its bio-
administered cells were significantly higher than those of cells logical activities [19]. To identify the involvement of AM receptors
exposed to hypoxia alone (Figure 3B). in regulating hypoxia-induced injury, 1 µM of AM 22-52, an AM
receptor antagonist, was added with AM/AMBP-1 to SH-SY5Y
Effects of AM/AMBP-1 on hypoxia-induced apoptosis cells before exposure to hypoxia. After hypoxia, ATP levels in the
We next examined whether the protective effect of AM/AMBP-1 cells treated with AM 22-52 and AM/AMBP-1 were 56% less than
on hypoxia-induced cell injury was associated with apoptosis. After those in the cells treated with AM/AMBP-1 alone (0.56±0.044 vs.
hypoxia, SH-SY5Y cells were subjected to TUNEL assay to detect 1.00±0.11, P<0.05). We then examined whether cAMP could mediate
the DNA fragmentation of apoptotic cells. There were very few AM/AMBP-1 in regulating cellular responses to hypoxia. As shown
green fluorescence cells in normoxia condition (Figure 4A). Hypoxia in Figure 5, cAMP levels in SH-SY5Y cells exposed to hypoxia
resulted in an 8.5-fold increase of TUNEL positive cells in com- were decreased 41.9% in comparison to the normoxia controls,
parison to cells grown in normoxia condition (Figure 4B). In the while administration of AM/AMBP-1 prevented the reduction of
presence of AM/AMBP-1, the number of TUNEL positive cells was cAMP levels after hypoxia. To further examine the effect of cel-
80.2% less than that in the cells exposed to hypoxia alone (Figure lular levels of cAMP on hypoxia-induced cell injury, SH-SY5Y cells
4B). To further confirm the occurrence of apoptosis in SH-SY5Y were pre-treated with forskolin before hypoxia. Forskolin directly
cells after hypoxia, we used Western blot analysis to determine the activates adenylate cyclase and raises cAMP levels in a wide variety
activation of caspase-3, a critical executioner of cell apoptosis [19]. of cell types, including neurons [20]. In the presence of 1 µM of
As shown in Figure 4C, cleaved caspase-3 at 19 kDa was barely
Figure 5. Effect of AM/AMBP-1 on levels of Figure 6. Effect of forskolin on cellular ATP levels and cleavage of caspase-3 after
intracellular cAMP after hypoxia. Differenti- hypoxia. Differentiated SH-SY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h,
ated SH-SY5Y cells were incubated in normoxia or in the presence of vehicle or forskolin (1 mM). (A) ATP levels were determined in total cell lysate.
hypoxia (1% O2) for 20 h, in the presence of vehicle (B) The cleaved caspase-3 was determined by Western blotting as described in Figure 4. Data are
or AM/AMBP-1 (100/50 nM). cAMP levels in to- presented as means ± SE (n=4-10). *p<0.05 vs. Normoxia; #p<0.05 vs. Hypoxia+Vehicle.
tal cell lysates were determined. Data are presented
as means ± SE (n=8-10). *p<0.05 vs. Normoxia;
#p<0.05 vs. Hypoxia+Vehicle.
Neuroscience
forskolin, ATP levels in hypoxia-treated cells were significantly hypoxia were comparable to those in cells grown in normal condi-
higher than those in cells exposed to hypoxia alone (Figure 6A). tion. Although we doubled amount of AM/AMBP-1 administration
We also observed that cells pre-treated with forskolin resulted in from 100/50 nM to 200/100 nM, we didn’t observe further improve-
a significant decrease of cleaved caspase-3 levels after hypoxia, as ment of cell injury, indicating that AM/AMBP-1 at 100/50 nM was
demonstrated by Western blotting (Figure 6B). These results indicate an optimal dose for treating SH-SY5Y cells to protect them from
that the protective effect of AM/AMBP-1 on neural cells under the hypoxia-induced injury. Furthermore, AM co-administered with
hypoxia condition may be due to their ability of elevating intracel- AMBP-1 had a better protective effect on hypoxia-induced injury
lular cAMP levels through activation of AM receptors. than administration of AM alone in SH-SY5Y cells, which is agreed
with a previous study shown an enhancement of AM activity in
Regulation of PKA by AM/AMBP-1 under hypoxia combined with AMBP-1 [18]. Administration of AMBP-1 alone had
It is well known that cAMP can activate PKA [21]. After observ- no protective effect on the cell injury induced by hypoxia.
ing the changes of cAMP levels, we then assessed the PKA activity Although the small intestine is the major source of AM produc-
in SH-SY5Y cells. Under hypoxia, there was a 21% reduction of tion under both physiological and pathophysiological conditions
PKA activity in comparison to the normoxia controls (Figure 7). [22], AM is also produced in the central nervous system (CNS)
In the presence of AM/AMBP-1, the reduction of PKA activity after at a relative low amount [23,24]. AM plays important roles in the
hypoxia was prevented (Figure 7). To further confirm the involve- maintenance of homeostasis through central mechanisms [25].
ment of PKA activity in the protection of hypoxia-induced injury The primary biosynthesis site of AMBP-1 is the liver [26]; however,
by AM/AMBP-1, we treated SH-SY5Y cells with 1 µM of KT5720, a AMBP-1 can be detected in the rat brain by immunohistochemical
PKA inhibitor. As shown in Figure 8, the cleaved capase-3 levels in staining [27]. Under inflammation and ischemia/reperfusion injury
the cells treated with KT5720 and AM/AMBP-1 were the same as in the gut, the expression of AMBP-1 is down-regulated [13,28].
those treated with vehicle alone after hypoxia. In contrast, admin- Administration of AM/AMBP-1 has been shown to prevent tis-
istration of AM/AMBP-1 could effectively prevent the cleavage of sue damage in animal models of intestinal and hepatic ischemia/
caspase-3 in cells exposed to hypoxia (Figure 4C). These results reperfusion injury [13,29]. In view of that, the observation of the
indicate that blocking PKA activity can diminish the protective protective effect of AM/AMBP-1 in cultured neural cells exposed to
effect of AM/AMBP-1 on hypoxia-inducing apoptosis. hypoxia provides a rational strategy of using AM/AMBP-1 to treat
brain damage caused by ischemia. There is a concern in crossing the
blood-brain barrier (BBB) for AM/AMBP-1 to reach the therapeutic
targets. It is well known that stroke can disrupt the BBB [30,31].
Discussion A previous study has demonstrated that distribution of AM in the
brain increases significantly in sepsis [32], suggesting that brain
In this study, we used human neuroblastoma SH-SY5Y cells permeability increases under disease conditions. In this scenario,
differentiated to neuron-like cell type, followed by hypoxia (1% we expect that BBB permeability to human AM and AMBP-1 will
O2) exposure, to simulate brain ischemia. We first demonstrated be markedly enhanced after ischemic stroke.
that hypoxia caused damage of SH-SY5Y cells shown a reduction After demonstrating that AM/AMBP-1 has neuroprotective
of cellular ATP levels and an increase of LDH released into super- properties under hypoxic conditions, we then elucidated the poten-
natant. By administration of AM/AMBP-1, the cell injury induced tial mechanism responsible for this novel protective effect. As it is
by hypoxia was significantly alleviated. The cellular ATP levels widely accepted that hypoxia promotes cell apoptosis [33], we first
and release of LDH in the AM/AMBP-1-treated cells exposed to tested whether AM/AMBP-1 can decrease hypoxia-induced neural
www.thurj.org 37
Figure 7. Effect of AM/AMBP-1 on PKA ac- Figure 8. Effect of PKA inhibitor on cleav- Figure 9. Model of AM/AMBP-1 in regulat-
tivity after hypoxia. Differentiated SH-SY5Y age of caspase-3 after hypoxia. Differenti- ing hypoxia-induced apoptosis through
cells were incubated in normoxia or hypoxia ated SH-SY5Y cells were incubated in normoxia the cAMP-PKA pathway. Exogenous AM/
(1% O2) for 20 h, in the presence of vehicle or or hypoxia (1% O2) for 20 h, in the presence AMBP-1 binds onto the CRLR/RAMP receptor
AM/AMBP-1 (100/50 nM). PKA activity in to- of vehicle or AM/AMBP-1 (100/50 nM) plus complex (i.e., AM receptors) and activates AC
tal cell lysates was determined and normalized KT5720 (1 mM). The cleaved caspase-3 was to generate cAMP for stimulating PKA activity,
to protein concentration. The PKA activity of determined by Western blotting as described resulting in protection of neural cells from hy-
the normoxia was considered to be 1. Data are in Figure 4. Data are presented as means ± SE poxia-induced apoptosis. AC, adenylate cyclase;
Neuroscience
presented as means ± SE (n=6-8). #p<0.05 vs. (n=4). *p<0.05 vs. Normoxia. AM, adrenomedullin; AMBP-1, AM binding pro-
Hypoxia+Vehicle. tein-1; cAMP, 3’-5’-cyclic adenosine monophos-
phate; CRLR, calcitonin receptor-like receptor;
RAMP, receptor activity-modifying protein; PKA,
protein kinase A.
cell apoptosis. By using TUNEL assay and detection of cleaved apoptosis. We first demonstrated that cAMP levels were decreased
caspase-3 from Western blotting, we confirmed an induction of in SH-SY5Y cells after hypoxia and AM/AMBP-1 treatment could
apoptosis in SH-SY5Y cells under hypoxic condition of 1% O2. effectively prevent the reduction of cAMP levels. Correspondingly,
When AM/AMBP-1 was administered, the number of apoptotic cells by adding forskolin, a stimulator of cAMP production, cell dam-
and levels of cleaved caspase-3 were significantly reduced in neural age and activation of caspase-3 induced by hypoxia were reduced
cells exposed to hypoxia. The protective effect of AM on apopto- in SH-SY5Y cells. To further identify whether the protective effect
sis has also been observed in cultured rat endothelial cells under by the elevated cAMP levels is PKA-dependent, we demonstrated
serum deprivation [34]. Apoptosis has pathological consequences that down-regulation of PKA activity was prevented by adminis-
on immune and other cell function under various detrimental tration of AM/AMBP-1 after hypoxia. Furthermore, we inhibited
circulatory conditions such as ischemia/reperfusion injury [35,36]. the PKA activity by treating cells with KT5720, a pharmacological
Moreover, inhibition of cell apoptosis has proven to be beneficial inhibitor of PKA. KT5720 overturned the protective effect of AM/
in reducing inflammation [37]. Thus, an inhibition of neural cell AMBP-1 on hypoxia-induced injury and activated caspase-3 to the
apoptosis not only can directly reduce brain injury but also can levels as same as hypoxia alone. These mechanistic studies involved
prevent the damage caused by inflammation after ischemia. in the regulation of hypoxia-induced apoptosis by AM/AMBP-1
AM and AMBP-1 are extracellular molecules. The next ques- in neural cells are summarized in Figure 9. This proposed model
tion is how intracellular apoptotic cascade in SH-SY5Y cells can indicates that the beneficial effect of AM/AMBP-1 on protecting
be regulated by AM/AMBP-1 during hypoxia. AM mediates its neural cells from hypoxia-induced cell injury is mediated by the
activities via heterodimeric receptors that are composed of a seven cAMP-PKA pathway.
transmembrane calcitonin-receptor-like receptor (CRLR) [38] and In summary, by using an in vitro cell culture system with dif-
a receptor activity modifying protein (RAMP) [39,40]. The RAMP ferentiated human neuroblastoma cells, the novel neuroprotective
family comprises three members (RAMP1, RAMP2 and RAMP3) effects of AM/AMBP-1 in hypoxia were discovered. This neuropro-
[39,40]. AM receptors are widely distributed in most cell popula- tection is mediated by a reduction of hypoxia-induced apoptosis.
tions, including neurons [39,40]. In this study, we demonstrated Furthermore, the attenuation of apoptosis by AM/AMBP-1 is
that treatment of AM receptor antagonist diminished the protective through activation of the cAMP-PKA pathway in neural cells under
effect of AM/AMBP-1 on hypoxia-induced injury, indicating that hypoxia. These novel findings have considerable, potential biomedi-
the interaction between AM and its receptors was required for this cal implications for developing AM/AMBP-1 as therapeutic agents
protection. Several signaling pathways can be stimulated by AM in to reduce temporary and/or permanent brain damage caused by
various cell types [41] and the cAMP-PKA signaling pathway has oxygen depletion. Further studies are underway to determine the
been shown to mediate many of AM effects, including cell prolifera- efficacy of AM/AMBP-1 in animal models of stroke, brain trauma,
tion and migration [42,43], cell protection [44], and cell regeneration and brain ischemia.
[45]. It has been demonstrated that activation of the cAMP-PKA
pathway can protect neuronal cells against apoptosis and improve
survival [46,47]. Therefore, we examined whether the cAMP-PKA
pathway could mediate AM/AMBP-1 in regulating hypoxia-induced
Acknowledgements cell death, Nat. Cell Biol. 9 (2007) 550-555.

18. R. Wu, M. Zhou, P. Wang, Adrenomedullin and adrenomedullin binding
This work was supported by Faculty Practice Plan Research protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer
Fund of North Shore-Long Island Jewish Health System and funds cells, Regul. Pept. 112 (2003) 19-26.
from TheraSource. The authors greatly appreciate the constructive 19. K. Yata, G.A. Matchett, T. Tsubokawa, J. Tang, K. Kanamaru, J.H. Zhang,
suggestions and comments from Allyson Weseley, Ph.D. We also Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after
thank Haiyun Deng, MD for his kind help with the experiment. neonatal hypoxia-ischemia in rats, Brain Res. 1145 (2007) 227-238.
20. P.A. Insel, R.S. Ostrom, Forskolin as a tool for examining adenylyl cyclase
expression, regulation, and G protein signaling, Cell Mol. Neurobiol. 23
(2003) 305-314.
References 21. R. Waltereit, M. Weller, Signaling from cAMP/PKA to MAPK and synaptic
plasticity, Mol. Neurobiol. 27 (2003) 99-106.
1. P.M. Kochanek, R.P. Berger, H. Bayir, A.K. Wagner, L.W. Jenkins, R.S. Clark, 22. M. Zhou, I.H. Chaudry, P. Wang, The small intestine is an important source
Biomarkers of primary and evolving damage in traumatic and ischemic of adrenomedullin release during polymicrobial sepsis, Am. J. Physiol Regul.
brain injury: diagnosis, prognosis, probing mechanisms, and therapeutic Integr. Comp Physiol 281 (2001) R654-R660.
decision making, Curr. Opin. Crit Care 14 (2008) 135-141. 23. J. Serrano, L.O. Uttenthal, A. Martinez, A.P. Fernandez, d. Martinez, V, D.
2. K.P. Doyle, R.P. Simon, M.P. Stenzel-Poore, Mechanisms of ischemic brain Alonso, M.L. Bentura, M. Santacana, J.R. Gallardo, R. Martinez-Murillo, F.
damage, Neuropharmacology 55 (2008) 310-318. Cuttitta, J. Rodrigo, Distribution of adrenomedullin-like immunoreactivity
3. S.H. Graham, J. Chen, Programmed cell death in cerebral ischemia, J Cereb. in the rat central nervous system by light and electron microscopy, Brain
Neuroscience
Blood Flow Metab 21 (2001) 99-109. Res. 853 (2000) 245-268.
4. F.J. Northington, D.M. Ferriero, D.L. Flock, L.J. Martin, Delayed neurode- 24. J. Shan, T.L. Krukoff, Distribution of preproadrenomedullin mRNA in the
generation in neonatal rat thalamus after hypoxia-ischemia is apoptosis, J rat central nervous system and its modulation by physiological stressors, J
Neurosci. 21 (2001) 1931-1938. Comp Neurol. 432 (2001) 88-100.
5. H. Kato, K. Kogure, Biochemical and molecular characteristics of the brain 25. J. Shan, T. Stachniak, J.H. Jhamandas, T.L. Krukoff, Autonomic and
with developing cerebral infarction, Cell Mol. Neurobiol. 19 (1999) 93-108. neuroendocrine actions of adrenomedullin in the brain: mechanisms for
6. J.D. Newcomb, C.T. Ajmo, Jr., C.D. Sanberg, P.R. Sanberg, K.R. Penny- homeostasis, Regul Pept. 112 (2003) 33-40.
packer, A.E. Willing, Timing of cord blood treatment after experimental 26. W. Schwaeble, J. Zwirner, T.F. Schulz, R.P. Linke, M.P. Dierich, E.H. Weiss,
stroke determines therapeutic efficacy, Cell Transplant. 15 (2006) 213-223. Human complement factor H: expression of an additional truncated gene
7. I. Ferrer, Apoptosis: future targets for neuroprotective strategies, Cerebro- product of 43 kDa in human liver, Eur. J Immunol. 17 (1987) 1485-1489.
vasc. Dis. 21 Suppl 2 (2006) 9-20. 27. J. Serrano, J.M. Encinas, A.P. Fernandez, S. Castro-Blanco, D. Alonso,
8. K. Kitamura, K. Kangawa, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, P. Fernandez-Vizarra, A. Richart, M.L. Bentura, M. Santacana, F. Cut-
T. Eto, Adrenomedullin: A novel hypotensive peptide isolated from human titta, A. Martinez, J. Rodrigo, Distribution of immunoreactivity for the
pheochromocytoma., Biochem. Biophys. Res. Commun. 192 (1993) 553-560. adrenomedullin binding protein, complement factor H, in the rat brain,
9. J.P. Hinson, S. Kapas, D.M. Smith, Adrenomedullin, a multifunctional Neuroscience 116 (2003) 947-962.
regulatory peptide, Endocr. Rev. 21 (2000) 138-167. 28. R.A. Brooimans, A.A. van der Ark, W.A. Buurman, L.A. van Es, M.R. Daha,
10. T.H. Elsasser, S. Kahl, A. Martinez, L.M. Montuenga, R. Pio, F. Cuttitta, Differential regulation of complement factor H and C3 production in human
Adrenomedullin binding protein in the plasma of multiple species: char- umbilical vein endothelial cells by IFN-gamma and IL-1, J Immunol. 144
acterization by radioligand blotting, Endocrinol. 140 (1999) 4908-4911. (1990) 3835-3840.
11. R. Pio, A. Martinez, E.J. Unsworth, J.A. Kowalak, J.A. Bengoechea, P.F. 29. A.J. Dwivedi, R. Wu, E. Nguyen, S. Higuchi, H. Wang, K. Krishnasastry, C.P.
Zipfel, T.H. Elsasser, F. Cuttitta, Complement factor H is a serum binding Marini, T.S. Ravikumar, P. Wang, Adrenomedullin and adrenomedullin
protein for adrenomedullin. The resulting complex modulates the bioactivi- binding protein-1 prevent acute lung injury after gut ischemia-reperfusion,
ties of both partners, J. Biol. Chem. 276 (2001) 12292-12300. J. Am. Coll. Surg. 205 (2007) 284-293.
12. E. Zudaire, F. Cuttitta, A. Martinez, Regulation of pancreatic physiology 30. W. Pan, A.J. Kastin, Tumor necrosis factor and stroke: role of the blood-
by adrenomedullin and its binding protein, Regul Pept. 112 (2003) 121-130. brain barrier, Prog. Neurobiol. 83 (2007) 363-374.
13. G.J. Carrizo, R. Wu, X. Cui, A.J. Dwivedi, H.H. Simms, P. Wang, Adre- 31. S.A. Menzies, A.L. Betz, J.T. Hoff, Contributions of ions and albumin to
nomedullin and adrenomedullin-binding protein-1 downregulate the formation and resolution of ischemic brain edema, J Neurosurg. 78
inflammatory cytokines and attenuate tissue injury after gut ischemia- (1993) 257-266.
reperfusion, Surgery 141 (2007) 245-253. 32. D.A. Ornan, I.H. Chaudry, P. Wang, Pulmonary clearance of adrenomedul-
14. R. Wu, W. Dong, M. Zhou, X. Cui, H.H. Simms, P. Wang, A novel approach lin is reduced during the late stage of sepsis, Biochim. Biophys. Acta 1427
to maintaining cardiovascular stability after hemorrhagic shock: benefi- (1999) 315-321.
cial effects of adrenomedullin and its binding protein, Surgery 137 (2005) 33. P. Maiti, S.B. Singh, B. Mallick, S. Muthuraju, G. Ilavazhagan, High altitude
200-208. memory impairment is due to neuronal apoptosis in hippocampus, cortex
15. A. Adem, M.E. Mattsson, A. Nordberg, S. Pahlman, Muscarinic receptors and striatum, J Chem. Neuroanat. (2008).
in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester 34. G.S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S.I. Said,
and retinoic acid-induced differentiation, Brain Res. 430 (1987) 235-242. W. Spielman, B.D. Uhal, Regulation of apoptosis by vasoactive peptides,
16. P. Garnier, W. Ying, R.A. Swanson, Ischemic preconditioning by caspase Am. J. Physiol Lung Cell Mol. Physiol 281 (2001) L749-L761.
cleavage of poly(ADP-ribose) polymerase-1, J Neurosci. 23 (2003) 7967-7973. 35. V. Kohli, M. Selzner, J.F. Madden, R.C. Bentley, P.A. Clavien, Endothelial
17. C.P. Baines, R.A. Kaiser, T. Sheiko, W.J. Craigen, J.D. Molkentin, Voltage- cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion
dependent anion channels are dispensable for mitochondrial-dependent injury in the rat liver, Transplantation 67 (1999) 1099-1105.
www.thurj.org 39
36. T. Scarabelli, A. Stephanou, N. Rayment, E. Pasini, L. Comini, S. Curello, 42. D.J. Withers, H.A. Coppock, T. Seufferlein, D.M. Smith, S.R. Bloom, E.
R. Ferrari, R. Knight, D. Latchman, Apoptosis of endothelial cells precedes Rozengurt, Adrenomedullin stimulates DNA synthesis and cell prolifera-
myocyte cell apoptosis in ischemia/reperfusion injury, Circulation 104 tion via elevation of cAMP in Swiss 3T3 cells, FEBS Lett. 378 (1996) 83-87.
(2001) 253-256. 43. K. Miyashita, H. Itoh, N. Sawada, Y. Fukunaga, M. Sone, K. Yamahara, T.
37. R.S. Hotchkiss, K.C. Chang, P.E. Swanson, K.W. Tinsley, J.J. Hui, P. Klender, Yurugi, K. Nakao, Adrenomedullin promotes proliferation and migration
S. Xanthoudakis, S. Roy, C. Black, E. Grimm, R. Aspiotis, Y. Han, D.W. of cultured endothelial cells, Hypertens. Res. 26 Suppl (2003) S93-S98.
Nicholson, I.E. Karl, Caspase inhibitors improve survival in sepsis: a critical 44. E.N. Chini, C.C. Chini, C. Bolliger, M. Jougasaki, J.P. Grande, J.C. Burnett,
role of the lymphocyte, Nat. Immunol. 1 (2000) 496-501. Jr., T.P. Dousa, Cytoprotective effects of adrenomedullin in glomerular
38. D.R. Poyner, P.M. Sexton, I. Marshall, D.M. Smith, R. Quirion, W. Born, cell injury: central role of cAMP signaling pathway, Kidney Int 52 (1997)
R. Muff, J.A. Fischer, S.M. Foord, International Union of Pharmacology. 917-925.
XXXII. The mammalian calcitonin gene-related peptides, adrenomedul- 45. K. Miyashita, H. Itoh, N. Sawada, Y. Fukunaga, M. Sone, K. Yamahara, T.
lin, amylin, and calcitonin receptors, Pharmacol. Rev. 54 (2002) 233-246. Yurugi-Kobayashi, K. Park, K. Nakao, Adrenomedullin provokes endothelial
39. L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N. Thompson, R. Akt activation and promotes vascular regeneration both in vitro and in vivo,
Solari, M.G. Lee, S.M. Foord, RAMPs regulate the transport and ligand spec- FEBS Lett. 544 (2003) 86-92.
ificity of the calcitonin-receptor-like receptor, Nature 393 (1998) 333-339. 46. S. Jiao, Z. Liu, W.H. Ren, Y. Ding, Y.Q. Zhang, Z.H. Zhang, Y.A. Mei, cAMP/
40. S. Kamitani, M. Asakawa, Y. Shimekake, K. Kuwasako, K. Nakahara, T. protein kinase A signalling pathway protects against neuronal apoptosis
Sakata, The RAMP2/CRLR complex is a functional adrenomedullin recep- and is associated with modulation of Kv2.1 in cerebellar granule cells, J
tor in human endothelial and vascular smooth muscle cells, FEBS Lett. Neurochem. 100 (2007) 979-991.
448 (1999) 111-114. 47. X. Wang, X. Tang, M. Li, J. Marshall, Z. Mao, Regulation of neuroprotective
Neuroscience
41. L.L. Nikitenko, S.B. Fox, S. Kehoe, M.C. Rees, R. Bicknell, Adrenomedullin activity of myocyte-enhancer factor 2 by cAMP-protein kinase A signal-
and tumour angiogenesis, Br. J Cancer 94 (2006) 1-7. ing pathway in neuronal survival, J Biol. Chem. 280 (2005) 16705-16713.

SQIF setup for measurements of extremely

low absolute magnetic field
Anne Polyakov ‘12
Physics Department, Stony Brook University
Information in superconductor circuits that are to be used in prospective super fast computers is presented by the
value or sign of the stored magnetic flux. As an unavoidable drawback, these unique circuits are highly sensitive
to the ambient magnetic field that ideally should be less than approximately 0.2 nT for their correct operation.
Sophisticated magnetic shields are currently employed for shielding of the Earth’s magnetic field. However, the
very low temperatures that are required for these circuits make it difficult to monitor and therefore compensate
the residual magnetic field with proper accuracy. In this work, the magnetometer setup was built, which utilizes
an ability of the Superconducting Quantum Interference Filter (SQIF) to be used for absolute (without offset)
zero-field detection. The setup is capable of measuring absolute magnetic fields with a record accuracy of 3.5 nT,
which is 25 times better than the accuracy of known counterparts. The setup is optimized for real-time monitor-
ing and active compensation of residual magnetic field in the vicinity (less then 1 mm) from the superconductor
circuit under test. Using the setup, the residual magnetic field and shielding capabilities of magnetic shields
made of µ-metal, Bi-2223 High-Tc superconductor ceramic and lead were measured and compared. It was also
shown that the setup can be used for direct measurements of the magnetic field produced by magnetic vortices
trapped in a superconductor chip. This feature presents a novel way of monitoring and avoiding the trapping of
magnetic fluxes in superconductor chips.
can be used in this situation is a Superconducting Quantum Inter-

Introduction ference Device (SQUID) [5], which is now widely used in sensitive
magnetometers, with a typical sensitivity of about 10 fT/Hz1/2 [6].
Superconducting analog and digital circuits, for example, Rapid The most common type is a DC SQUID [5], which consists of a
Single Flux Quantum (RSFQ) devices dramatically outperform superconducting loop with two Josephson junctions (designated
Physics
conventional silicon counterparts in speed and energy dissipation as JJ in Figure 1A).
[1]. Unfortunately, they operate at very low (helium) temperature of A Josephson junction is a weak coupling between two supercon-
about 4.2K and are extremely sensitive to ambient magnetic fields. ducting wires, with a very small area of 1-5 µm2. According to the
When such devices are cooled down below the critical tempera- Josephson effect [7], such a junction remains superconducting until
ture of niobium, in the presence of even very small magnetic field, the current through it is less than the critical current Ic, which is
magnetic vortices [2] are formed and can become trapped in the usually chosen in the range 10 – 50 µA. When the SQUID is biased
superconductor material of the chip, preventing it from operating by the current slightly exceeding 2∙Ic (because there are two junc-
correctly. This effect is known as “flux trapping” and is considered tions connected in parallel), quantum interference occurs between
the main obstacle to practical applications of highly integrated the two junctions [7,8]. The voltage drop Vs across the SQUID then
superconductor circuits [3]. Usually, the influence of an external
magnetic field is significantly reduced by placing the supercon-
ductor chip inside of a magnetic shield, either a superconducting
shield or one made of a metal with high magnetic permeability [4].
Unfortunately, it was found that flux trapping still occurred in the
majority of cooling cycles, which indicates that the magnetic field
that affected the chip was still high enough to cause flux trappings.
To guarantee that fluxes will not be trapped at all, the magnetic
field B must be smaller than a critical field Bcrit that can create flux
equal to a fundamental magnetic flux quantum Φ0 = t-15 Wb,
through the typical area of a superconductor chip A ≈ 10 mm2. This
gives the following estimation of the critical field:
Bcrit = Φ 0 / A ≈ 2 10−10 T = 0.2nT (1)
However, even the measurement of such weak fields at low tem- Figure 1. Schematic diagrams of: (A) DC SQUID with two Josephson
peratures is difficult. This is because the only magnetic sensor that junctions (JJ); (B) Series SQIF; (C) Parallel SQIF.
www.thurj.org 41
becomes a periodic function of the flux Φ through the SQUID loop, This tradeoff between measurement accuracy and range is
with a period of Φ0 and minima corresponding to Ltϒ0, where avoided in a circuit containing several SQUIDs with different
k= 0, 1, 2 … (Figure 2A). This dependence is called a “flux-voltage sensitivities to magnetic field and connected in series (Figure 1B).
characteristic” or “modulation characteristic” of the SQUID. It This kind of circuit is known as the Superconducting Quantum
makes it possible to use the SQUID as a magnetometer or a device Interference Filter (SQIF) [9, 10, 11]. When loop areas of individual
sensitive to applied magnetic fields. SQUIDs are chosen in a such a way that none are multiples of each
The periodicity itself is in fact a major drawback of the SQUID. other, the result is a non-periodic voltage modulation characteristic
Because of it, the SQUID cannot distinguish between input fluxes, with a sharp dip at B=0 (Figure 2C). The absence of periodicity
which differ by the period Φ0. To be able to measure input fluxes in allows for dramatically increasing the range of measured DC mag-
a higher range, a special feedback electronics has to be used, which netic fields. Therefore, the SQIF can be used for direct, non-FLL
keeps the SQUID voltage Vs at the point of maximum slope dVs/dΦ measurements of absolute magnetic fields with high accuracy and
by applying the corresponding feedback flux [7]. This method of a large magnetic field range.
SQUID readout is referred to as FLL (Flux Locked Loop) and is As an absolute magnetic field sensor, the SQIF has been used
widely used in many practical applications including biomagnetism, mostly to measure relatively high magnetic fields, like the Earth’s
susceptometors, nondestructive evaluation, geophysics, scanning magnetic field of about 25 µT [12]. The measurement accuracy in
SQUID microscope, nuclear magnetic resonance and quantum [12] was reported to be restricted by a residual field of about 0.15 µT
computing [5, 8]. However, the FLL method can only measure from the magnetic shields, and flux trapping of about 0.5 µT in the
relative changes of magnetic field, and cannot provide any informa- SQIF itself during cooling cycles. Another source of error was a
tion about the field itself, or as it is frequently called, the absolute magnetic field created by the SQIF bias current, which limited the
magnetic field. accuracy of the SQIF to 0.1 µT in [13]. Authors have mentioned the
On the other hand, the absolute magnetic field can be measured possibility to compensate this error by using a so called bias reversal
directly by determining a position of the main minimum (at k=0) technique that switches the bias current polarity [10], but did not
on the modulation characteristics recorded by applying the test use it in their work. A SQIF magnetometer in [9] was designed
magnetic field. This method, however, will obviously work only to minimize the field from the SQIF bias current to well below
when the position of the minimum is within ±Φ0, or else it will be the measurement accuracy of 0.1 µT, which was yet limited at this
impossible to tell which peak the position B=0 belongs to. The range value by the low curvature of the voltage peak tip. Besides, they
of measured magnetic fields can be widened by increasing the volt- also observed an unexpected shift of about 1 µT due to residual
age modulation period, which can be achieved by using a SQUID field inside their shields. In many applications of SQIF magnetom-
with a smaller loop area. However, this will cause a corresponding eters the accuracy is not important at all; rather, magnetometers
reduction in measurement accuracy, because the voltage minimum are optimized for best noise limited sensitivity, which can reach
will become less sharp and it will be more difficult to determine its 10 fT/Hz1/2 or better values.
position on the B axis. As it can be seen, the accuracy of available SQIF magnetometers
Physics
Figure 2. Conventional sine-wave modulation characteristics of: (A) DC

SQUID; (B) Individual SQUIDs with different loop areas in a SQIF; (C)
Resultant modulation curve of a SQIF with a minimum near B = 0. Figure 3. The layout of the SQIF circuit.

Figure 4. SQIF mounted on the chip holder.
is not enough for monitoring the magnetic field in the vicinity of

superconductor chips, which can trap flux and become non-opera-
tional if the ambient magnetic field is greater than the Bcrit= 0.2 nT Figure 5. Rotation angle measurement device
given by (1). The goals of this research are: a) to create the SQIF
magnetometer setup optimized for best possible accuracy of mag-
netic field measurement, ideally to be able to measure absolute We installed the SQIF sensor on the underside of the fiberglass
magnetic fields down to 0.2nT, and b) investigate the effectiveness chip holder board (Figure 4) of a low-temperature test probe used
of shields used to protect superconductor circuits from external in the lab for testing superconductor circuits. The probe consisted
magnetic fields. of a stainless steel tube with all wiring situated inside, a connector
Physics
box on its upper end and a chip holder on its lower end. The probe
was cooled down by insertion into liquid helium in a non-magnetic
Dewar (model Cryofab-100). To be able to measure the magnetic
Methods and Results field at a very close proximity to the RSFQ chip, some of the fiber-
glass material was removed with a milling machine. This allowed
for enough space to install the sensor so that the RSFQ chip could
Experimental setup be located parallel, at a distance of 0.5 mm from the SQIF surface.
As the sensitive element of our magnetometer setup, we used a The chip holder had several 16 mm diameter coils containing 8
SQIF sensor designed in our laboratory and fabricated by a third turns each, used for imposing a specified magnetic field by applying
party company. The SQIF sensor contained two identical SQIF cir- a calculated DC current to the coils. The main coil was mounted on
cuits, fabricated with multi-layer niobium technology on a silicon the surface of the chip holder (XY plane in Figure 4), and it created
substrate with dimensions 2.5 x 2.5 mm (Figure 3). The technology a magnetic field in the Z direction. The holder also had Helmholtz
uses niobium for all on-chip wiring and a silicon dioxide as an coils (pairs of coils used to create a field with improved uniformity)
insulator between layers. installed to create a magnetic field in X and Y directions.
The SQIF itself was built from eleven SQUIDs connected in The chip was protected from external magnetic fields by a shield
series. For additional increase of the output voltage level, four such consisting of three nested shielding cans with open tops, oriented
SQIFs were connected in series to form a single 44 SQUID array. in the Y direction. The shield was cooled down to liquid helium
The SQUID areas have been selected as a geometric progression temperatures together with the chip holder. Two outer cylinders
with a common ratio equal to 0.92. The SQIF chip also contained were made of µ-metal, an alloy with a very high magnetic perme-
two heaters (Figure 3) made of molybdenum films, each with a ability (µ ≈ 1∙105) that contains 75% nickel, 15% iron, copper and
resistance of about 330 Ω . The heaters were used to perform a molybdenum. The inmost cylinder in our experiment was chosen to
“thermal deflux” procedure, or removal of trapped fluxes by heating be either a high-Tc superconductor (Bi-2223), low-Tc superconductor
the chip above the niobium critical temperature of 9.3K for about (lead) or a µ-metal cylinder. The residual magnetic field inside the
4 seconds. After this procedure the heater turned off, allowing the shield was created by two major sources: penetration of external
chip to cool down to its original temperature of 4.2 K. magnetic field through the shield and a magnetic field created by
www.thurj.org 43
Figure 7. Simulated modulation characteristics for best bias

current. (A) DC SQUID, (B) Different loop-area size SQUID magnetom-
eters; (C) Resultant modulation characteristic of a SQIF with dip at B = 0;
(D) Expanded view of the dip for the best bias current.
Figure 6. Typical modulation characteristics of a DC SQUID for
different bias currents. The SQUID was similar to the one used in our
SQIF.
Physics
Figure 9. SQIF modulation characteristics for positive and nega-

Figure 8. Modulation characteristics of a SQIF in a wide range tive polarities of the bias current varied from 34.5µA to 37.5µA
of magnetic fields for different bias currents. The sharpest peak is with step 0.5µA. The sharpest peak achieved at Is ≈ 37µA. Dashed line
shows the trend of the minimum positions with Is.
achieved at Is ≈ 35 µA.
the shield itself. Dewar flange during the experiment (Figure 5). We also attached
Since the chip surface in our configuration was orthogonal to the individual pointers to both the connector box (which rotated with
Z-axis, the flux trapping was determined only by the Z-component the chip holder) and the shield tube, so that we were able to set the
of the magnetic field. This presented us with an interesting oppor- chip holder and shield angles independently, anywhere between 0
tunity to separate contributions of external magnetic field and a and 360° with an accuracy of about ±3°.
shield-generated field. For this purpose, the shield was mounted on The measurements were conducted using a multi-channel auto-
a separate stainless steel tube, which was concentric with the chip mated system, which is used in the lab for performing measurements
holder tube and had a slightly larger diameter. This made it pos- of superconductor chips. The system can apply DC currents and
sible to manually perform independent rotation around the Y axis measure voltages at up to 64 independent channels. All operations
of either the chip holder or the shield. With such an arrangement, were programmed in XLISP language which makes it possible for
when the shield was rotated, the chip experienced variations of the users to create their own programs of controlling the measure-
Z-component of the magnetic field produced by the shield only. On ment process. A nice feature of the system was that all intermediate
the other hand, rotation of the chip holder tube caused both the measurement data and a log file with all commands of the current
external field and the shield field to contribute to variations of the session were automatically saved and could be retrieved in the future
Z-component of the field. for the purpose of reviewing, or to be used as examples. This was
To precisely control the rotation angles of the chip holder and the very useful for a quick and easy understanding of the XLISP lan-
shield, we made a special protractor dial, which was attached to the guage and principles behind controlling the measurements.
Figure 10. The shape of SQIF’s positive and negative minima.

The minimum for negative current was reflected against the axis origin to
be on the same scale with positive current minimum. Dots – experimental Figure 11. Response of SQIF to external magnetic step of 0.1 nT.
data. Solid lines – fitting with 4th order polynomial. Measured standard deviation σ ≈ 13 pT.
Physics
Figure 12. Rotation of the shield (µ-metal inner cylinder) and Figure 13. Response of SQIF in µ-metal shield, to placing a per-
the chip holder. The difference between the two is barely noticeable. manent magnet. (A),(C) – placing North Pole to cryostat. (E),(G) –
This points to the fact that the contribution of Earth’s magnetic field is placing South Pole to cryostat. (B),(D),(F) – removing from cryostat. Shift
negligible. in baseline characterizes magnetization of a µ-metal shield by the magnet.
Optimization of the SQIF setup for measurements of absolute progression with a factor of 0.92. To simulate distortions of sinu-
magnetic field soidal shape we have approximated a modulation curve by the
To provide the output voltage Vs (Figure 1), the SQIF has to be exponent of the cosine wave in the form:
biased with a constant current Is that slightly exceeds the critical
current. The choice of the bias current is very important as it deter- y ! exp( A Cos( x )) (2)
mines the shape of the flux-voltage characteristic of the SQIF. A
typical modulation characteristic for an individual SQUID similar By varying the parameter A we were able to simulate all different
to the one used in our SQIF is shown on Figure 6. shapes of modulation characteristics shown in Figure 6. The result
It can be seen that at Is > 30 µA, the shape is almost sinusoidal. of simulation is shown in Figure 7.
Below 30 µA, the sign wave becomes distorted and turns almost We have found that the general shape of the SQIF modulation
rectangular at Is ≈ 26 µA. To figure out what shape is better for the characteristics (Figure 7C) did not change much with variations in
SQIF magnetometer, we have simulated the formation of SQIF shape of the SQUID’s modulation curve. The SQIF still exhibited
modulation characteristic in Matlab as a sum of periodic curves the single main minimum at zero magnetic field, and the ampli-
for all eleven SQUIDs, with periods distributed in a geometric tude of other unwanted minima never exceeded 1/3 of the main
www.thurj.org 45
Figure 14. Rotation of the shield (High-Tc inner cylinder) and the Figure 15. Response of the SQIF in High-Tc superconductor shield to:
chip holder. Non-symmetric shape is due to non-uniformity of the field (A) placing permanent magnet, (B) removing magnet, (C) applying 2 nT
created by the flux trapped in the shield. The two graphs were barely dis- calibration field with the main coil, and (D) removing field. Unlike the
cernable, pointing again to the fact that the Earth’s magnetic field barely case with µ-metal shield, the baseline didn’t shift, which demonstrated the
influenced the measurements of the SQIF. absence of shield magnetization.
minimum at all possible shapes shown in Figure 6. However, the position of a peak was determined by taking measurements at about
sharpness of the main peak varied significantly with the shape of the 20 points located around the minimum on the B axis, with a very
SQUID’s modulation curve (Figure 6). Therefore, for better accuracy fine step of about 0.4 nT. To average the measurement noise and to
of determination of peak position we must choose a bias current of avoid inaccuracy due to a finite measurement step, the data was then
the SQIF that provides the sharpest peak shape, which is necessary fitted with a 5th order polynomial and a polynomial minimum was
for improved accuracy of absolute magnetic field measurements. calculated (Figure 10). This method made it possible to maximize
For initial evaluation of operation of our SQIF, we recorded the precision of determination of the position of voltage minimum.
its modulation characteristics at different bias currents in a wide The whole algorithm was then programmed in XLISP language as
range of magnetic fields from -15 µT to +15 µT (Figure 8). As it a magnetic field measurement function that automatically measured
was expected, the SQIF demonstrated a single dip around zero positions of both positive and negative minima and calculated their
magnetic field, which had the sharpest shape at the bias current mean value to compensate for the contribution of the SQIF bias
Physics
Is ≈ 35 µA. For more accurate determination of the best bias cur- currents. The function returned the value of the actual magnetic
rent, we repeated the same recording with a smaller step and in a field, meaning that the SQIF could now operate as a real magne-
narrower range of magnetic fields from -0.3 µT to 0.3 µT (Figure tometer of absolute magnetic field. Each single measurement of
9). We have used here both positive and negative bias currents to such a magnetometer involved about 120 voltage measurements
evaluate operation in bias reversal mode. It can be seen that the at different currents through the main magnetic coil, which in all
position of the voltage peak strongly depends on the bias current took about 15 s. In the future, the time of this measurement can be
of the SQIF and is located symmetrically around B ≈ 0 for positive noticeably reduced by using a faster voltmeter and special electronic
and negative current values. hardware to find the positions of voltage minima.
This means that the position of the peak on the B axis is mainly We checked the stability of our SQIF magnetometer by mon-
determined by the parasitic magnetic field created by the bias cur- itoring a background magnetic field for a long time (about 200
rent Is of the SQIF. It can be also noticed that this dependence measurements) and calculating the standard deviation of measure-
(dashed line in Figure 9) is very non linear, and not proportional to ments which was found to be about 13 pT. We then repeated the
Is as one may expect. This behavior is most probably explained by same stability test but in a more harsh condition, by executing a
the non-uniformity of the magnetic field created by the SQIF bias thermo recycling after each unit measurement. Surprisingly, the
current. In this case, the change in Is can create different changes standard deviation was found to be of about the same order, 16 pT,
in magnetic field for different loops of the SQIF, which will cause which means that our SQIF sensor did not trap any magnetic fluxes
shape distortions leading to an additional shift of a peak position. during the cooling down process.
In any case, since this effect is intrinsically symmetric with respect To demonstrate the magnetometer sensitivity, we have recorded
to the sign of the bias current, then it can be easily eliminated by a response of the SQIF magnetometer to a 0.1 nT step-like magnetic
taking a middle point between the positive and negative peaks as field (Figure 11). The magnetic field step was generated by applying
a measure of the actual magnetic field. an additional current to the same main coil which was used for
Using data on Figure 9, we chose Is = 37 µA as the best bias cur- recording modulation characteristic of the SQIF.
rent, because it provided the sharpest minimum and thus ensured
the best accuracy of determination of the peak position on the Measurement of magnetic field with SQIF magnetometer
B axis. For further improvement of measurement accuracy, the Using the SQIF magnetometer setup we were able to measure
estimation of the shielding factor we have recorded the response of

the magnetometer to placing a permanent magnet in the vicinity
of the Dewar at a distance of about 40 cm from the SQIF sensor
location. The direction of the field was orthogonal to the surface of
the SQIF for maximum sensitivity. Prior to placement, the field of
this magnet was measured with the flux gate magnetometer and was
about 240 µT at a 40 cm distance. The recorded response is shown
in Figure 13. It was noticed that after the placing and removing of
the magnet, the magnetic field baseline acquired some shift which
remained constant at subsequent placements of the magnet in the
same polarity. However, when the magnet was placed in the oppo-
site polarity, the baseline was shifted to the opposite direction in a
similar way. This shift can be explained by partial re-magnetization
of µ-metal shields with the magnet, which affected the residual
magnetization acquired by the shield during the cooling down pro-
cess. The average response from the magnet was about 2 nT, which
leads to an estimated shielding factor of about 1.2∙105. Such a high
shielding factor is a very important property of the used µ-metal
Figure 16. Response of SQIF installed in the vicinity of the su- material, which was not originally specified by the manufacturer
perconductor chip, to rotation of the High-Tc superconductor for application at liquid helium temperatures.
shield. Upper curve – after first cooling cycle. Lower two curves – second We have also recorded similar data for the inner shield made
cooling cycle. Predicted traces are shown by dashed lines.
of a High-Tc superconducting ceramic (Figure 14). Again, the con-
tribution of the Earth’s magnetic field was negligible with respect
the absolute magnetic field inside of our shields and to compare the to the field produced by the shield. The upper and lower parts of
effectiveness of magnetic shielding provided by different materials. the graph were not that symmetric as in the case of the µ-metal
For all experiments, we used the same set of two outmost shielding shield. This distortion of the graph can be explained by a higher
cylinders made of µ-metal. The third, inmost cylinder was chosen non-uniformity of the field produced by the shield. Because the
between µ-metal, High-Tc or Low-Tc superconductor. First, we inves- shield was superconductive, the magnetic field measured by the
tigated the shielding configuration with a µ-metal inner shield. All SQIF was produced by flux trapped in a small area on the shield
three µ-metal shields had been demagnetized before experimenta- wall. The value of the magnetic field produced by this frozen flux
tion on a special setup, which subjected the shield to an oscillating was around 20 nT, which was about the same as in the case of the
7 Hz sine-wave magnetic field with gradually decreasing amplitude. µ-metal shield.
The initial amplitude was about 10 mT to ensure saturation of the The response from the permanent magnet is shown in Figure 15,
µ-metal material. It was then exponentially reduced with a time con- from which we calculated the shielding factor to be about 1∙106. The
Physics
stant of about 40 s. The whole demagnetization procedure took about shielding factor of a BSCCO 22 12 phase High-Tc superconducting
5 min and was performed inside of a bigger tri-layer µ-metal shield. cylinder in [14] had a maximum of 1∙106, which matches our value
After demagnetization, the residual magnetic field was checked very well. This value is significantly higher compared to the µ-metal
with the flux-gate magnetometer. Special attention was given to shield and is still probably limited by the quality of the HTS ceramic
the inner µ-metal shield, which we always tried to demagnetize to shield, which had a small crack in the wall.
less than 3 nT. The dependence of magnetic field on the rotation Lastly, we measured the Low-Tc shield, which was made of lead.
angles of the µ-metal shield and the chip holder is shown in Figure The value of the field produced by this shield was about 7 nT, which
12. The data was recorded by setting the angle from 0º to 360º with is noticeably better than values of the previous two shields. The
an increment of 10º and performing a measurement of magnetic measured shielding factor was 1.2∙107, which is roughly 10 times
field at each position. We must note here again that the graph of better than that of a High-Tc shield. Thus, a lead shield can be con-
the shield rotation (blue curve) represents only the contribution sidered to provide the best shielding in combination with the two
of magnetic field produced by the shield, while the graph of chip outer µ-metal shields.
holder rotation (green curve) contains contributions of magnetic Finally, we used our SQIF magnetometer setup to measure mag-
field from both the shield and an external source (Earth’s magnetic netic fields in the vicinity of a real superconductor chip. For this
field). It can be seen that both graphs coincide with the measure- purpose we chose the superconductor Quantum Computer chip,
ment accuracy, which means that the contribution of the Earth’s the operation of which was expected to be extremely sensitive to the
magnetic field is negligible in comparison to the residual magne- ambient magnetic field. When we rotated the shield (with a High-Tc
tization of the shield. From Figure 12 we can estimate the Earth inner cylinder), we observed jumps in the measured magnetic field
magnetic contribution not to exceed 1 nT, which gives an estimation baseline between several almost equidistant levels (Figure 16). This
of the shielding factor to be higher than 2.4∙104. Another interesting indicated that our SQIF has directly measured the magnetic field of
observation was an unexpected increase of the residual magnetiza- fluxes trapped in the superconductor chip. Also, it was noticed that
tion of the µ-metal shield from about 2 nT at room temperature to the magnetic field produced by the High-Tc superconductor shield
21 nT at liquid helium temperature. This increase might have been was reduced by a factor of about 7, obviously due to the shielding
induced by the thermal stress in the shield material and must be effect of the ground plane of the superconductor chip.
taken into consideration when using the µ-metal shield. For better This unique capability of the SQIF to directly measure the
www.thurj.org 47
magnetic field of flux trapped in superconductor chips can be

extremely valuable for future applications of the SQIF magnetom- References
eter setup for monitoring magnetic environments in the vicinity
of tested superconductor circuits. 1. K. Likharev. “Superconductor Devices for Ultrafast Computing. In H.
Weinstock, ed., Applications of Superconductivity. Kluwer, Dordrecht, 1999.
2. P. Caputo, J. Oppenländer, Ch. Haeussler, J. Tomes, A. Friesch, T. Traeuble,
N. Schopohl, “High performance magnetic field sensor based on Super-
Conclusions conducting Quantum Interference Filters,” Applied Physics Letters 85
(2004): 1389-1391.
We have built a setup capable of measurement of absolute mag- 3. J.R. Kirtley and M.B. Ketcehn, “Magnetic imaging of moat-guarded super-
netic fields at liquid helium temperatures with 3.5 nT accuracy that conducting electronic circuits,” Applied Physics Letters 67 (1995): 1769-1771.
is about 25 times better than in the best known counterparts [9, 4. Katsuhiro Fukuoka and Mitsuo Hashimoto, “AC magnetic shielding char-
12, 13]. The achieved accuracy allowed us to investigate magnetic acteristic of a YBCO superconductor using magnetic field visualization
shielding properties and compare effectiveness of shields made measurement,” International Journal of Applied Electromagnetics and
of: µ-metal, High-Tc (Bi-2223) and Low-Tc (lead) superconductors. Mechanics 14 (2001): 95 – 99.
We discovered that the residual magnetization of the inner 5. Gordon B. Donaldson, “SQUIDs - ultimate magnetic sensors”, Physica
µ-metal shield at helium temperature was about 21 nT, which was Status Solidi (c) 2 (2005): 1463 – 1467.
much worse than the expected 2 nT value as measured at room 6. D.F. He and H. Itozaki, “Measuring the absolute magnetic field using high-
temperature. As a result, similar shields (which are now commonly Tc SQUID,” Journal of Physics: Conference Series 43(2006): 1227-1230.
used) could not be recommended for prospective superconductor 7. R. Kleiner, D. Koelle, F. Ludwig, J. Clarke, “Superconducting quantum
devices. Lead shield provided much smaller residual field of about interference devices: State of the art and applications,” Proceedings of the
7 nT, which was caused by trapped fluxes and can be significantly IEEE 92 (2004): 1534 – 1548.
reduced in practical applications by thermal defluxing. 8. Hong-Chang Yang, Jau-Han Chen1, Shu-Yun Wang, Chin-Hao Chen1,
We also showed that the tri-layer shielding configuration with Jen-Tzong Jeng1, Ji-Cheng Chen1, Chiu-Hsien Wu1, Shu-Hsien Liao and
lead inner cylinder yields a record shielding factor of 1.2∙107 achieved Herng-Er Horng, “Superconducting Quantum Interference Device: The
with a simple cylindrical shield with an open top. The shielding Most Sensitive Detector of Magnetic Flux,” Tamkang Journal of Science
factor of High-Tc and µ-metal shields was much worse, being equal and Engineering 6 (2003): 9-18.
to 1∙106 and 1.2∙105 correspondently. 9. J. Oppenländer, T. Träuble, C. Häussler, and N. Schopohl, “Superconducting
We have also demonstrated that our setup presents a novel way Multiple Loop Quantum Interferometers,” IEEE Transactions on Applied
of direct monitoring of magnetic field trapped in superconductor Superconductivity 11 (2001): 1271-1274.
chips. This is especially valuable for controlling the magnetic envi- 10. V. Schultze, R. IJsselsteijn, H. -G. Meyer, J. Oppenländer, C. Häussler, and
ronment when testing numerous superconductor circuits, like RSFQ N. Schopohl, “High-Tc superconducting quantum interference filter for
or components of prospective superconductor quantum computers, sensitive magnetometers,” IEEE Transactions on Applied Superconductiv-
which can operate only in extremely low ambient magnetic fields. ity 13 (2003): 775-778.
Physics
There are several ways for further improving the accuracy of 11. J. Oppenlander, C. Haussler and N. Schopohl, “Non-Φ0-periodic macro-
the setup toward the targeted field of 0.2 nT, below which magnetic scopic quantum interference in one-dimensional parallel Josephson junction
fluxes will be not trapped at all. At first, the currently achieved arrays with unconventional grating structure,” Phys. Rev. B 63 (2001):
accuracy was totally determined by a constant offset of 3.5 nT, which 024511-1 – 024511-9.
was most probably produced by a microscopic piece of magnetic 12. P. Caputo, J. Tomes, J. Oppenlander, C. Haussler, A. Friesch, T. Trauble,
material accidentally deposited close to the SQIF circuit. Other N. Schopohl, “Superconducting Quantum Interference Filters as Absolute
possible reasons for this offset include the flux trapping in the nio- Magnetic Field Sensors,” IEEE Transactions on Applied Superconductivity
bium layout of the SQIF itself, and also the finite accuracy of the 15 (2005): 1044 – 1047.
bias reversal operation. These assumptions will be verified when 13. J. Oppenlander, P. Caputo, Ch. Haussler, T. Trauble, J. Tomes, A. Friesch, and
we try the newer SQIF design, which is currently in the produc- N. Schopohl, “Effects of magnetic field on two-dimensional superconducting
tion facility. The new design is also expected to generate much less quantum interference filters,” Applied Physics Letters 83 (2004): 969-971.
magnetic field by the bias current, which can significantly simplify 14. H.Matsuha, A.Yahara and D.Irisawa, “Magnetic Shielding Properties of
and speed up the field measurement procedure. HTc Superconductor,” Superconductor Science and Technology 5 (1992):
With all the above improvements, we consider the accuracy S432-439.
of 0.2 nT realistically achievable, which will make it possible to
minimize the magnetic field below the flux trapping threshold Bcrit
and ensure reliable operation of highly integrated superconductor
circuits.

Searching for dark matter

beyond the WMAP haze
Lauren Weiss ‘10
Harvard College, weiss@fas.harvard.edu
One of the current mysteries in galactic astronomy is the WMAP haze [1], an excess in microwave radiation from
within 20 degrees of the galactic center. The WMAP haze reveals an overall “hardening” of synchrotron radiation
in the range of 23-94 GHz, where the specific intensity spectrum goes as ν−0.5 compared to ν−1 elsewhere in the
Galaxy. One potential mechanism for producing such hard synchrotron radiation is a model of self-annihilating
cold dark matter creating ultra-relativistic e+e− pairs, which accelerate in the galactic magnetic field and produce
synchrotron radiation. This research models the synchrotron radiation that results from dark matter annihila-
tions throughout the Milky Way Galaxy with a parameter space that enables the adjustment of DM particle mass,
DM particle cross-section, galactic magnetic field, electron energy injection, and electron energy diffusion. We
use IDL 7.0 to integrate the volume emissivity along each line of sight to compare our model to ARCADE data,
which identifies a 23 mK synchrotron excess at 3 GHz [2]. We investigate the possibility that the WMAP haze
and the ARCADE excess have a common origin in dark matter annihilation. We find that (a) when normalized
to the haze, vanilla WIMP annihilation model under-predicts the ARCADE data by a factor of ~ 20, and (b)
this model produces a spectral index of 0.9, which is softer than the ARCADE spectral index by 0.33. Although
a vanilla WIMP model cannot explain the discrepancy between the ARCADE and WMAP excesses, invoking a
more exotic DM particle or considering a non-DM phenomenon such as e+e− pair production from local pulsars
could explain the ARCADE data.
Introduction Methods
Recent data from the WMAP satellite reveal an excess of syn-
chrotron radiation at 23-94 GHz [1], which has been dubbed the Constructing the model
WMAP “Haze”. The source of the synchrotron excess is unknown, The number of DM annihilations per unit volume per unit time is
Physics
but its hardened spectrum (index ~ 0.5) indicates that it does not 2
originate in the usual supernova mechanism (index ~ 1.0). One ρ 
 NFW  σv
 m  (1)
possibility is that ultra-relativistic electrons and positrons produced  χ 
by cold dark matter self-annihilation (e.g., from neutralinos) could
create a synchrotron excess with a hardened spectrum. Although where ρNFW is the Navarro-Frenk-White (NFW) [6] profile (see §2.2),
the WMAP Haze is described well with models of self-annihilating mχ is the WIMP mass and ‹σν› is the annihilation cross section.
dark matter [1],[4],[5], our lack of knowledge about dark matter The number of electrons produced per dark matter particle per
limits the extent to which we can constrain such models. unit time is
A new opportunity has arisen to test models of dark matter Emax dN
self-annihilation. The balloon mission ARCADE reports a syn- ¨ Emin dE
dE (2)
chrotron excess at 3-10 GHz [2]. Not only did ARCADE observe
synchrotron excess in a different frequency range from WMAP, where Emax and Emin are the maximum and minimum electron ener-
but it also observed a different part of the sky. The ARCADE foot- gies produced in annihilations, dN
dE
is the number density of electrons
print is roughly an annulus centered at ~ (l, b) = (70, 0) with inner per unit volume in a given energy bin and E is energy.
radius ~ 20 degrees and outer radius ~ 40 degrees [2]. Because the The synchrotron power from a single electron is
region ARCADE observed is removed from the galactic center, and
because it reveals a synchrotron excess in a previously un-modeled 3q3 v ∞
2
B(r )( )∫ v K 5/3 (η ) d η (3)
frequency range, fitting models of dark matter self-annihilation to me c vc vc sin α
the ARCADE data could reveal new constraints on the parameters
of dark matter self-annihilation processes (see Figure 1). For these where q is the electron charge, me is the electron mass, c is the speed
models, we consider “vanilla” WIMPs (χ), which do not experience of light, B(r) is the radially dependent magnetic field, α is the pitch
any large-scale forces other than gravity, although more exotic DM angle between the magnetic field and the electron velocity, νc is
particles exist and should be considered in the future. the critical frequency as defined in [7] and K is a modified Bessel
function.
With dimensional analysis and proper integration over pitch
www.thurj.org 49
Figure 1. Schematic of ARCADE data within the larger body of

knowledge. If the ARCADE data fits the spectrum described by WMAP
and Haslam, it could agree with dark matter annihilation models. Incon-
sistencies in the power law of the spectrum or the specific intensity at 3
GHz could rule out basic dark matter annihilation as the source of the
ARCADE excess.
Figure 2. The Haze. Top left: Model of synchrotron excess from vanilla
WIMP annihilation at 3.3 GHz, parameters 101. Top right: same model,
with a mask for all sources with extinction greater than 1 mag. Bottom: A
previous example of the observed haze [3], with the region of ARCADE
observation overlaid in red and the haze boxed in green.
Figure 3. jν as a function of electron energy. The synchrotron flux

Physics
is calculated for two values of ν: ν1 = 3.3 GHz (ARCADE) and ν2 = 22.5

GHz (WMAP). The synchrotron distributions peak at different energies:
10 GeV for ARCADE and 30 GeV for WMAP. These differing synchrotron
distributions reflect the different energy loss rates for electrons at different
frequencies.
Figure 4. The ARCADE footprint and calculated annulus. Left: The

angle and energy, we can combine the above equations to create an region of the sky observed by ARCADE, Figure from Kogut et. al. [2].
Color corresponds to the total antenna temperature. Right: The calculated
expression for the synchrotron power produced per unit volume at annulus is overlaid in red.
a given frequency,
1 ρNFW 2 3q3 Emax dN π/2 v ∞
jv = ( ) σv × B(r )∫ ∫ sin α( )∫ v K 5/3 (η ) d ηdαdE (4)
4 π mχ me c 2 Emin dE 0 vc vc sin α the Milky Way Galaxy at 3.3 GHz (the lowest ARCADE frequency
band) and 22.5 GHz (the lowest WMAP band). To model dark
To find the specific intensity Iν from synchrotron radia- matter self-annihilation, we considered several parameters that
tion in a given direction, we integrate along the line of sight. could affect the synchrotron excess.
smax
Distribution of dark matter.
Iv = ∫ jv ds (5)
0 We assumed an NFW profile:
S0
where ds is a distance element along the line of sight and we take SNFW (r ) =
r r
(1 + )2 (6)
smax = 30 kpc. A model of the synchrotron excess at 3.3 GHz and a rs rs
real map of the haze are shown in Figure 2.
where ρ(r = 8.5 kpc) = 0.3 GeV/cm3), rs = 20 kpc is the scale radius
Model parameters [1], ρ0 is the density of dark matter at the galactic center, and r is
the radius from the galactic center.
We used IDL 7.0 to generate a model of the synchrotron excess in
Parameters bf WMAP offset χ2 bfARCADE bfARCADE/ bf WMAP
000 7.63023 0.392911 17.171836 5.9899180 0.78502458
001 8.91677 0.552084 20.393415 18.915372 2.1213256

010 20.8165 0.392777 17.171847 52.040299 2.4999543
011 22.6527 0.489442 18.800070 97.316993 4.2960438
100 2.63142 0.446227 21.185211 36.030924 13.692579
101 2.05719 0.199734 21.105665 41.469781 20.158460
110 4.35786 0.338744 24.607047 102.29569 23.473834
111 3.43724 0.0169221 25.541085 88.401674 25.718797
101, mχ = 300 5.08966 0.154001 21.789670 105.47548 20.723481
101, mχ = 1000 15.1392 0.118970 22.305505 314.73431 20.7894
101, index = -0.9 2.05719 0.199734 21.105665 27.965467 13.5940
Table 1. ARCADE vs. WMAP Boost Factors. For each permutation of parameters, the best χ2 fit was found by varying bf WMAP and offset. The boost
factor and offset to the WMAP data for each fit are listed. Boost factors for the ARCADE and WMAP data were compared for each permutation of the
parameters. From left to right, the parameter code indicates diffusion, energy injection, magnetic field (see §2.2 for a description of each switch). AR-
CADE boost factors are approximate (to a factor of 10 without diffusion, a factor of 2 with diffusion).
Dark matter mass and annihilation cross section. ARCADE data. Magnetic fields that depend on galactic latitude as
We assumed a particle mass of mχ = 100 GeV unless otherwise well as radius should be tested in the future.
noted. To match the observed dark matter density Ωm = 0.3, the
annihilation cross section should be roughly ~ 3×10−26 cm3/s for a Distribution of electron energy density
100 GeV neutralino, in accordance with the physics of “freeze-out” Because the synchrotron spectrum of a single electron depends
during the cooling of the universe. on the electron energy, the electron energy distribution affects the
total spectrum. We consider two energy injections: a delta function
Magnetic field variation at the energy of the DM particle mass, and a power law of
We allowed two different magnetic field models, one with con- dN
∝ E −3/2 (8)
stant magnetic field of B0, and one with exponential decay described
Physics
by dE
r
Emax dN
[4]. The normalization constant is set so that ∫ E E dE dE = mDc
2
−
B(r ) = B0e rscale (7) min
. In diffusion models, the energy distribution is modified by a dif-
where B0 = 10μG is the field at the galactic center and rscale = 10kpc fusion code to achieve the steady state solution.
is the scale radius. These equations assume a spherically symmetric
magnetic field, whereas the true galactic magnetic field probably has Diffusion of electron energy
a stronger disk component. Because ARCADE observes up to 40 Ultra-relativistic electrons lose their energy through (a) synchro-
degrees above and below the galactic plane, the spherical symmetry tron radiation and (b) inverse Compton scattering. These losses are
assumed in our model might not be a good approximation for the accounted for in a diffusion code, which solves for the steady-state
system. Models without diffusion were considered as well; for these
simpler models we assumed that an electron lost its energy as a step
function at one million years for an electron of frequency ν = 23
Gal. coordinates bf (normalized) GHz, and at 10 million years for ν = 3 GHz (see Figure 3).
We used several binary “switches” in the modeling process, vary-
(70,30) 20.715328 ing the model to allow for a step function energy decay (0) versus
steady-state diffusion (1), a power law electron energy injection (0)
(100,0) 39.900335 versus a delta function injection at 100 GeV (1), and a constant mag-
netic field (0) versus an exponentially decaying magnetic field (1).
(30,0) 3.1558273
Boost factors
(70,120)* 25.543292 For each set of parameters, we found the boost factor, which is
the ratio of the observed flux to the modeled flux, for both ARCADE
Table 2. Boost Factors at different coordinates at 3 GHz. The and WMAP. For WMAP, we were also able to calculate an offset.
boost factor was calculated at three points within the ARCADE ring one
point (*) outside the ARCADE ring (see Figure 4).
I v ,WMAP = bfWMAP × I v ,model + offset (9)
www.thurj.org 51
Figure 6. Comparative spectra. Left: The distribution of electron en-

ergies from 1-100 GeV produced by a power-law injection and subjected
to energy diffusion. Right: The spectra of the model (solid) and the AR-
CADE data (dotted) from 1-100 GHz. Whereas the ARCADE data claims
a power law of Iν ν−0.57 [2], the vanilla WIMP model produces a power
law of Iν ν−0.90 at 3 GHz, and Iν ν−1.0 at 23 GHz. Note that antenna tem-
perature goes as ν2Iν, which is why the spectral index is ~ 0.5 as opposed
Figure 5. Preferred fit of WMAP Haze. This model allows for energy to ~ 2.54.
diffusion, a power law energy distribution and an exponentially decaying
magnetic field (parameters 101 in Table 1). The dashed line is the mod-
el; the vertical bars are the WMAP data. bf = 2.05719, offset = 0.199734, 23 mK
χ2=21.1056665. bf ARCADE = (12)
I v (v = 3.3 Ghz )× 2.9939 mK per kJy /sr
The best fit for the WMAP data was determined by finding the where bfARCADE is the boost factor to the ARCADE data and Iν is the
reduced χ2 over bfWMAP and offset. Because we did not have data intensity at 3.3 GHz in kJy/sr.
from ARCADE, we could only approximate the boost factor (see
Equation 12). The normalized boost factor,
bf ARCADE
bf !
bfWMAP
(10) Results
describes the ratio of the synchrotron excess from ARCADE to
a model that fits the WMAP data. Brightness of the ARCADE excess
Although the value of bf WMAP and bfARCADE are sensitively depen- In our most reasonable models, we found the ARCADE excess
Physics
dent on the WIMP mass, the ratio between them is not (see Table at (l, b) = (70, 30) to be a factor of ~ 20 higher than the excess gen-
1). The boost factor can be interpreted as either an enhancement erated through the annulus our model after normalizing to the
of the cross section above the assumed 3 × 10−26 cm3/s value, or an WMAP boost factor (see Table 1). The model that produces this
enhancement in the mean of the dark matter density squared ‹ρ2›/‹ρ2› result involve energy dissipation through diffusion, electron energy
(e.g., via substructure). The boost factors required to fit various injection as a power law, and an exponentially decaying magnetic
models to the WMAP and ARCADE data are shown in Table 1. field (parameters 101, see Figure 5).
Data Comparative spectra

We used data from the ARCADE balloon survey in the 3.3 GHz We compared the spectral indices of ARCADE and WMAP data
band. Problems with the 8 and 10 GHz bands made these data to our model at 3 GHz and 23 GHz. A spectral index is defined by
unreliable. Because we did not have raw data from the ARCADE I v ∝ v −s (13)
team, we calculated the intensity for points witin an annulus of inner
radius 20o and outer radius 40o centered on (l, b) = (70, 0), which where s is the spectral index [7]. The model shows indices of 0.90
approximates the region of the sky seen by ARCADE (see Figure at 3.3 GHz and 1.0 at 22.5 GHz. ARCADE quotes a spectral index
4). We approximated a value for the excess antenna temperature of 0.57 at 1 GHz [2], which is “harder” (skewed toward higher fre-
based on the relation [2]. quencies) than the model by a spectral index of 0.33 (see Figure 6).
Texcess v −2.57
= 500× (11)
[mK] [Ghz]
Discussion and Conclusions
This equation yields an antenna temperature of 23 mK in the ring
at 3.3 GHz. We used the conversion factor 2.9939 mK / kJy to convert Although the ARCADE boost factors nicely match the WMAP
from antenna temperature to kJy/sr. We were able to compare this boost factors for some of the non-diffusion cases, the non-diffusion
value to the average flux through the ARCADE footprint (see Table model is too uncertain to be conclusive. Furthermore, the diffusion
1) and at four sample points (see Table 2) in our model. At 3.3 GHz, model is a more accurate representation of the physical processes in
the galaxy, and so its results are more believable. The most physically 2. Dark matter particle interactions are more complex than the
accurate model is the parameter set 101, which includes energy dif- vanilla WIMP model permits.
fusion, a power law energy injection, and an exponentially decaying Our model was built on the assumption that neutralinos only
magnetic field. This model requires a normalized boost factor of interact via gravity on large scales. Some models for dark matter
~ 20 to fit the model to the ARCADE data. Is a normalized boost allow the particles to attract or repel each other through other forces,
factor of ~ 20 reasonable? In Table 2, the boost factors at individual which could drastically change the annihilation rates of the DM
points within the ARCADE footprint are shown. The normalized particles. In particular, attractive particles in over-dense regions of
boost factors vary from ~ 3 to ~ 40, indicating a broad range of boost substructure could produce far more synchrotron radiation than
factors within the ARCADE annulus. Note that at (l, b) = (30, 0) the simple “vanilla” WIMPs considered in our models. More exotic
(the sampled point nearest to the galactic center), the boost factor dark matter particles will be considered in future simulations.
is the smallest at ~ 3; the model approaches the WMAP portion of
the haze here. However, at (l, b) = (100, 0) the boost factor is ~ 40,
indicating that the model drops off too quickly at large radii to
explain the synchrotron excess. This is consistent with the data from
PAMELA, a satellite that observed the positron-to-electron excess References
and found that dark matter annihilation requires high boost factors
[8]. Thus, the synchrotron excess that ARCADE and PAMELA see 1. Finkbeiner, Douglas. “WMAP Microwave Emission Interpreted as Dark
is simply too bright to be explained by vanilla dark matter annihi- Matter Annihilation in the Inner Galaxy.” AstroPh preprint, 2005.
lation alone. Although any number of explanations could justify 2. Kogut et. al. “ARCADE 2 Observations of Galactic Radio Emission.”
this discrepancy, two come to mind easily: AstroPhpreprint, 2008.
3. Dobler and Finkbeiner. “Extended Anomalous Foreground Emission in the
1. The ARCADE synchrotron excess is dominated by a non-DM WMAP 3-Year Data. AstroPh preprint, 10/09/06.
source. 4. Blasi, Olinto,and Tyler. “Detecting WIMPs in the microwave sky.” Astropar-
Local pulsars that produce electron-positron pairs could boost ticle Physics, 18 (2003) 649-662.
the local synchrotron signal, creating the excess observed by 5. Bertone, Hooper, and Silk. “Particle Dark Matter: Evidence, Candidates
ARCADE. A pulsar-created synchrotron excess will be modeled and Constraints”. Fermilab, 2004.
to determine whether there are enough local pulsars to explain the 6. Navarro, Frenk, White. “A Universal Density Profile from Hierarchical
ARCADE and PAMELA excesses. However, it is important to note Clustering.” ApJ, 490:493-508,1997 December 1.
that finding a working model of pulsar-driven synchrotron excess 7. Rybicki, Lightman. Radiative Processes in Astrophysics. Wiley and Sons,
does not rule out dark matter annihilation mechanisms. In particu- Inc., 1979.
lar, the WMAP haze, which is well fit by a vanilla WIMP model, 8. Cholis et. al. “High Energy Positrons from Annihilating Dark Matter.”
cannot be explained by pulsars because the spatial distribution of arXiv:0809.1683v1 [hep-ph]
pulsars in the galaxy is inconsistent with the haze morphology.
Physics
www.thurj.org 53
Engineering recombinase enzymes to

emulate the CCR5-∆32 mutation conferring
resistance to HIV-1 infection
Meera Atreya ‘09*, Kevin M. Esvelt, and David R. Liu
*Harvard University , meera@atreya.us
The CCR5-∆32 deletion mutation is a naturally-occurring genetic polymorphism that confers strong resistance
to HIV-1 infection. I propose the use of chimeric recombinase enzymes, capable of site-specifically excising
DNA, as a gene therapy method to safely emulate the natural mutation in CCR5 and therefore confer resistance
to HIV-1. I designed and created enzymes consisting of the catalytic domain of Hin-H107Y, a hyperactive mutant
DNA invertase, fused via a flexible linker sequence to engineered zinc finger DNA-binding domains. Individu-
ally, the engineered enzymes show both activity and substrate specificity with regards to their target sequences
on CCR5. To optimize enzyme activity and specificity individually, I developed and validated a substrate-linked
protein evolution selection scheme. I generated enzyme libraries in E. coli, selected active mutants, and performed
recombinase activity assays side-by-side with starting pool samples. Future experiments will use the starting
libraries for iterative rounds of directed evolution. After determining efficient recombinase variants individually,
I will test the evolved enzymes for recombinase-mediated excision on CCR5, first in E. coli and subsequently in
human cells. These engineered recombinase enzymes have the potential to provide protection from HIV-1 in a
manner suitable for delivery in developing nations.
binds to the immune cell-surface molecule CD4 receptor (Janeway

Introduction et al. 2008). Gp120 undergoes a conformational change upon CD4
binding, allowing the virus to bind to a chemokine receptor in the
cell membrane as a co-receptor. Co-receptor binding allows viral
glycoprotein gp41 to fuse the cell membrane and viral envelope
together, allowing the HIV-1 genome and proteins to enter the cell
Chemokine receptors (Allen et al. 2007; Janeway et al. 2008).
CCR5 is a required co-receptor for HIV-1 infection of mac-
In response to foreign antigens, the body’s immune system rophages in the first (M-tropic) phase. The corresponding HIV-1
concentrates specific white blood cells at the site of infection. Cell variants are deemed “R5.” Almost all primary HIV-1 isolates
migration and activation in this process is mediated by signal- are “R5” viruses, as this is the dominant viral phenotype of new
ing molecules known as chemokines (chemoattractant cytokines)
which are secreted early in the immune response (de Silva and
Stumpf 2004).
There are four subfamilies of chemokines and their respective
receptors, distinguished by patterns of cysteine residues (abbrevi-
ated “C”) in the former. For example, the nomenclature “CCR5”
refers to the 5th chemokine receptor of the CC subfamily. Several
Cellular Biology
Molecular and
ligands bind to each chemokine receptor, and many ligands bind to

multiple receptors. Due to this redundancy, a chemokine receptor
knockout rarely results in phenotypic changes (Allen et al. 2007).
CCR5 and HIV infection

Chemokine receptor 5, encoded by gene CCR5, plays a central
role in cellular infection by the human immunodeficiency virus
(HIV). CCR5 is expressed on the surface of numerous cells of the
immune system, including macrophages and CD4+ T cells (Berger et
al. 1999). Infection of these cells by HIV-1 is often described in two
consecutive stages, the M-tropic and T-tropic phases, correspond- Figure 1. HIV-1 (top) infects a macrophage cell (bottom) via binding
ing to the target cells: macrophage or T-lymphocyte cells. During of viral protein gp120 to cell-surface molecule CD4 and to co-receptor
viral infection, a glycoprotein on the surface of the virus, gp120, CCR5, poising gp41 for membrane fusion (BBC 2007).
infections (Janeway et al. 2008). It is estimated that “R5” viral strains donor and was effectively cured. As a result of transplantation, the
account for 90% of all HIV-1 infections (O’Brien and Moore 2000). patient’s genotype changed from heterozygous to homozygous for
the CCR5-∆32 allele. Following the transplant, the patient discon-
The CCR5-∆32 mutation tinued highly active antiretroviral therapy (HAART), HIV-1 RNA
In studies of disease progression following exposure to HIV-1, levels in the patient’s blood and bone marrow became negligible, and
a genetic mutation in CCR5 has been linked to resistance to HIV-1 CD4+ T cell counts steadily increased. At the time of this writing,
infection (de Silva and Stumpf 2004). The CCR5-∆32 mutation is more than 20 months post-transplant, no detectable replicating,
a naturally-occurring deletion polymorphism in CCR5 that yields active HIV-1 has been reported in the patient (Hutter et al. 2009).
a non-functional cellular receptor. Thirty-two nucleotides of the The CCR5-∆32 allele appears to not only provide resistance to initial
CCR5 coding region are absent in the mutation, resulting in a frame- infection, but also potentially to treat current infections.
shift and premature stop codon (Janeway et al. 2008). The resulting Homozygous allele carriers of the CCR5-∆32 mutation are
truncated protein is not transported to the cell surface and instead immunologically healthy. The allele has no known serious side-
accumulates in the cytoplasm (de Silva and Stumpf 2004). Conse- effects (Carrington et al. 1997), although some studies indicate that
quently, individuals homozygous for the CCR5-∆32 allele do not heterozygous CCR5-∆32 allele carriers may have increased risks of
have detectable CCR5 receptors on lymphoid cell surfaces (Car- cervical cancer development (Singh et al. 2008) and susceptibil-
rington et al. 1997) and heterozygous allele carriers express fewer ity to symptomatic West Nile virus infection (Glass et al. 2006).
functional receptors on their cell surfaces (Y. Huang et al. 1996). Chemokine-receptor functional redundancy has been hypothesized
Although rare in Africans and Asians, up to 20% of Caucasians to compensate for the lack of CCR5 in those individuals (Premack
are heterozygous Δ32 allele carriers. In addition to having fewer and Schall 1996).
functional CCR5 receptors due to Δ32 heterozygosity, the trun-
cated protein may reduce wild-type CCR5 and CXCR4 cell-surface A strategy for conferring resistance to HIV-1 infection
expression by forming heterodimers with them, which are retained Given the promising phenotype of the CCR5-∆32 mutation,
in the endoplasmic reticulum (de Silva and Stumpf 2004; Agrawal many researchers have striven to mimic its effects. Recently, zinc
et al. 2007). As “R5” HIV-1 variants require co-receptor CCR5 for finger nucleases have been targeted to CCR5 to replace the wild-
infection, heterozygous carriers of the CCR5-∆32 allele experience a type allele with the ∆32 by homologous recombination (Perez et al.
delayed onset of AIDS by two to three years if infected with HIV-1. 2008). These genome-editing enzymes introduce a double-stranded
Furthermore, the 1% of Caucasians who are homozygous for the DNA break which is corrected via homologous recombination with
allele are nearly fully resistant to infection by the HIV-1 virus (Y. a provided ∆32 template strand. As a therapy, this method requires
Huang et al. 1996; Stephens et al. 1998). adoptive transfer of ex vivo-expanded, zinc finger nuclease-modified
Although extremely rare, there are 12 documented cases of CD4+ T cells (Perez et al. 2008), which is not a feasible treatment for
individuals homozygous for the CCR5-∆32 allele who have been use in developing nations. Zinc finger nucleases have been shown
successfully infected with HIV-1 (D. Oh et al. 2008). The viral strains to induce toxicity as a result of DNA cleavage at non-target sites,
in these individuals appear to use CXCR4 as a co-receptor during resulting in cell death and apoptosis, and so would be ill-suited for
infection (Agrawal et al. 2007; Lama and Planelles 2007). A contrast- in vivo delivery to target cells (D. Carroll 2008; T. Cathomen and
ing example, however, involves a patient infected with HIV-1 who J. K Joung 2008). Furthermore, DNA double-strand break repair
received a stem cell transplant from a homozygous CCR5-∆32 allele systems have been linked to human genome instability and cancer
(van Gent et al. 2001). This represents a major limitation for zinc
finger nuclease-mediated gene therapy to become suitable for use
in humans.
Using the natural ∆32 deletion mutation as a model, I aim to
emulate the CCR5-∆32 mutation at the DNA level in the genomes
of individuals lacking the allele. Specifically targeting CCR5 on
chromosome three, I will use engineered, genome-editing enzymes
capable of safely excising DNA to create a deletion in CCR5 which
results in a similarly-truncated receptor. If successful, this project Cellular Biology
Molecular and
Figure 3. Serine recombinase enzyme (γδ-resolvase) dimer

Figure 2. Site-specific recombinase-mediated excision. The se- bound to DNA. Catalytic domains in yellow, interdomain linkers in or-
quence of interest (arrow) between recombinase enzyme recognition sites ange, DNA-binding domains in green, and DNA in gray (Akopian et al.
(boxes) is excised, creating a freed loop of DNA (Akopian et al. 2003). 2003).
www.thurj.org 55
will further efforts to confer resistance to HIV-1 infection, or to 2007). These domains are physically separated and connected by
possibly treat infected individuals by generating a reserve of resis- a flexible linker.
tant cells. Furthermore, recombinase enzymes are unlikely to suffer Because sequence recognition and catalysis functions of a recom-
the same disadvantages as zinc finger nuclease enzymes because binase can be specified by unrelated protein domains, it is possible
recombinase-mediated DNA excision does not rely on the genera- to replace the enzyme’s native helix-turn-helix DNA-binding motif
tion of DNA damage as a means to effect a CCR5-null phenotype. with a zinc finger DNA-binding motif (Akopian et al. 2003).
Given an appropriate delivery mechanism, this therapy should be
safe for direct in vivo application, which is a necessity for conditions Zinc finger DNA-binding motifs
in the developing world. A zinc finger is a protein domain that binds to DNA via sequence-
specific base contacts by an α-helix in the major groove (Segal et
Serine recombinase enzymes al. 1999). Proteins containing zinc fingers are common in eukary-
Site-specific recombinase enzymes are ideal for genome modi- otes; in humans they comprise approximately two percent of all
fication applications due to their ability to recognize precise DNA genetically-encoded proteins. Many zinc finger-containing proteins
sequences and remove, replace, or invert the flanked sequence are involved in gene regulation, conferring sequence specificity to
(Gordley et al. 2007). transcription factors and other DNA-localized proteins (Matthews
The serine family of recombinase enzymes (named for their and Sunde 2002).
active-site serine residues), catalyze excision or inversion depending Of all zinc finger classes in the human genome, Cis2His2 zinc
on the orientation of a two-base pair overhang at the center of the fingers, named for pairs of cysteine and histidine residues which
recombination sites. If the sites are in direct repeat, the sequence interact with the zinc ion, are the most prevalent (Matthews and
between them is excised. Similarly, a sequence can be integrated Sunde 2002). Each zinc finger generally interacts with three DNA
into a recombination site via the reverse reaction. Inverse repeats base pairs. Unlike helix-turn-helix DNA-binding domains, which
lead to inversion of the intervening sequence (Gordley et al. 2007). often dimerize to recognize symmetric DNA sequences, zinc finger
Recombinase-mediated excision or inversion in the serine DNA-binding domains are modular and can be combined to recog-
recombinase family occurs via an enzyme tetramer. Enzyme dimers nize extended, asymmetric sequences (Segal et al. 1999; Pavletich
recognize the two sites to be recombined, interact to form a catalytic and Pabo 1991). For example, three or more zinc fingers together
tetramer between crossover sites, and coordinately cleave all four can confer DNA sequence specificity to a protein, allowing it to
DNA strands with catalytic serine residues, covalently attaching target a particular gene (Matthews and Sunde 2002).
each recombinase monomer to the DNA backbone (Gordley et al. Several researchers have categorized the zinc finger protein
2007). The dimers then exchange partners by 180° rotation about a sequences needed to recognize specific triplets of DNA base pairs.
flat, hydrophobic interface, preventing dissociation. Subsequently, These functionally modular sequences can be combined to make
the four 3’ hydroxyls attack the serine esters, forming new phos- proteins that can bind with nanomolar affinity to DNA sequences
phodiester bonds and ligating the strands without DNA loss (W.
Li et al. 2005; Gordley et al. 2007).
The serine recombinase family is attractive for protein engineer-
ing applications due to the enzymes’ structural modularity. One
domain is responsible for DNA binding, while a distinct, catalytic
domain mediates all subsequent enzymatic steps (Gordley et al.
Cellular Biology
Molecular and
Figure 4. Illustration depicting the full-length CCR5 gene (pro-

tein-coding sequence marked by the blue arrow) within a segment of ge-
nomic DNA (light and dark blue horizontal lines). The Δ32 deletion region
(purple bracket), recombinase-mediated deletion region (green bracket),
and central dinucleotides of both recombination sites (green arrows and
“TT,” “AA”) are indicated. Chimeric recombinase enzyme target sequences Figure 5. Illustration of genomic DNA before and after recombi-
are marked by their respective zinc finger DNA-binding domain recog- nase-mediated excision of the C-terminal region of gene CCR5.
nition sites (orange, pink, gray, turquoise brackets). Although not distin- Central dinucleotides (marked by “TT” and “AA” in white and yellow)
guished in the illustration, the zinc finger domains of chimeric recombi- are in direct repeat. The two-base pair overhangs base pair with exchange
nase enzymes A and C bind to the CCR5 coding strand, while those for partners during recombinase-mediated excision. Stop codon introduced
B and D bind to the non-coding DNA strand. Stop codons for the Δ32 by the resulting frameshift denoted by a red bracket. The resulting protein
mutant, wild-type, and recombinase-modified CCR5 versions are repre- will lack the proper C-terminal region and will instead contain a segment
sented by red brackets. of amino acids derived from non-coding genomic DNA.
Figure 6. CCR5 coding sequence excerpts of wild-type, Δ32

deletion mutant, and recombinase-treated CCR5 versions. Se- Figure 7. Selection plasmid RecX-SitesY construct.
quence continuation is abbreviated as an ellipsis. Three of the four chime-
ric recombinase enzyme target sequences are marked by locations of their
respective zinc finger DNA-binding domain recognition sites (orange,
pink, and turquoise underlines). Zinc fingers for B and D bind to the non-
coding DNA strand, not pictured. The target sequence for recombinase C
is not marked as it is excised during recombinase treatment. The “TAG”
stop codon, introduced by the recombinase-mediated excision and result-
ing frameshift, is followed by another stop codon “TAA” just six base pairs
downstream, ensuring that the translated protein will be truncated.
up to 18 base pairs in length (Segal et al. 1999). Not all triplets can
be recognized; while there is nearly complete coverage of 5’-GNN-3’,
5’-ANN-3’, and 5’-CNN-3’ DNA-recognition sequences (Segal et al.
1999; Dreier et al. 2001; Dreier et al. 2005), the 5’-TNN-3’ sequences
are largely unknown. Furthermore, not all characterized sequences
exhibit specific binding, and reported success rates of the strict
modular assembly methods vary considerably. Library selection for
improved binders is often required (Ramirez et al. 2008).
As mentioned previously, replacement of the helix-turn-helix
DNA-binding motif of a serine recombinase with a zinc finger
DNA-binding motif changes the specificity of the resulting chi- Figure 8. Recombinase selection scheme. Active enzymes excise
meric enzyme to a site recognized by the zinc finger (Akopian et the region subjected to restriction endonuclease digestion, while inactive
al. 2003). Chimeric enzymes of the serine recombinase family have library members are cleaved. After complete digestion, only active library
members yield PCR products. In the event of incomplete digestion, re-
been retargeted to sites precisely specified by Zif268 zinc finger combined products can be gel-purified before subsequent amplification
DNA-binding domains (Akopian et al. 2003). Importantly, such and diversification steps.
chimeras are capable of recombining their newly-specified target
sites in mammalian cells (Gordley et al. 2007).
selected sites will excise the entire C-terminal coding sequence of
the CCR5—a targeted deletion of 571 base pairs of genomic DNA.
Cellular Biology
Molecular and
Materials and Methods Creation of chimeric recombinase enzymes with designed zinc
finger DNA-binding domains
Hin DNA invertase is a serine recombinase with a similar size
Selection of zinc finger protein domains necessary to bind to and organization as γδ-resolvase and Tn3-resolvase (Figures 3 and
enzyme target DNA sequences S3) (Grindley et al. 2006). The hyperactive Hin mutant H107Y, which
After identifying candidate CCR5 recombination sites and is able to catalyze efficient DNA inversions and deletions without
recombinase target sequences (see Supplementary Text), I evaluated the Fis regulatory protein, recombinatorial enhancer sequence,
each of the candidate recombination sites and chose the two sites or supercoiled DNA substrate required by the wild-type enzyme,
yielding the four enzyme target sequences with the most promising and was selected as the catalytic core (Sanders and Johnson 2004).
designed-zinc finger binding affinities and least competition (abil- Oligonucleotides (Integrated DNA Technologies, Coralville,
ity of other zinc fingers to bind to the sequences) (Segal et al. 1999; IA) encoding the designed zinc fingers were assembled and ligated
Dreier et al. 2001; Dreier et al. 2005). Recombination between the to generate DNA sequences encoding the respective zinc finger
www.thurj.org 57
Figure 9. Selection plasmid Rec-Chl-SitesY construct to facili-

tate backbone purification following HindIII and EcoRI digests.
Figure 11. Left: 1Kb+ DNA ladder image (Ingenesys, Co.). Right: Aga- Figure 10. Test constructs used to assay engineered chimeric recom-
rose gel depicting the presence or absence of PCR products, reflecting en- binase activity in E. coli for enzymes A (top) and B (bottom). Upon re-
zyme activity on target recombination sites or lack there-of. Lanes 1 and combinase-mediated inversion, primer 2 inverted and could form a PCR
15: 1Kb+ DNA Ladder. Lanes 2-4 and 9-11: PCR templates were plasmids product with primer 1.
with the correct target recombination sites but different chimeric recom-
binase zinc finger regions as controls. No product was expected. Lanes 5-8
and 12-15: PCR templates were plasmids with the correct recombination Recombination sites used in these assays had the following
sites and respective zinc finger regions. Expected products were ~770 base sequences:
pairs long for A and ~700 base pairs long for B. Site A: 5’-!"##$$#$$"""!!$#$!$!!$$$#$"$#"!""!""!!$#-3’
Site B: 5’-!!!!$$#$""""!!$#$!$!!$$$#$"$#"!$"!""####-3’
domains. These zinc fingers were cloned into Hin-H107Y hyperac- Site C: 5’-####"###$"""!!$#$!$!!$$$#$"$#"!"!!!$!!!!-3’
tive recombinase mutants, replacing their C-terminal domains, in Site D: 5’-$"$$!$#"$"""!!$#$!$!!$$$#$"$#"!"$!"#""$"-3’
a manner analogous to that reported by Stark and coworkers using For excision assays, both recombination sites were identical.
hyperactive mutant Tn3 resolvase. The flexible peptide sequence For inversion assays, the second recombination site was the reverse
linking the catalytic and engineered DNA-binding domain of the complement of the sequence listed above. Enzyme activity on target
most active reported chimeric recombinase was retained (Akopian recombination sites in E. coli was determined using PCR and gel
Cellular Biology
Molecular and
et al. 2003). These clones yielded chimeric recombinase enzymes electrophoresis (see Supplementary Text).
designed to target CCR5.
Generation of a selection system for directed evolution of
Assaying the activity of each engineered chimeric recombinase engineered chimeric recombinase enzymes
enzyme individually As enzymes capable of acting upon the plasmid encoding them,
Enzymes were assayed individually using substrate DNA plas- recombinases are suitable for substrate-linked protein evolution.
mids with target recombination sites containing a total of four Using a selection scheme similar to that demonstrated by Buchholz
identical zinc finger DNA binding sequences. Each enzyme acted and coworkers, albeit adapted to zinc finger-recombinase recog-
a homotetramer to carry out recombinase-mediated DNA inver- nition sequences, I constructed recombinase selection plasmids
sion or excision. This strategy allowed determination of the activity (Buchholz and Stewart 2001).
of each individual engineered recombinase enzyme, permitting I first crafted DNA plasmids containing a gene for a chimeric
individual optimization prior to testing the four variants together recombinase X (A, B, C, or D) as well as two recombination sites for
for heterotetramer activity on CCR5. recognition by chimeric recombinase Y (a different recombinase).
Figure 12. Test constructs used to assay engineered chimeric recombi-

nase activity in E. coli for enzymes C (top two plasmids) and D (bottom
two plasmids). Upon recombinase-mediated inversion one primer bind-
ing site was inverted, permitting PCR product formation.
By design, such plasmids would be stable and show no recombina-

tion. Pairings were as follows: recombinase A with recombination
sites C, recombinase B with recombination sites D, recombinase C
with recombination sites B, and recombinase D with recombina-
tion sites A. Figure 13. Agarose gel depicting the results of an enzyme activ-
ity assay for chimeras C and D. Lane 1: 1Kb+ DNA ladder. Lanes 3 and
The selection plasmid for a given recombinase enzyme con- 5: PCR on plasmid DNA collected from cells transformed with the proper
tained several restriction endonuclease sites flanked by recombinase recombination sites plasmid only. No products were expected. Lanes 4 and
recognition sites. Adjacent to this segment was the recombinase 6: PCR on plasmid DNA collected from cells transformed with both the
gene itself. Upon transformation into competent E. coli, plasmids proper recombination sites plasmid and recombinase plasmid. A 375-base
carrying an active recombinase library member would remove the pair long product was expected to result from recombinase-mediated in-
version and those bands are surrounded by a maroon box. All other bands
restriction endonuclease sites by recombinase-mediated excision. are unknown.
After DNA isolation and restriction digest, active library members
would remain uncut, while inactive members would be digested.
The subsequent amplification with PCR primers flanking the exci- Restriction digests cleave the zinc finger region, or chloram-
sion region and the recombinase gene should positively select for phenicol resistance gene, from the backbone vector (containing
library members active on the desired site. In the event of incom- the recombination sites and the Hin-H107Y recombinase catalytic
plete restriction endonuclease digests, a successfully recombined domain). Subsequent ligation reactions pair each recombinase zinc
product would still be distinguishable by size via gel electrophoresis, finger region with the catalytic domain on plasmids containing the
significantly reducing the background. appropriate recombination sites. The chimeric enzymes recom-
With these plasmids, I used HindIII and EcoRI restriction bine the plasmid DNA encoding them, excising a large fragment.
enzymes (New England Biolabs, NEB, Ipswich MA) to cleave the Therefore, active variants were identifiable as those with proper
zinc finger regions and therefore separate the DNA-binding domain DNA deletions. Chimeric recombinase libraries were created and
Cellular Biology
Molecular and
of recombinase X from the backbone containing sites Y. However, analyzed as described in the supplementary text.
only purification of the zinc finger region, not the backbone, was
feasible due to the similar sizes of digested, backbone DNA and
undigested vector.
To facilitate backbone gel-purification, I created four new plas- Results
mids expressing the comparatively large chloramphenicol resistance
gene in place of the short zinc finger region. This resulted in plas-
mids encoding a gene for the chimeric recombinase catalytic domain Engineered chimeric recombinase enzymes have activity on
fused to the chloramphenicol resistance gene. Like the zinc fin- target recombination sites
ger region, the resistance gene was flanked by HindIII and EcoRI After creating chimeric recombinase enzymes engineered to
restriction sites. These plasmids, too, would be unable to recombine bind to CCR5, I assayed the enzymes individually in E. coli for
due to the recombinase-chloramphenicol fusions and lack of zinc homotetramer activity on their respective sites. To evaluate the
finger DNA-binding domains. first two chimeras, A and B, I expressed the plasmids shown below:
www.thurj.org 59
Figure 14. Selection plasmid

RecX-SitesX (left) and RecX-
Library-SitesX (right) con-
structs resulting from cloning
X zinc finger region inserts into
backbone vector containing X
recombination sites.
Recombination sites on the plasmids were aligned for recombi- subjected to mutagenic PCR to introduce diversity. Ligations pro-
nase-mediated DNA inversion. To assay for enzyme activity, PCR duced RecX-SitesX plasmids or RecX-Library-SitesX plasmids,
reactions were performed using primers which bind in the same depicted below.
orientation unless recombinase enzymes can invert the segment The library of engineered zinc finger-recombinase enzyme
containing the one primer’s binding sequence. PCR product forma- mutants appeared to be of substantial size for all four enzyme librar-
tion was indicative of active recombinase enzymes as no product ies as determined by sample dilutions (Fig S7). Based on those
was expected from non-inverted substrates. plates, library sizes ranged from 107 to 108. Sixteen random library
As confirmed via sequencing, recombinase-mediated inversion members were sequenced to determine the mutation rate for each
occurred with engineered chimeric enzymes A and B. library, which appeared to be fairly low (Table S1).
To assess the activities of recombinases C and D, I transformed E.
coli with two plasmids instead of one. In this activity assay, substrate Library analysis
plasmids were separate from those that encoded the recombinase Libraries of engineered zinc finger-recombinase enzyme mutants
enzymes. The two sets of plasmids expressed for activity assays of were generated in order to isolate mutant enzymes with improved
chimeras C and D are depicted below: activity and specificity via iterative rounds of selection. Due to time
The activity assays described above support the conclusion constraints, here I report analyses of the initial libraries only, not
that I successfully conferred CCR5 binding specificity to the four those after multiple rounds of directed evolution. Further rounds
chimeric recombinase enzymes. All engineered enzymes were will be completed in the near future.
active on their target recombination sites. Activity levels cannot Recombinase-mediated excision on normal RecX-SitesX and
be directly compared because PCR amplification bias renders this RecX-Library-SitesX plasmids was assayed using PCR and gel elec-
a non-quantitative assay. Given that the four engineered chimeric trophoresis. Normal and library plasmid samples were analyzed
recombinases displayed some level of activity, all were suitable after either four or 21 hours in cells, and with or without restriction
candidates for directed evolution. endonuclease digestion to cleave substrates that did not recombine.
Results are shown below:
Successful enzyme library generation The above gels show that the original engineered recombinase
Following purification of insert X zinc finger regions and back- enzymes show some activity, and that library members may show at
bone vector containing X recombination sites, DNA was ligated least as much activity, if not more. Furthermore, PCRs performed
and transformed into cells. Inserts for library generation were first on restriction endonuclease-digested templates help bias the reac-
tions towards producing the shorter, recombined bands. Therefore,
digestion is an important step in the selection scheme, illustrat-
ing that recombinase activity levels are still quite low. Data also
Figure 15. Agarose gel de- show that recombination events were significantly more prevalent
picting PCRs on normal
when plasmids had been in cells for 21 hours as opposed to only
Cellular Biology
RecX-SitesX plasmids and

Molecular and
RecX-Library-SitesX plasmids for four hours. Finally, excision catalyzed by engineered recombi-
after four hours in E. coli cells nases appears to be sequence specific; RecX-SitesY plasmids do not
but without restriction en- recombine (Figure 16).
donuclease digestion. Lanes
1 and 10: 1Kb+ DNA lad-
der. Lanes 2-5: RecA-SitesA,
Selection of active library members and subsequent selected
RecB-SitesB, RecC-SitesC, and library analysis
RecD-SitesD. Lanes 6-9: RecA- Active recombinase enzyme sequences were selected via gel
Library-SitesA, RecB-Library- purification of the recombined, 1017-base pair long band. Zinc finger
SitesB, RecC-Library-SitesC, regions were amplified by PCR, cleaved with restriction enzymes,
and RecD-Library-SitesD. Un-
recombined plasmids are 2037
and cloned back into the proper vector backbone. For each library,
base pairs long, while recom- 16 of these selected RecX-Library-SitesX members were compared
bined plasmids are 1017 base to two normal RecX-SitesX plasmids in PCR-based recombination
pairs long. assays. All were sequenced to aid in the categorization of interesting
Figure 16. Agarose gels depicting PCRs on normal RecX-SitesX plasmids

and RecX-Library-SitesX plasmids after four hours in E. coli cells and after Figure 17. Agarose gel depicting PCRs on normal RecX-SitesX plasmids
restriction endonuclease digestion. Left lanes 1 and 6: 1Kb+ DNA ladder. and RecX-Library-SitesX plasmids after 21 hours in E. coli cells but with-
Left lanes 2-5: RecA-SitesA, RecB-SitesB, RecC-SitesC, and RecD-SitesD. out restriction endonuclease digestion. Lanes 1 and 10: 1Kb+ DNA lad-
Right lanes 1 and 6: 1Kb+ DNA ladder. Right lanes 2-5: RecA-Library-Site- der. Lanes 2-5: RecA-SitesA, RecB-SitesB, RecC-SitesC, and RecD-SitesD.
sA, RecB-Library-SitesB, RecC-Library-SitesC, and RecD-Library-SitesD. Lanes 6-9: RecA-Library-SitesA, RecB-Library-SitesB, RecC-Library-Si-
Un-recombined plasmids are 2037 base pairs long, while recombined plas- tesC, and RecD-Library-SitesD. Un-recombined plasmids are 2037 base
mids are 1017 base pairs long. pairs long, while recombined plasmids are 1017 base pairs long.
mutants. As these variants were the result of only a single diversifi- To perform directed evolution experiments, all that is required
cation and selection round, however, this selected library pool was is initial activity, regardless of how minimal. As all four candi-
not expected to significantly exceed normal enzyme activity. Addi- dates satisfied this requirement, I was able to develop and validate
tionally, the gels below are not quantitative; a darker band may be a substrate-linked protein evolution selection scheme to select for
attributed to differing enzyme activity, PCR bias or a greater amount variants with improved enzymatic activity and specificity.
of DNA being loaded into the lane. However, to make samples as I was unable to perform iterative rounds of selection due to time
comparable as possible, the PCR reactions were completed after constraints, but the analyses of my initial libraries showed promise.
only 20 amplification cycles. Ranging from 107 to 108 in size, recombinase enzyme libraries were
The supplementary material contains gel and sequence analyses large, although mutation rates per base pair within the mutated zinc
that are representative of the first round of directed evolution. The finger region were rather low at less than 0.5%. As a whole, library
data are presented at this time as an illustration of the current state members from this first round of mutagenesis appeared to be more
of the project. active than the starting pool; this observation was particularly
pronounced with library A, which was more active to start.
After selection of successful recombinants, I compared the activ-
ity of 16 selected variants to normal, non-mutagenized starting pool
Discussion enzymes. Both starting pool and library members demonstrated
successful recombination, although I did not identify any mutants
that were definitively more active than normal variants. This is
Significance of results no surprise, given that only 16 selected variants from each library
My experimental results demonstrate that chimeric recombinase were analyzed, and all were the result of just one round of selection.
Cellular Biology
Molecular and
enzymes can be successfully engineered to recognize target sites, Within the libraries, there appears to be significant background
using modular zinc fingers constructed as described by Barbas III recombinase activity resulting from normal, non-mutagenized
and coworkers (Segal et al. 1999; Dreier et al. 2001; Dreier et al. variants as many of the selected library members had no mutations
2005). Furthermore, the enzymes appear to only recombine their in the zinc finger region.
specific targets; they have not demonstrated promiscuity in my
analyses using RecX-SitesY plasmids. Upcoming experiments
One notable limitation of the presented data is the absence of Given the consistent activity of the chimeric enzymes as origi-
quantification. PCR bands visualized on agarose gels indicate the nally designed, in subsequent experiments, I will need to give the
presence or absence of recombination events, but tell little about enzymes significantly less time in cells to enrich for the most active
what fraction was recombined. Based on the data, it appears that variants. This strategy will be adopted with all forthcoming experi-
in all cases only a small portion of substrate DNA was successfully ments. For library enrichment, I will isolate active mutants, clone
recombined; quantitative PCR reactions could be done to verify them into backbone vector, give them two hours to recombine,
this estimate. and repeat. Following two rounds of enrichment, I will amplify
www.thurj.org 61
Figure 18. Agarose gel depicting PCRs on normal RecX-SitesX plasmids

and RecX-Library-SitesX plasmids after 21 hours in E. coli cells and after
restriction endonuclease digestion. Lanes 1, 10, and 15: 1Kb+ DNA lad-
der. Lanes 2-5: RecA-SitesA, RecB-SitesB, RecC-SitesC, and RecD-SitesD.
Lanes 6-9: RecA-Library-SitesA, RecB-Library-SitesB, RecC-Library-
SitesC, and RecD-Library-SitesD. Lanes 11-14: Rec-Chl-SitesA, Rec-Chl-
SitesB, Rec-Chl-SitesC, and Rec-Chl-SitesD after restriction endonuclease
digestion serve as the negative controls. Un-recombined plasmids are 2037
base pairs long, while recombined plasmids are 1017 base pairs long.
selected library members via mutagenic PCRs and perform itera-

tive rounds of directed evolution, adding additional enrichment
steps in between.
If necessary, negative selection against a particular undesired
target site can be achieved by incorporating “false” recognition sites,
differing slightly from the correct site, into the selection plasmids.
Recombination using “false” sites surrounding a PCR primer bind-
ing site would prevent amplification of less-specific recombinase
variants, allowing selection against promiscuous variants.
After evolving highly-effective copies of the four zinc finger-
recombinase enzymes individually, I will verify their concerted
activity on the CCR5 gene sequence in E. coli to determine if the
enzymes can effect a CCR5 deletion. I have already constructed a Figure 19. Agarose gel depicting PCRs on RecX-SitesY plasmids after
plasmid designed to assay activity in bacteria by cloning the CCR5 restriction endonuclease digestion as negative controls. Lanes 1 and 6:
gene into a bacterial vector. The four evolved recombinase genes will 1Kb+ DNA ladder. Lanes 2-5: RecA-SitesC, RecB-SitesD, RecC-SitesB,
be cloned onto one expression plasmid to facilitate transformation and RecD-SitesA. Un-recombined plasmids measure 2037 base pairs long,
into cells containing the CCR5 test plasmid. Only cells containing while recombined plasmids measure 1017 base pairs long.
both constructs will be selected. A restriction endonuclease digest
of the CCR5-containing plasmid will enable detection of the proper cells harboring a CCR5 deletion.
deletion via gel electrophoresis, with the relative band intensities
yielding an estimate of excision efficiency (Thomson and Ow 2006). Engineered recombinase enzymes as gene therapy agents
Even more accurate quantification of activity could be achieved conferring protection from HIV-1
by assays in vitro. To quantitatively evaluate tetramer activity in The deletion in CCR5 created by the engineered recombinase
vitro, recombinase enzymes could be purified and mixed with the enzymes is expected to be as effective as the natural Δ32 deletion
substrate selection vector. DNA after various time periods could be conferring HIV-1 resistance because the enzymes will generate a
digested with restriction endonucleases and electrophoresed on aga- truncated protein in an analogous manner. Although a number of
rose gels to detect the proper deletion. This assay would illuminate amino acids are added to CCR5 prior to the frameshift-induced stop
the time-frame and efficiency of recombinase-mediated excision. codon, they are not especially hydrophobic. There is no reason to
Should only weak recombinase-mediated excision of CCR5 be expect that they would allow efficient CCR5 membrane insertion,
observed, further directed evolution will be performed to select but this requires verification in human cells. CCR5 Δ32 and the
for stronger activity of the enzyme tetramer. The method will be recombinase-treated protein version should be directly compared
similar to before, using restriction endonucleases that will cut at in human cells to determine if the mutation was properly emulated
Cellular Biology
Molecular and
sites inserted in the middle of CCR5. and resistance to HIV-1 conferred.

Following successful CCR5-segment excision in bacteria and in The development of safe, efficient recombinase enzymes for
vitro, in vivo assays will be conducted with human cells. Given the genome modification would represent a significant step towards
results of Barbas III and coworkers, there is reason to expect some gene therapy; however, it is only one component of a successful
degree of activity in human cells (Gordley et al. 2007). An empty treatment. A method for safe and reliable enzyme delivery to the
selection vector modified for human cells will be co-transfected appropriate cells is equally necessary and one being pursued by
into human cells with a vector containing all four recombinase numerous researchers (Lu et al. 2004; S. D. Li and L. Huang 2006).
genes, downstream of mammalian promoters. Both vectors will At present, there are few genome modification methods with
also encode different fluorescent protein genes, enabling isolation the potential to generate a precise deletion in a gene such as CCR5.
of co-transfected cells by fluorescence-activated cell sorting (FACS). To date, the only enzymes capable of performing such a dele-
After fixed time intervals, co-transformants will be selected via tion are zinc finger nucleases (Perez et al. 2008), which have been
FACS and their DNA extracted. Recombinase activity in human demonstrated on this specific target. However, their potential for
cells will be assayed using qPCR to detect the percent of transfected development into an efficacious therapy is doubtful due to the
necessity of generating a double-strand break in the target cell, Nile virus infection. J Exp Med, 203(1), 35-40.
which leads to non-homologous end-joining and the potential for Gordley, R.M. et al., 2007. Evolution of programmable zinc finger-recombinases
deleterious and possibly carcinogenic chromosomal rearrangements with activity in human cells. J Mol Biol, 367(3), 802-13.
in at least a fraction of the modified cells (van Gent et al. 2001). Grindley, N.D., Whiteson, K.L. and Rice, P.A., 2006. Mechanisms of Site-
While very effective in modifying cells in culture, which could Specific Recombination. Annu Rev Biochem.
conceivably be cultured and transferred to the patient, this therapy Huang, Y. et al., 1996. The role of a mutant CCR5 allele in HIV-1 transmission
is unlikely to be suitable for direct delivery. As such, the develop- and disease progression. Nat Med, 2(11), 1240-3.
ment of safe and specific enzymes for direct gene modification in Hutter, G. et al., 2009. Long-Term Control of HIV by CCR5 Delta32/Delta32
patients remains an open problem. Stem-Cell Transplantation. N Engl J Med, 360(7), 692-698.
A more ideal method, suitable for delivery of engineered recom- Ingenesys, Co., 1Kb+ DNA Ladder. Available at: http://www.ingenesys.co.kr/
binase enzymes, is direct viral vector injection. For example, the product_data/solgent/c_0203_4.gif [Accessed April 6, 2009].
HIV-1-based lentivirus vector VRX496 might be modified to deliver Janeway, C. et al., 2008. Janeway’s immunobiology 7th ed., New York, NY:
the engineered chimeric recombinase genes (encoded with a viral or Garland Science.
human cell promoter) (Lu et al. 2004). This method would naturally Lama, J. and Planelles, V., 2007. Host factors influencing susceptibility to HIV
target all of the relevant immune cells, delivering the recombinase infection and AIDS progression. Retrovirology, 4, 52.
therapy precisely where it is needed. Li, S.D. and Huang, L., 2006. Gene therapy progress and prospects: non-viral
The purpose of this protein engineering research is to advance gene therapy by systemic delivery. Gene Ther, 13(18), 1313-9.
efforts to prevent HIV-1 infection; however, the project only Li, W. et al., 2005. Structure of a synaptic gammadelta resolvase tetramer cova-
addresses the gene-modification aspect of gene therapy—one of lently linked to two cleaved DNAs. Science, 309(5738), 1210-5.
many important challenges. Engineered chimeric recombinase Lu, X. et al., 2004. Antisense-mediated inhibition of human immunodeficiency
enzymes show promise towards the generation of an HIV-1 gene virus (HIV) replication by use of an HIV type 1-based vector results in
therapy that would be safe for use with direct delivery. Therefore, severely attenuated mutants incapable of developing resistance. J Virol,
such a therapy could be suitable for treatment of individuals in 78(13), 7079-88.
developing countries, where a more involved gene delivery method Matthews, J.M. and Sunde, M., 2002. Zinc Fingers--Folds for Many Occasions.
is not a feasible option and where need for HIV-1 protection is dire. IUBMB Life, 54(6), 351-355.
Mecsas, J. et al., 2004. Evolutionary genetics: CCR5 mutation and plague pro-
tection. Nature, 427(6975), 606.
Moreno, C. et al., 2005. CCR5 deficiency exacerbates T-cell-mediated hepatitis
References in mice. Hepatology, 42(4), 854-62.
O’Brien, S.J. and Moore, J.P., 2000. The effect of genetic variation in chemo-
Agrawal, L. et al., 2007. CCR5{Delta}32 Protein Expression and Stability Are kines and their receptors on HIV transmission and progression to AIDS.
Critical for Resistance to Human Immunodeficiency Virus Type 1 In Vivo. Immunological reviews, 177, 99.
J. Virol., 81(15), 8041-8049. Oh, D. et al., 2008. CCR5Δ32 Genotypes in a German HIV-1 Seroconverter
Akopian, A. et al., 2003. Chimeric recombinases with designed DNA sequence Cohort and Report of HIV-1 Infection in a CCR5Δ32 Homozygous Indi-
recognition. Proc Natl Acad Sci U S A, 100(15), 8688-91. vidual. PLoS ONE, 3(7), e2747.
Allen, S.J., Crown, S.E. and Handel, T.M., 2007. Chemokine: receptor structure, Pavletich, N.P. and Pabo, C.O., 1991. Zinc finger-DNA recognition: crystal
interactions, and antagonism. Annu Rev Immunol, 25, 787-820. structure of a Zif268-DNA complex at 2.1 A. Science, 252(5007), 809-17.
Berger, E.A., Murphy, P.M. and Farber, J.M., 1999. Chemokine receptors as Perez, E.E. et al., 2008. Establishment of HIV-1 resistance in CD4+ T cells by
HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev genome editing using zinc-finger nucleases. Nat Biotechnol, 26(7), 808-16.
Immunol, 17, 657-700. Premack, B.A. and Schall, T.J., 1996. Chemokine receptors: gateways to inflam-
Buchholz, F. and Stewart, A.F., 2001. Alteration of Cre recombinase site specific- mation and infection. Nature Medicine, 2(11), 1174-1178.
ity by substrate-linked protein evolution. Nat Biotechnol, 19(11), 1047-52. Ramirez, C.L. et al., 2008. Unexpected failure rates for modular assembly of
Carrington, M. et al., 1997. Novel alleles of the chemokine-receptor gene CCR5. engineered zinc fingers. Nat Meth, 5(5), 374-375.
Am J Hum Genet, 61(6), 1261-7. Sanders, E.R. and Johnson, R.C., 2004. Stepwise dissection of the Hin-catalyzed
Carroll, D., 2008. Progress and prospects: zinc-finger nucleases as gene therapy recombination reaction from synapsis to resolution. J Mol Biol, 340(4),
agents. Gene Ther, 15(22), 1463-8. 753-66.
Cellular Biology
Molecular and
Cathomen, T. and Joung, J.K., 2008. Zinc-finger nucleases: the next generation Segal, D.J. et al., 1999. Toward controlling gene expression at will: selection
emerges. Mol Ther, 16(7), 1200-7. and design of zinc finger domains recognizing each of the 5’-GNN-3’ DNA
Company, B.B., 2007. The Process of HIV Infection, BBC. target sequences. Proc Natl Acad Sci U S A, 96(6), 2758-63.
Dreier, B. et al., 2001. Development of zinc finger domains for recognition of de Silva, E. and Stumpf, M.P., 2004. HIV and the CCR5-Delta32 resistance
the 5’-ANN-3’ family of DNA sequences and their use in the construction allele. FEMS Microbiol Lett, 241(1), 1-12.
of artificial transcription factors. J Biol Chem, 276(31), 29466-78. Singh, H. et al., 2008. CCR5-Delta32 polymorphism and susceptibility to cer-
Dreier, B. et al., 2005. Development of zinc finger domains for recognition of vical cancer: association with early stage of cervical cancer. Oncol Res,
the 5’-CNN-3’ family DNA sequences and their use in the construction of 17(2), 87-91.
artificial transcription factors. J Biol Chem, 280(42), 35588-97. Stephens, J.C. et al., 1998. Dating the origin of the CCR5-Delta32 AIDS-
van Gent, D.C., Hoeijmakers, J.H.J. and Kanaar, R., 2001. Chromosomal sta- resistance allele by the coalescence of haplotypes. Am J Hum Genet, 62(6),
bility and the DNA double-stranded break connection. Nat Rev Genet, 1507-15.
2(3), 196-206. Thomson, J.G. and Ow, D.W., 2006. Site-specific recombination systems for
Glass, W.G. et al., 2006. CCR5 deficiency increases risk of symptomatic West the genetic manipulation of eukaryotic genomes. Genesis, 44(10), 465-76.
www.thurj.org 63
8JUIQBSUJDVMBSUIBOLTUP%FBO'MJFS %S'SFFENBO 1SPWPTU)ZNBO
'BDVMUZPG"SUTBOE4DJFODFT '"4
%FBO4NJUI BOE'"4%FBOGPSUIF
1IZTJDBM4DJFODFT+FSFNZ#MPYIBNGPSUIFJS
JOWBMVBCMFTVQQPSUJOQVCMJTIJOHUIJTJTTVF

THURJ Vol. 3 Issue 1

Загружено:

Сведения о документе

Исходное описание:

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

THURJ Vol. 3 Issue 1

Загружено:

Авторское право:

Доступные форматы

9RO,VVXH6SULQJ

Dear Harvard Community:

The Harvard Undergraduate Research Journal

Dear Harvard Community,

10 Organismic and Evolutionary Biology

12 A review of Richard Dawkins’s latest

14 Developments in reprogramming cell

17 Harvard professors develop new tools

20 At the heart of an effort to merge

Managing Editor of Content Executive Board Design Chair

Boards Faculty Advisory Board

8 The Harvard Undergraduate Research Journal

a personal connection, and as a professor at Harvard, he

elementary schools to the sole medical school in the coun-

Photos courtesy of Hopi Hoekstra

H opi Hoekstra likes mice. A professor in Harvard’s

Photos courtesy of Hopi Hoekstra

Illustration from Wikimedia Commons

16 The Harvard Undergraduate Research Journal

A lthough not completely accepted

global climate is fast becoming chlorofluorocarbons (the main

default rate on a mortgage is to the financial structure. It What Now?

The Synergistic Combination of

at the HARVARD CLINICAL

C ollaborative efforts between the Harvard Medi-

One night in the spring of 2007, in the fluorescent white confines of

To the co-founders of The Harvard Undergraduate Research Journal,

2007 2008 2009

Spring 2008 Spring 2009

24 The Harvard Undergraduate Research Journal

Fall 2009 February 2010

September 2009 October 2009 Spring 2010

Social stress and scapegoatism:

Introduction This paper analyzes scapegoatism from the perspective of the

values Scapegoat Model

t It is bounded Simulation Results and Analysis

13 This is a simplification made to avoid issues with members whose social

yields a statistically significant result at the 5% significance level23

Distribution of Social Values p n ^

The effect of a leader in the social group28

23 See Appendix B for the full statistical test.

a scapegoat event may decrease social value of the group as a whole,

Social role and scapegoatism

32 The Harvard Undergraduate Research Journal

Circulating hormone adrenomedullin and

The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA

acids, acts as a circulating hormone to induce various biological

of medium per well, followed by 5 min shaking. Subsequently, 50 µl

34 The Harvard Undergraduate Research Journal

5% nonfat dry milk in 10 mM Tris-HCl with 0.1% Tween-20, pH 7.5 Results

Acknowledgements cell death, Nat. Cell Biol. 9 (2007) 550-555.

40 The Harvard Undergraduate Research Journal

SQIF setup for measurements of extremely

Physics Department, Stony Brook University

can be used in this situation is a Superconducting Quantum Inter-

Figure 2. Conventional sine-wave modulation characteristics of: (A) DC

42 The Harvard Undergraduate Research Journal

Figure 4. SQIF mounted on the chip holder.

is not enough for monitoring the magnetic field in the vicinity of

Figure 7. Simulated modulation characteristics for best bias

Figure 9. SQIF modulation characteristics for positive and nega-

Figure 10. The shape of SQIF’s positive and negative minima.

estimation of the shielding factor we have recorded the response of

9RO,VVXH6SULQJ