THURJ

The Harvard Undergraduate Research Journal

Vol. 2 Issue 2 Fall 2009

A Renaissance Man of
the Genomics Era p. 13
Do you know your own
mind? p. 16
Victory by Association:
DNA Measuring Coattail
Glycosylase: Effects p. 26
Still Images of a Variations in HIV-1
Motion Picture p. 55 Epitope Production p.45
Volume 2 Issue 2 | Fall 2009 LETTERS

HARVARD COLLEGE

EVELYNN M. HAMMONDS UNIVERSITY HALL, FIRST FLOOR

DEAN OF HARVARD COLLEGE CAMBRIDGE, MASSACHUSETTS 02138
BARBARA GUTMANN ROSENKRANTZ TELEPHONE (617) 495-1560 FAX (617) 496-8268
PROFESSOR OF THE HISTORY OF SCIENCE EVELYNN_HAMMONDS@HARVARD.EDU
AND OF AFRICAN AND AFRICAN AMERICAN STUDIES

November, 2009

Dear Harvard Community,

November 11, 2009
WithHarvard
Dear this issueCommunity,
of the Harvard Undergraduate Research Journal we continue to see the impressive
scientific accomplishments of Harvard College students.

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intellectual individuality Journal show that our
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to engage with faculty in across a
wide variety of disciplines. They are also building and sustaining their own scientific communities
cutting edge research is one of the highest priorities of the College.
while engaged in this work. I look forward to many more issues of this outstanding journal.
The articles in this issue of the Harvard Undergraduate Research Journal show
Sincerely, undergraduates are already making impressive contributions to new scientific
that our
knowledge across a wide variety of disciplines. They are also building and sustaining
their own scientific communities while engaged in this work. I look forward to many
more issues of this outstanding journal.
Evelynn M. Hammonds, PhD
Sincerely,
Barbara Gutmann Rosenkrantz Professor of the History of Science and of African and African
American Studies &
Dean of Harvard College
Evelynn M. Hammonds, PhD
Barbara Gutmann Rosenkrantz Professor of the History of Science and of African and
African American Studies &

Dean of Harvard College

www.thurj.org 1
LETTERS Volume 2 Issue 2 | Fall 2009

The Harvard Undergraduate Research Journal

November, 2009

Dear Harvard Community,

It is with great pleasure that we present to you the fourth issue of The Harvard Undergraduate
Research Journal (THURJ), which showcases undergraduate work in the natural and social sciences.
This issue represents our continued commitment to original intellectual pursuit as part of the
undergraduate experience at Harvard. We are proud to spotlight research that spans topics across the
academic spectra, from economics to public health to molecular biology. This year, our prize-winning
research article from Oluwatobi Ogbechie investigates peptide degradation in certain immune cell
types in response to HIV-1 infection, which has implications for the way our immune system targets
and clears infected cells.

In addition to highlighting undergraduate research, our staffers also bring science endeavors in
our community to the forefront. In this issue our feature writers report on the Concord Field Station’s
incredible work on the mysteries of navigational motion in everything from mammals to insects. We
look into the way George Church’s projects in genomics will affect our lives, the way our own Program
for Research in Science and Engineering (PRISE) affects the lives of undergraduate researchers, and
the way a web application reveals our the demographic biases of our society. We hope these articles

As always, this issue has been made possible by the dedicated work of our staff, and they cannot go
unmentioned. We thank our peer reviewers, Harvard faculty, graduate students, and associates who
reviewed submissions to ensure this publication’s scientific and written caliber. Moreover, our issue
could not have been possible without our staffers on the Content, Design, Business, and Social and
Public Relations Boards. A special thanks goes out to Harvard College Dean Evelynn Hammonds,
HMS Dean Jeffrey Flier, Professor Steven Freedman, Provost Steven Hyman, FAS Dean Michael Smith,
Harvard, FAS Dean for the Physical Sciences Jeremy Bloxham, Harvard University, and Harvard
College for their unending support . And thanks to you, the reader, whom we hope to delight and
fascinate as you flip through the following pages. We hope you enjoy the read!

Sincerely,

John Mei Lisa Rotenstein

Co-Editor-in-Chief Co-Editor-in-Chief

2 The Harvard Undergraduate Research Journal

 Contents
Features
6 How do animals gallop, jump, and fly with such
Research
18 Measuring the immune system’s response to HIV
speed and skill? Learn how one team of scientists are with peptidases.
trying to unravel the secrets behind motion. Tobi Ogbechie ‘09
Sarah Zhang ‘11
28 Victory by ‘coattails’: Hidden influences in
10 Students spend a summer “getting their science Congress on critical election years.
on” as PRISE research fellows. S. Travis May ‘09
Alissa D’Gama ‘11
34 Salt of survival: Startling information on iodine
13 Putting your DNA on the web: How one man shortages in India.
envisions the future of genomics. Shubha Bhat ‘09
Isha Jain ‘12
39 New technology allows scientists to ‘see’ odors as
16 Are we more open minded than our parents? they are processed in the brain.
Uncovering hidden biases in the mind. David Blauvelt ‘09
Jen Gong ‘12
47 Two proteins that regulate neuron growth offer
clues on brain development.
Stephanie Mok ‘09

55 Snapping stills of DNA repair.

Kimberly Oo ‘09

:
edit
a g e cr kwith
Im r Bec
Noo
CONTENTS Volume 2 Issue 2 | Fall 2009

Managing Editor of Content Executive Board Design Chair

Fernando Racimo ‘11 Co-Editors-in-Chief Francis Deng ‘12
Managing Editor of Peer Review and Lisa Rotenstein ‘11 and John Mei ‘12 Manager for Social and Public
Submissions Business Manager Relations
John Liu ‘11 Tengbo Li ‘12 Hyunje Grace Cho ‘12

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
Eric Chen ‘12 - Associate Manager Biology Biology
Varun Bansal ‘13 Paul Bamberg, Ph.D Efthimios Kaxiras, Ph.D
Anne Polyakov ‘12 Senior Lecturer on Mathematics Gordon McKay Professor of Applied Physics
Jeanine Sinanan-Singh ‘13 Michael Brenner, Ph.D and Professor of Physics
Roy Zhang ‘13 Glover Professor of Applied Mathematics and George Lauder, Ph.D
Applied Physics Professor of Biology and Alexander Agassiz
Content Myron Essex, D.V.M., Ph.D Professor of Zoology
Alissa D’Gama ‘11 - Associate Manager Mary Woodard Lasker Professor of Health Richard Losick, Ph.D
Sophie Wharton ‘11 - Associate Manager Sciences in the Faculty of Public Health Maria Moors Cabot Professor of Biology
Jen Jian Gong ‘12 Brian Farrell, Ph.D L. Mahadevan, Ph.D
Isha Jain ‘12 Professor of Biology Lola England Professor of Applied
Sarah Zhang ‘11 Jeffrey Flier, M.D. Mathematics
Dean, Harvard Medical School, and George C. David Mooney, Ph.D
Peer Review and Submissions Reisman Professor of Medicine Gordon McKay Professor of Bioengineering
Meng Xiao He ‘11 - Associate Manager Nicole Francis, Ph.D Hongkun Park, Ph.D
Monica Liu ‘12 - Associate Manager Associate Professor of Molecular and Cellular Professor of Chemistry and of Physics
Charlotte Seid ‘10 - Associate Manager Biology Steven Pinker, Ph.D
Jessica Zeng ‘12 - Associate Manager Steven Freedman, M.D., Ph.D Johnstone Family Professor of Psychology
Helen Yang ‘11 - Head Copy Editor Associate Professor of Medicine Tobias Ritter, Ph.D
Lisa Chen ‘12 - Copy Editor Robin Greenwood, Ph.D Assistant Professor of Chemistry and Chemical
Darius Li ‘12 - Copy Editor Associate Professor of Business Administration Biology
Jacob Cedarbaum ‘12 Guido Guidotti, Ph.D Eugene Shakhnovich, Ph.D
Andrew Chen ‘12 Higgins Professor of Biochemistry Professor of Chemistry and Chemical Biology
Eric Chen ‘12 David Haig, Ph.D Irwin Shapiro, Ph.D
George Putnam Professor of Organismic and Timken University Professor
Sway Chen ‘12
Evolutionary Biology Zhigang Suo, Ph.D
Francis Deng ‘12
Marc Hauser, Ph.D Allen E. and Marilyn M. Puckett Professor of
Ben Dobkin ‘12 Professor of Psychology Mechanics and Materials
Eva Gillis-Buck ‘12 Dudley Herschbach, Ph.D David Weitz, Ph.D
Jen Gong ‘12 Frank B. Baird Jr. Professor of Science Mallinckrodt Professor of Physics and of
Johnny Hu ‘11 John Hutchinson, Ph.D Applied Physics
Edward Kogan ‘12 Abbott and James Lawrence Professor of
Shravani Mikkilineni ‘12 Engineering and Gordon McKay Professor of
Briana Prager ‘12 Applied Mechanics
Nicholas Tan ‘12
Jacob Weatherly ‘12
Ke Xu ‘11 Faculty Reviewers
Vanisha Yarbrough ‘10 Charles Stiles, Ph.D R. Paul Johnson, M.D.
Chi Zhang ‘12 Professor of Microbiology and Molecular Associate Professor of Medicine
Genetics Filipe Campante, Ph.D
Design Farhan Ali, BS Assistant Professor of Public Policy
Ritchell van Dams ‘11 - Associate Chair Ph.D Candidate in Organismic and David Fisher, M.D., Ph.D,
Ryan Neff ‘13 - Associate Chair Evolutionary Biology Margaret M. Dyson Professor of Pediatrics
Amanda Lu ‘13 Sandeep Robert Datta, M.D., Ph.D, Anthony Letai, M.D., Ph.D
Esther Moon ‘12 Postdoctoral Fellow - Axel Lab Assistant Professor of Medicine
Allen Shih ‘13 Frank Wensheng Fan, Ph.D Paul Moorcroft, Ph.D
Shelun Tsai ‘13 Program Coordinator - Harvard School of Professor of Organismic and Evolutionary
Public Health China Initiative Biology
Rachelle Gaudet, Ph.D Karim Kassam, MSc
Social and Public Relations Associate Professor of Molecular and Cellular Ph.D Candidate in Psychology
Janet Song ‘13 - Associate Manager Biology Nancy Lou Conklin-Brittain, Ph.D
Preya Shah ‘13 - Associate Manager Amitinder Kaur, M.D. Lecturer on Human Evolutionary Biology
Caroline Huang ‘13 Assistant Professor of Medicine Guy Crosby, Ph.D
Shannon Purcell ‘12 Daniel Kavanagh, Ph.D Associate Professor of Nutrition
Jeanine Sinanan-Singh ‘13 Instructor in Medicine

The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009
Contact
General: contact@thurj.org
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Subscribing: subscriptions@thurj.org
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Website: http://www.thurj.org
Copyright 2009 The Harvard
Undergraduate Research Journal.
No material appearing in this publication
may be reproduced without written
permission of the publisher, with the
exception of the rights of photographs
which may only be granted by the
photographer. The opinions expressed
in this magazine are those of the
contributors and are not necessarily
shared by the editors. All editorial rights
are reserved.
www.thurj.org
CONTENTS
About Us
The Harvard Undergraduate Research Journal (THURJ) showcases
peer-reviewed undergraduate student research from all science and
quantitative social science disciplines. As a biannual publication,
THURJ familiarizes students with the process of manuscript
submission and evaluation. Moreover, it provides a comprehensive
forum for scientific discourse on the cutting-edge research that impacts
our world today.
At its core, THURJ allows students to gain insight into the peer
review process, which is central to modern scientific inquiry. All
THURJ manuscripts are rigorously reviewed by the Peer Review Board
(consisting of Harvard undergraduates), and the top manuscripts
that they select are further reviewed by Harvard graduate students,
post-doctoral fellows, and professors. This process not only stimulates
faculty-student collaboration and provides students with valuable
feedback on their research, but also promotes collaboration between
the College and Harvard’s many graduate and professional schools. In
addition to publishing original student research papers, THURJ is also
an important medium for keeping the Harvard community updated on
science research-related news and developments.
FEATURE Volume 2 Issue 2 | Fall 2009

On the
Move:
Move:
Move:
Biomechanics at the
Concord Field Station
MCZ’s oversized collections. The field station’s ample
size—65 acres adjoining Harvard’s 650 acre Estabrook
Woods—gives the scientists enough lab space to con-
struct wind tunnels and outdoor insect habitats. Re-
search at the field station focuses on the biomechanics
of animal movement, which explains the presence of
the rather exotic emu in Bedford, Massachusetts.

From Missile Silo to Zoological Lab

The facilities of the Concord Field Station origi-
nally belonged to a missile base built during the Cold
War. When Harvard University acquired the land and
surrounding woods in 1966, the field station was born.
“Until recently there was a whole question of whether
missiles were ever actually at this base,” says Biewener,
the Lyman Professor of Biology and Director of the
Concord Field Station, as he pulled up an aerial photo-
graph to prove that there were indeed missiles here.
The base’s buildings have been repurposed for
science: the barracks became the field station’s main
By Sarah Zhang ‘11 building, housing offices, an animal surgery room, ani-
Photography by Noor Beckwith ‘11 mal care facilities, and, closer to the building’s original
purpose, a small apartment for visiting researchers. Re-

H
arvard may not boast the reputation of “Ani- minders of the field station’s past are most apparent in
mal House”, but just seventeen miles away the underground bunkers now used as storage. Inside
from campus, the Concord Field Station the bunkers, equipment originally used to hoist mis-
could quite literally take the name. The research facil- siles above ground is still visible amidst the dozens of
ity in Bedford is home to animals ranging from guinea whale, dolphin, and porpoise skeletons that make up
fowls to frogs to African pygmy goats. A retired emu— the MCZ’s oversized cetacean collection. It is obvious
the last from a former study on emu locomotion—still why the whale skeletons, from whales that had beached
struts its stuff behind the field station’s main office. themselves, have to be stored off-site: the jawbone of
Affiliated with Harvard’s Museum of Comparative each whale easily surpasses the height of a grown man.
Zoology (MCZ) and the Department of Organismic More interesting than the skeletons are the live an-
and Evolutionary Biology, the Concord Field Station imals that inhabit the field station. The late C. Richard
accommodates the labs of Professors Andrew Biewener Taylor, Biewener’s predecessor and founder of the Con-
and Stacey Combes and serves as storage space for the cord Field Station, was a pioneer in the field of animal
locomotion. It was under his direction that the field
6 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
station once housed kangaroos, wal-
labies, antelope, llamas, wolves, coy-
otes, ponies, and chimpanzees for
the purpose of studying locomotion.
The original treadmill from Taylor’s
days in the 1970s—the first treadmill
to have force plates embedded in it
and the first to be used by a kan-
garoo—is still used in locomotion
studies on larger animals, including
goats and humans.
Lights, Camera, Flight
Today, Biewener’s research
interests are primarily focused on
smaller animals, particularly on avi-
an flight. When he came to Harvard
from the University of Chicago in
1999, a new wind tunnel, essentially
an aerial treadmill, was constructed
to study birds in flying. The birds,
usually cockatiels, are released and
photographed in the wind tunnel,
which can generate winds up to 65
miles per hour.
Although the wind tunnels and
treadmills have factored extensively
www.thurj.org
into research at the field station,
Biewener stresses the importance of
studying animal movements in more
natural environments. The repetitive
walking motion on a treadmill, after
all, is quite different from navigating
a rocky, winding path, and the same is
true of the constant speeds in a wind
tunnel versus natural flight. One of
the ongoing projects in Biewener’s
lab studies the take-off and landing
of birds, in which case wind tunnels
are not very useful. Instead, doves are
set up indoors to fly back and forth,
and the displacement of air is tracked
by laser imaging. In the same
way that sunlight reveals
dust in the air, laser lights up
a thin sheet of air, and when
a mist is sprayed, particles in
the laser light can be tracked
by a camera to calculate the
flow field around the bird.
Transducers, tiny devices
that measure the flexion and
extension of muscles, are
also implanted in the birds to
study how they use muscles
in different maneuvers.
The same laser imag-
ing set-up is used to study 90
degree and 180 degree turns
in flight, the mechanism of
which is still poorly under-
stood. 180 degree turns are
especially interesting because
they involve the bird almost
FEATURE
stopping in midair at the instant
they are changing direction. Flying
at slower speeds actually requires the
bird to generate more force, which is
why hovering is so difficult for most
birds. Biewener hopes that laser im-
aging can shed some more light on
the mechanisms behind these tricky
turns.
However, avian flight is only
one area of research in Biewener’s
lab. Other active research proj-
ects include guinea fowl walking,
frog swimming, and locomotion in
goats.
Chasing Goats
“The immediate plan is to chase
a goat around!” laughs doctoral stu-
dent Carlos Moreno when asked
about his work on African pygmy
goats, another project supervised by
Biewener. “It’s pretty nonscientific.
You just holler and clap and make a
lot of noise. And they run away down
the corridor,” Moreno says about his
goat-chasing techniques. The goal
is to make the goat perform evasive
maneuvers in order to study the bio-
mechanics of turning and dodging
movements. Chasing the goat is only
the easy part, though probably the
most physically strenuous.
Prior studies on goats at the
field station have looked at bone
bending in turns. Strain gauges are
glued to the bone to detect bending,
7
FEATURE
What about their horns? “Handles
growing out of their heads,” says
Moreno.
and the goat is shooed around an en-
closed space. Results showed that of
two bones in the leg, the radius has
less variability in bending than the
metacarpal. The radius is a curved
bone, so bending mostly happens
in one direction, in contrast to the
straight metacarpal that can bend
every which way.
The remnants of a “cinderblock
mountain” once implanted with
force plates, stand next to the goat’s
runway as a reminder of past climb-
ing studies. Current studies con-
tinue to focus on the biomechanics
of 90-degree turns. A goat is chased
down a plywood runway embedded
with force plates that detect the force
exerted by the goat on the ground.
Additionally, a camera captures the
movement of the goat’s joints, spe-
cially labeled with joint markers.
The act of chasing a goat down
a plywood runway may sound silly
in itself, but it simulates evolution-
arily relevant behaviors by mimick-
8 The Harvard Undergraduate Research Journal
ing the act of a gazelle running from
a cheetah. A tiny slippage can be the
difference between life and death for
the gazelle. In contrast to a treadmill,
these more variable environments in
which goats run allow for more in-
sight into how animals turn and ac-
celerate, as a gazelle running from a
cheetah would.
Goats are chosen for the stud-
ies because they are representa-
tive quadrupeds, and also because
they’re quite harmless. They don’t
bite or kick, and are easily made to
run. What about their horns? “Han-
dles growing out of their heads,” says
Moreno. Chasing goats also seems
to be a favored activity of Duchess,
a pet dog who sometimes makes in
appearance at the field station. She
stands in contrast to the dozens of
other nameless animals that popu-
late the field station, since usually,
the scientists here are careful to keep
a certain distance from their lab
animals, labeling them by numbers
Volume 2 Issue 2 | Fall 2009
instead of names. The animals are
well cared for, especially by Pedro
Ramirez, the animal care technician
who has worked at the field station
for [twenty] years – “He’s the reason
that science can happen around here”
says Moreno – but it’s inevitable that
most animals will eventually reach
their terminal surgery.
Technologies developed for
these studies have also spilled into
experiments on human movement
and evolution. Professor Dan Lie-
berman, from the department of
Human Evolutionary Biology, has
adapted the goats’ force plates to an-
alyze the dynamics of barefoot hu-
man running.
Bees Can’t Fly? Unraveling
Insect Flight
Just outside the Station’s main
lab space is a large tent-like struc-
ture with a running pond and a few
plants. In early May, this greenhouse
is rather empty, but as summer rolls
around, the plan is to bring in local
dragonflies to study how they chase
prey. “Dragonflies do amazing aerial
chases,” says Professor Stacey Comb-
es, Assistant Professor of Organis-
mic and Evolutionary Biology, who
heads the second lab at the field sta-
tion studying the biomechanics and
behavioral ecology of insect flight.
Combes came to Harvard and
the field station in 2008 from the
Miller Institute at Berkeley, where
she studied the flight of wild orchid
bees in Panama. A wind tunnel,
smaller than the one for birds, is also
planned to be built for her study of
insect flight. However, Combes too
is interested in behaviors beyond
smooth flight in a wind tunnel. “In
all the years of studying flight no-
body has ever looked at how tur-
bulence affects flight,” says Combes
highlighting her research interests.
In Panama, Combes studied bees
Volume 2 Issue 2 | Fall 2009
Whether or not an insect can fly higher
in the tree canopy, where there is
turbulence, may affect its success at
mating or evading capture.
with a fan blowing out into the open
air, which causes air to curl in and out
creating turbulence. There she found
that instead of retracting their large
legs to reduce drag at high speeds,
the bees actually stick out their legs
to reduce rolling and pitching, much
like a figure skater sticking out his
arms to slow a spin.
Combes’ research builds on
foundational work done in the past
few decades. Until twenty years ago,
the aerodynamics of insect flight
was a mystery. Insect flight works
differently from the more familiar
aerodynamics of a bird or airplane,
in which air flows faster over the top
than the bottom of the wing to cre-
ate lift, and the myth once circulated
that “insects can’t fly.” The mystery
was solved by studying robotic wings
in mineral oil, which found that in-
www.thurj.org
sects beat their wings so fast as to
create a negative vortex around their
wings, thus generating lift. Combes
is interested in taking the study of
insect flight outside of controlled lab
spaces like a vat of mineral oil and
marrying biomechanics with ecol-
ogy, thus the dragonfly habitat out-
side. Turbulence, of course, is found
in any natural environment, and an
insect’s ability to fly in turbulence is
likely to have evolutionary impor-
tance. Whether or not an insect can
fly higher in the tree canopy, where
there is turbulence, may affect its suc-
cess at mating or evading capture.
Additionally, Combes stud-
ies the shape and material proper-
ties of insect wings, which bend and
flop remarkably in flight, though the
aerodynamic effects of this flexibil-
ity are unclear. Some of this work is
FEATURE
done in collaboration with Professor
Rob Wood’s Microrobotics Lab at the
School for Engineering and Applied
Sciences, which builds miniature ro-
botic insects.
The use of robotics is a theme
that runs through other research
projects too. Until recently, Biewen-
er’s lab group worked in collabora-
tion with Boston Dynamics, the
biorobotics company famous for Big
Dog, an amazingly agile robot dog.
Data from the goat studies as well
as other dog studies at the Concord
Field Station went into figuring out
the mechanics of Big Dog’s move-
ments.
Researchers at the Concord
Field Station continue to undertake
a wide range of studies on the biol-
ogy and biomechanics of movement.
The insights they obtain have appli-
cations in diverse fields, like robotics
and human locomotion. Throughout
its forty year history, the Station has
harbored countless animals and sci-
entists, and today it continues to be a
center for novel research. Harvard’s
‘Animal House’ parties on.
9
FEATURE Volume 2 Issue 2 | Fall 2009

Get Your Science On!

with the Harvard Program for Research
in Science and Engineering (PRISE)
By Alissa D’Gama ‘11,
THURJ Staff

H
arvard students have a lot
of answers to the ques-
tion “What did you do
over the summer?” Some may say
“intensive language study in Japan”,
“an internship with Goldman Sachs
in NYC” or “planning events in the
White House,” but there are about
one hundred who will always en-
thusiastically reply “PRISE!”

Put to Task
What is PRISE? The simple
answer to Harvard’s love of acro-
nyms is the Program for Research
in Science and Engineering. The Credit: Kate Xie THURJ ‘09

actual answer begins back in Janu-
ary 2005, when then Harvard Uni-
versity President Lawrence Sum-
mers made some widely criticized
remarks about gender and science
at the National Bureau of Econom-
ics Meeting. He shortly thereafter
appointed a faculty task force on
Women in Science and Engineer-
ing, which decided one of its pri-
orities was to develop a “summer
scientific research community” for
undergraduates.
In September 2005, Dean of
the College Evelyn Hammonds,
then the Senior Vice Provost for
Faculty Development and Diver-
sity, and Provost Steven Hyman,
provided three years of pilot fund-
ing for such a summer research
program. With no time to lose if
they were to get the program up
and running by the next summer,
the steering committee—newly ap-
pointed director Gregory Llacer,
Dean of Administration Georgene
Herschbach, and Fiona Chen from
the Provost’s Office—set out to de-
sign what would become PRISE.
Now transitioned from pilot
program status and having already
completed a successful four years,
PRISE is a staple for the undergrad-
uate research community at Har-
vard College, providing not only
housing and food over the summer,
but also friendships that last well
beyond the closing dinner.
Summer In Boston
Over the summer, PRISE
Fellows live together in Leverett
House, one of Harvard’s upper-
classman residential buildings.
They are hosted by House Masters
Howard Georgi, well known as the
professor of Physics 16 and for his
Thursday Physics nights, and his
wife Ann Georgi, the Life Sciences
Undergraduate Research Adviser,
who has helped many PRISE Fel-
lows find their labs.
Unlike during the school year,
when the budding researchers must

10 The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009
also complete problem sets, take
orgo midterms, go to lecture, and
participate in a slew of other extra-
curricular activities, PRISE “creates
an environment for a concentrated
group of scientists to share their ex-
periences and support each other on
a daily basis,” said Llacer.
Besides living in Leverett, the
Fellows are provided with a stipend
for lunch and also eat dinner togeth-
er, where the chatter often revolves
around the important questions
“One of the special things about a
PRISE fellow is that you can have a dis-
cussion on the most random science
topics for hours at a time, even if it’s
about compost and fertilizer.”
of the day—including how to get
an overlapping three-piece PCR to
work and the good movies coming
out next week.
This may seem unusual, but Jer-
emy Hsu, a PRISE Fellow in Summer
www.thurj.org
’09, jokes that you can spot a PRISE
Fellow in a crowd because “They’re
the ones that simultaneously crowd
around both the cheesecake and the
science!”
Adds Carol Suh, PRISE Fellow
in Summer ’08 and Administra-
tive Fellow in Summer ’09, “Some
people have told me that one of the
special things about a PRISE fellow
is that you can have a discussion
on the most random science topics
for hours at a time, even if it’s about
compost and fertilizer.”
This kind of conversation is
just what Director Gregory Llacer
hopes for every summer; indeed,
his favorite part of PRISE is not just
the summer, but the entire year, as
FEATURE
the PRISE email list continues to be
filled with questions and the Fellows
often end up as lab partners or din-
ner dates, continuing to engage with
each other and with science.
“PRISE provides early interdis-
ciplinary opportunities to network
with peers to experience science
more broadly and to make connec-
tions outside more narrow or tra-
ditional definitions of scientific re-
search,” adds Llacer.
Outside of their labs, the Fel-
lows are able to propose and receive
funding for a number of science and
social activities—a unique aspect of
the program that allows the Fellows
to take the initiative.
As Hsu recalls, “There were
countless fun things PRISE did –
whale-watching, getting hit in the
eye at point blank range by a water
balloon that didn’t explode, try-
ing to figure out what exactly a 3-D
shadow of a 4-D object is, and ex-
ploring the creepy tunnels at Boston
Harbor Islands.”
This past summer, for example,
Hsu was part of the team that orga-
nized a trip to Tanglewood, the sum-
mer home of the Boston Symphony
Credit: Kate Xie THURJ ‘09
11
FEATURE Volume 2 Issue 2 | Fall 2009

Orchestra, to see them perform
with Yo-Yo Ma, and part of the team
that planned a canoeing trip down
the Charles river (cleverly named
‘PRISE takes over the Charles’).
a little nerve-wracking, is the first
time they get to learn in detail what
all their newfound friends had been-
working on over the past ten weeks:
“It was a remarkable experience to
volume of applicants, interviews are
not feasible, so the committee relies
on the written materials the appli-
cants present.
The ultimate question the

“There were countless fun things PRISE did – whale-

watching, getting hit in the eye at point blank range
by a water balloon that didn’t explode, trying to figure
out what exactly a 3-D shadow of a 4-D object is.”
The Fellows aren’t limited to hear the cumulation of so many committee looks to answer is “Will
just getting to know each other— summers of research from all your this person effectively contribute to
there are weekly lectures by distin- friends and peers, and to be able and benefit from being in the PRISE
guished faculty, optional “faculty to hear from such a wide variety of community?” said Llacer.
chats” where fellows can bring in subjects,” said Hsu. The chosen Fellows for next
their principal investigator for an summer certainly have a lot to look
informal meal, and an end of the Get Me In! forward to. While Llacer notes that
summer dinner for Fellows, their For Harvard students interest- it is hard to pick his favorite mem-
post-docs and graduate students, ed in PRISE, the application, which ory from PRISE, he admits that “If
and PIs. is due in the spring semester, con- I had to single out a specific event it
And of course, there are the sists of several parts: essays about would be sitting in the dunk tank at
presentations given by PRISE Fel- your proposed project and contri- the PRISE “carnival” of 2006. It was
lows at the end of the summer, de- butions to the community, letters of a beautiful, warm day, and getting
scribing their research and often recommendation, your transcript, dumped in the water over and over
attended by their lab mentors. For and your hopefully abundant enthu- actually was pretty fun.”
many Fellows, this event, although siasm for PRISE. Due to the sheer

Credit: Kate Xie THURJ ‘09

12 The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009 FEATURE

George
Church:
A ‘Renaissance
Man’ of the
Genomics Era
By Isha Jain ‘12, THURJ Staff

H
Image Credit - Edge Research
arvard Medical School Professor George Church guy who was working in biology in the computer science
is what many would call ‘a renaissance man’ in the department. Sophomore year I found the only guy in the
biological sciences. He has published papers in biology department doing work in computer science. I
fields ranging from synthetic biology to sequencing tech- worked on solving the crystal structure of tRNAs. One
nologies to bioethics, and he helped found the modern of the programs I wrote was still in use thirty years later
science of genomics. But aside from his groundbreaking and is only now fading away.
research, he has also been advisor to 27 different com-

“
panies. His latest endeavor is the Q: How did this lead to your
Personal Genome Project, which later work with sequencing and
involves sequencing the genome the Human Genome Project?
of 100,000 volunteers. Dr. Church Then I thought,
sat down with THURJ and told us how cool would it be if A: At one point we wanted to
about his life, his research and his know if the structure we had de-
views on science and technology we could type in the termined for our tRNA was ap-
in the genomics era. sequence of a human plicable to the other ones that
had been sequenced. I typed in all
Q: You have a strong back- being and have the nucleotide sequences of the
ground in both computer sci-
ence and biology. How did ‘fold
people known tRNAs and asked if they
could fold up to fit our deter-
these interests merge and de-
velop? up?’ mined structure. I thought it was
so cool that we could just type in
the sequence of a tRNA and have
A: When I was very young, I got it fold up. Then I thought, how
interested in computers. It was re- cool would it be if we could type
ally hard, because I wasn’t exactly in the sequence of a human being
in an intellectual hotbed. I would and have people “fold up”?
play with electrical parts that I
could scrounge from construc- Q: So you actually started
tion sites. By 9th grade I managed to get into a better edu- thinking about the Human Genome Project at that
cational system. We had some sort of a toy network that point?
connected Dartmouth to our high school and I learned a
lot. I was constantly looking for a way that I could con- A: Yeah, it was a very vague and immature way of think-
nect my interest in math and computers with biology and ing about it. Looking back it was kind of visionary, but
medicine. My freshman year of college I found the only it was really just stupid at the time. So I started becom-

www.thurj.org 13
FEATURE Volume 2 Issue 2 | Fall 2009

Making Your DNA Public

The Personal Genome Project

As its website explains,
the PGP aims at “recruiting
volunteers who are willing
to share their genome se-
quence and many types of
personal information with
the research community
and the general public, so
that together we will be
better able to advance our
understanding of genetic
and environmental con-
tributions to human traits
and to improve our ability
to diagnose, treat, and pre- participants in the near
vent illness.” future.
Ten notable names in
science, like Steven Pinker, For more infor-
Esther Dyson and George mation, go to:
Church himself, have al- h t t p : / / w w w.
ready volunteered to share personalge-
their entire genomic se- nomes.org/
quences with the world at
large. They are the mem- The
bers of PGP-10, the first 3D Model
phase of the project, of DNA
which may ultimate-
Image:
ly enroll 100,000 more Wikipedia

ing obsessed with how we would actually sequence all this
DNA. Sequencing struck me as very unsolved, but very
solvable. At that time no one used computers in biology,
except for maybe a little bit in crystallography or neurosci-
ence. There was actually a bit of a sidetrack at this point
that some people find amusing. I finished college two years
early and then flunked out of graduate school. I was work-
ing so hard on the crystal structure of tRNAs. I figured I
had already proven that I could take hard courses and do
well in them; did I have to do it again? So, I flunked out of
Duke and for some reason Harvard let me into graduate
school.
Q: What kind of interdisciplinary projects are you
working on now?
A: Probably the most interdisciplinary project we have is
the Personal Genome Project. We have actually had to in-
novate in these fields. We got to the point that we needed
a lot of holistic human data. Many previous studies had
been very “tunnel-vision”: looking at this liver enzyme or
this liver disease. By specifically recruitinh people that are
okay with public disclosure, We made a new paradigm in
medical research.
Q: What criteria were involved in selecting the PGP10
(the first 10 experimental subjects of the Personal Ge-
nome Project)?
A: They were board members of genomic companies or
a chief scientific officer or CEO of a sequencing instru-
mentation facility. The IRB (Institutional Review Board)
wanted it to be very likely that they knew what they were
getting themselves into by having published about genom-
ics or made public statements. We wanted them to be di-
verse within that definition, so that if I was missing some-
thing, they could provide another set of advisers. There
are certain things in science that you just can’t do yourself.
One of the problems with the old way of thinking was
that you would put your work in a vault and then only let
the people who think the same way you do, see it. Maybe
the person with the answers is a school teacher in England
- not American, and with no scientific background. The
most likely person to think outside of the box is the least
likely person to have the credentials.
Q: Perhaps I could ask you about some of your other
projects? Could you tell us something about your ‘ag-
ing project’?

The PGP-10 From left to right, George Church, John Halamka, Esther Dyson, Misha Angrist, Keith Bachelder, Steven Pinker , Kirk
Maxley, Rosalynn Gill, Stanley Lapidus , and James Sherley. (Credit: Personal Genomics, Inc.)

14 The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009 FEATURE

A: We have been working on the aging project for a few
years. We have one of the best comparative zoology da-
tabases. What we ideally want are two animals that are re-
ally close to each other in sequence but have very different
longevities. Two species that have very similar sequences
are the mouse and the naked mole rat. The mouse is an
ideal human surrogate. It turns out that the typical mouse
lives just over two years and the naked mole rat lives about
twenty years. So what we are doing is taking big chunks of
DNA from the mole rat and putting them into the mouse,
replacing the mouse gene with the mole rat gene to see if
we can increase its longevity.
Q: How direct is your involvement with businesses
and companies, and what is your motivation for de-
veloping biotech products?
A: YYou can choose to just publish [your research] and
hope that someone pays attention, which is increasingly
not the case. In the early days of molecular biology, com-
panies would make the enzymes for you [the academic
researcher]. And then that wasn’t enough. So then the
companies would make a kit for you. And then even that
wasn’t enough. And eventually they would build a device
that would implement the kit. This has made it easier for
scientists to use a wide variety of tools, but you can’t al-
ways hide in the ivory tower. You need to make sure that
you have enough control over whatever method you are
developing, and work with a company to ensure they don’t
botch it. You don’t want to micromanage your company
just like you don’t want to micromanage your lab, but you
do want to manage it.
Q: You have a very eclectic range of topics. How do
you keep track of them?
A: Well, to me they all fit into one thing. There are a rela-
tively small number of applications that make sense to me.
For example, I say if you have to pick a disease, what is the
thing that everyone dies of? When you are between the age
of 20 and 45, not much happens to your body. It is only
once you start aging that almost every organ system starts
fading. Aging is really the fundamental problem and all the
other things are just symptomatic.

genebook Home Profile Microarray cDNA 19 George Church Settings Logout Find Cures

George Church is making massive amounts of genomic data available to

doctors and researchers worldwide! twenty minutes ago clear

Wall Info Alleles Cures Research! +

What genes are you expressing right now? Post

View My Genes (8527)

All Posts Posts by George Posts by Others Settings
Analyze My DNA for Diseases
Today
Edit My Cures
Personalized RNA Sequence for Diabetes 7:30 AM

“Maybe the cure for cancer is 1 atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct
written... in my genes!” 61 aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaactt
121 gggaatcaaa atcagattga aacatgcaat caaagcgtca ttacttatga aaacaacact
181 tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg acagtcagtg
241 gtttccgtga aattagcggg caattcctct ctctgccctg ttagtggatg ggctatatac
301 agtaaagaca acagtataag aatcggttcc aagggggatg tgtttgtcat aagggaacca
361 ttcatatcat gctccccctt agaatgcaga accttcttct tgactcaagg ggccttgcta
421 aatgacaaac attccaatgg aaccattaaa gacaggagcc catatcgaac cctaatgagc
481 tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcagt cgcttggtca
541 gcaagtgctt gtcatgatgg catcaattgg ctaacaattg gaatttctgg cccagacaat
601 ggggcagtgg ctgtgttaaa gtacaacggc ataataacag acactatcaa ........
Image: “The Future of Medicine “ by Ryan Neff; George Church Picture: Personal Genomics, Inc.

www.thurj.org 15
 FEATURE
Uncovering
Implicit Biases
By Jen Jian Gong ‘12
M
any of us believe we are more impartial than
our historic predecessors, whether it be in
matters of race, gender or ethnic equality.
Landmark achievements like the Universal Declara-
tion of Human Rights, the end of apartheid or the rise
of a black president to the highest office in America
might seem like enough reason to believe that human-
ity’s biases are a thing of the past, at least in the de-
veloped world. Recent scientific discoveries, however,
suggest that they are not. Currently, Banaji’s research focuses on how implicit
Harvard psychology professor Mahzarin Banaji is a
leader in the field of implicit social cognition, which
investigates what she has termed “implicit biases”- un-
conscious prejudices that persist even as our explicit
attitudes evolve. She is the current Richard Clarke
Cabot Professor of Social Ethics and is one of the prin-
cipal investigators of “Project Implicit.” She is joined
in this project by Brian Nosek, from the University of
Virginia.
According to Nosek, the goal of Project Implicit is
“to understand thoughts and feelings that exist out-
side of awareness and control.” The project functions
as a virtual laboratory, where online visitors can assess
their own implicit attitudes using the so-called Implic-
it Association Test (IAT). The IAT requires subjects to
rapidly categorize pairs of stimuli, using differences in
response time as an indicator of implicit bias. If, for
example, a subject holds an unconscious bias against
Illustration by Amanda Lu
a certain group, then one would expect the subject to
have more difficulty associating positive stimuli with
images of that group’s members than with images of
members belonging to a preferred group. This diffi-
culty manifests itself in slower response times, making
the IAT a reliable indicator of bias.
This technique has been used to explore many forms
of prejudice, using group classifications like race, sex,
16
Volume 2 Issue 2 | Fall 2009
Do you know your
own mind?
age and religion to expose a myriad of implicit biases
These tests (located online at https://implicit.harvard.
edu/implicit/demo/) are an eye-opening experience
for many subjects. Since its launch in 1998, over 4.5
million online visitors have completed the web-based
version of IAT, generating an immense data set on the
prevalence of prejudice. The data yield a clear conclu-
sion: implicit biases not only exist, they are pervasive
across large sets of the general population.
attitudes and preferences first came to be. “We know
that many of them are learned by us after we are born…
but our minds seem infinitely ready to learn them, for
some reason.” This curious “readiness” has led Banaji
and her colleagues to conduct studies utilizing young
children, as well as adults, in order to look for pat-
terns in the appearance and development of implicit
attitudes. They studied children’s explicit attitudes to-
wards certain groups, but also measured their uncon-
scious biases using an IAT. They found that younger
children spoke more openly about their preferences,
while older children and adults tended to mask their
biases with more socially acceptable language. How-
ever, “on the IAT, if white American adults are show-
ing a certain level of preference for whites over blacks,
then young children in that group are showing the
exact same preference.” These IAT data seem to in-
validate the assumption “that young kids should not
really show some of those biases, because they are still
evolving.” In fact, implicit bias is present in what looks
to be exactly the same form in children (as young as
age 6) as it is in adults. Disturbingly enough, implicit
biases may begin to develop at a far younger age than
we previously thought.
In addition to trying to understand biases from a
developmental perspective, Banaji and her colleagues
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
have begun using neuroimaging techniques to under-
stand how our brain activity varies when we distinguish
between different groups of people. Collaborating with
her is Jason Mitchell, an Assistant Professor in the De-
partment of Psychology. As Banaji explains, “Most of us
feel that we treat people from different groups largely
the same, that our minds focus on them in the same
way. Our job is to look at how early in the sequence our
brains start doing something different when we look at
somebody.”
In one of their recent studies, published in the journal
Neuron, subjects heard descriptions of people of varying
political views and were asked questions about them.
The study showed that “many liberals
literally use a different set of neurons For Banaji, the an-
when they’re thinking about Joe, the
liberal, versus Jeff, the conservative.”
swer is a resound-
Most of the questions the researchers ing yes: we are
asked about the characters did not
touch on political beliefs, for exam- adaptable beings,
ple: ‘Do you think he does his laun-
dry every week?’ The neuroimaging
and these implicit
data from these studies showed that biases are not set
there was more activity in the ven-
tral medial prefrontal cortex (mPFC) in stone.
when subjects were asked to make judgments about
those with similar political leanings. When they were
asked questions about those with dissimilar views, there
was more activity in the dorsal mPFC.
This research is clearly relevant to our daily lives,
with far-reaching educational and judicial implications.
Banaji receives many phone calls about her work – and
not just from other psychologists. Individuals in busi-
ness and law want to know more about these uncon-
scious prejudices because they have the potential to play
an integral role in their own jobs. “People don’t know
that there is this stuff in their mind that ends up affect-
ing how they treat people,” she explains. “Because their
intention is to do the right thing, they become really
disturbed when they learn that maybe they’re not acting
in the interest of their patient.”
Banaji sees these implicit biases as “mental viruses”,
analogous to any other illness or physical virus we have
discovered. Since everyone is susceptible to implicit
bias, Banaji says, “when your role is to be public de-
fender or a social worker, you especially don’t think of
yourself as harming anyone. But imagine that you too
carry those viruses. Are you really able to do your job as
well as you might be able to?”
www.thurj.org
FEATURE
Scholars are also beginning to recognize the relevance
of this research to their own fields. Professor Jerry Kang,
from the University of California, Los Angeles Law
School, has collaborated with Banaji to explore the ways
that such scientific findings should shape the study and
practice of law. For example, one collaborative paper
addresses affirmative action policy and findings from
implicit social cognition that might affect its implemen-
tation. “Right now, civil rights and racial justice talk
seems to be stuck in the mud,” Kang says. Rather than
utilizing “new philosophical arguments about justice,”
perhaps the findings from research into the brain and
mind will eventually provide “insurmountable evidence
that we are not in fact ‘colorblind’.”
While it is true that a growing body
of psychology literature is revealing our
unconscious tendencies to think about
groups of people differently, the im-
portant question seems to be whether
or not these biases can be changed. Is
there a cure for the mental virus?
For Banaji, the answer is a resounding
yes: we are adaptable beings, and these
implicit biases are not set in stone. She
believes there is a “very hopeful mes-
sage that’s coming from a lot of work: that our systems
are highly adaptable, that we are very malleable.”
She suggests that a diverse campus like Harvard’s is
also a good laboratory in which to test the malleability
of our minds. Experiences with different groups of peo-
ple can challenge our deep-seated biases and, perhaps,
begin to change them. “If we’re indeed adaptable, then
these simple notions of who’s conservative and who’s
liberal will pose for us opportunities to see if our minds
can take that kind of leap.”
Thanks to the IAT and similar research endeavors, we
are more aware of implicit biases than past generations.
So, Banaji asks, “now that we know they exist…will we
do with this information what we do with new medi-
cal information?” We now “know that there are things
happening between us that we can’t see…and that it’s
costing our society.” Because we can no longer claim ig-
norance, she argues, “the standard for us is different.”
The first step towards combating these viruses, Bana-
ji firmly states, is “awareness, awareness, awareness.”
Since they operate outside conscious awareness, it is
not always easy to acknowledge our biases. But, they are
consequential nonetheless, affecting who we marry, be-
friend, hire or convict.
17
 RESEARCH Volume 2 Issue 2 | Fall 2009

Differential peptidase activity in

Immunology

CD4+ T-cells and monocytes results in

variations in HIV-1 optimal epitope
production and degradation
Oluwatobi Ogbechie
Harvard College ‘09, tobi9ogbe8@gmail.com

Human Immunodeficiency Virus 1 (HIV-1) infects several cell types, including CD4+ T-cells and monocytes. These
cells are important virus targets because they are substantially depleted during infection and can also store dormant
virus, which fuels the disease at later stages. A crucial part of the recognition and clearance of infected cells involves
their presentation of pieces of viral protein, called epitopes, at their surface for recognition by immune cells. These
pieces originate from the degradation of viral peptides by intracellular peptidases. It is unknown if cell types that
are infected by HIV present similar viral peptides to evoke analogous immune responses. Here, I examined whether
CD4+ T-cells and monocytes would exhibit differences in the production of viral peptides and if that could affect the
HIV-specific immune responses. I measured the activity and kinetics of intracellular peptidases involved in viral
peptide degradation in CD4 + T-cells and monocytes using an in vitro fluorescence-based hydrolytic activity assay.
Peptidases’ activities were higher and faster in monocytes. Speculating that these differences in activity might affect
viral peptide production, I measured the production of a 9-amino acid long HIV epitope (RK9) from a longer peptide
using an intracellular degradation assay where the longer peptide is incubated with cytosolic extract from CD4+ T-
cells and monocytes. I used RP-HPLC to identify degradation products. The longer peptide’s degradation and RK9
production were faster in monocytes. In addition, monocyte degradation products elicited greater epitope-specific
immune responses. I sought to determine the overall effect of these cell-type-specific differences in peptidase activity
on the intracellular degradation of randomly selected HIV epitopes using a similar intracellular degradation assay.
The epitopes’ half-lives were significantly shorter in monocytes than in CD4 + T-cells. These differences in antigen
processing could affect presentation of HIV-1 peptides to immune cells and might influence clearance of infected
CD4+ T-cells and monocytes.

Introduction Immune response to HIV-1 infection

HIV-1 infection
Human Immunodeficiency Virus 1 (HIV-1) is a retrovirus that
preferentially infects human nucleated cells with tropism towards
CD4+ T or monocyte-derived cell lines (Callaway et al., 1999; Sup.
Fig. A). After infection, the virus integrates its retro-transcribed
DNA into the host cell’s genome and can either reproduce numerous
active progeny or remain quiescent (Stevenson et al., 1990; Hauber et
al., 1987). Cells with dormant virus, known as viral reservoirs, can
linger in the host indefinitely (Chun and Fauci, 1999).
Of all the Peripheral Blood Mononuclear Cell (PBMC) subsets,
researchers implicated CD4+ T-cells and monocytes as key reservoirs
of virus during infection (Smith et al., 2003; Brenchley et al., 2004).
Severe depletion of CD4+ T-cells occurs during the acute stage of HIV
infection with around 40-50% lymphocyte loss (Hel et al., 2006; Sup.
Fig. B). However, monocytes do not drastically decline in numbers
as CD4+ T-lymphocytes do (Douek, 2007). During chronic infec-
tion, viral levels in the blood rise, the immune system weakens, and
consequently AIDS symptoms, like Kaposi’s sarcoma, occur (Douek,
2007). Progression to AIDS is faster among HIV-infected patients
with drawn out symptoms during acute infection due to a weaker
immune response (Pedersen et al., 1989).
18
Effective regulation of the acute and chronic phase by the host’s
immune system gives the patient a much better prognosis for the dis-
ease (Vanhems and Beaulieu, 1997). Patients with little loss of CD4+
T-cells have slow disease progression, hence validating the impor-
tance of an efficient immune response (Sheppard et al., 1993). For
this study, I was interested in aspects of the cell-mediated immune
response because cell types involved in this response that undertake
anti-HIV specific functions could increase the host’s capability to
control HIV-infection (Schmitz et al., 2001).
To initiate cell-mediated responses, circulating dendritic cells
(DCs) in the bloodstream can phagocytose the foreign pathogens,
process them, and present the viral peptides to immature CD4+ and
CD8+ T-cells in order to begin their maturation process (Randolph
et al., 2008). Mature CD8+ T-cells become cytotoxic T-lymphocytes
(CTLs) that target and kill infected cells or produce other antiviral
responses, and mature CD4+ T helper cells either assist in the recruit-
ment of CTLs, aid in the production of neutralizing antibodies, or
produce cytokines that enhance the immune response (Betts et al.,
2001; Hel et al., 2006). Thus, infection of CD4+ T-cells greatly weak-
ens the immune system. Also, it gives a rationale for the success of
long-term non-progressors who have highly efficacious T-cell-medi-
ated responses against HIV (Paroli et al., 2001).
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Antigen processing
One of the most important events in a cell-mediated immune re-
sponse is antigen processing, which encompasses the degradation,
transport, and presentation of both self and foreign proteins to CTLs.
There are two major antigen processing pathways: endogenous and
exogenous.
The endogenous pathway
Defective, newly synthesized, or intracellular proteins can be
tagged with multiple copies of ubiquitin, a protein that targets them
for proteolysis in the proteasome (Schubert et al., 2000). Three of the
proteasome’s beta subunits bear the protease’s active sites: caspase-
like, chymotrypsin-like, and trypsin-like, which cleave proteins after
acidic, hydrophobic, and basic amino acids, respectively (Kopp et al.,
1997).
After proteasomal processing, the endogenous pathway utilizes
peptidases in the cytosol, such as tripeptidyl peptidase II (TPPII),
thimet oligoendopeptidase (TOP), and aminopeptidases, to further
degrade the peptide products (Groothuis et al., 2005). Recently, an in
vitro intracellular experiment demonstrated that TPPII was neces-
sary for the generation of an HIV-1 epitope presented through the
endogenous pathway in DCs (Seifert et al., 2003). Like TPPII, TOP
can generate C-termini that are different from peptides produced by
the proteasome, but TOP prefers to cleave shorter peptides (Oliveira
et al., 2001). Saric and colleagues determined that TOP was a criti-
cal intracellular endopeptidase that degraded proteasomal products
of 9-17 amino acids (Saric et al., 2004). The large family of cytosolic
aminopeptidases further processes these peptide products (Hattori
and Tsujimoto, 2004). However, an experiment on the capability of
mice lacking leucine aminopeptidase to present viral peptides found
that that this peptidase was not necessary to generate peptides for the
MHC-I pathway (Towne et al., 2005). This evidence does not contra-
dict previous findings that implicate aminopeptidases as contribu-
tors to antigen processing. Rather, it suggests that many peptidases
contribute to the processing of viral peptides.
After cytosolic processing, peptides around 9 amino acids long
with a preferred C-terminal residue can be transported to the en-
doplasmic reticulum (ER) by transporter associated with antigen
processing (TAP) (Larsen et al., 2005). In the ER, degradation by
ERAP-1 may continue before the peptide is loaded onto an MHC
class I molecule. Stronger binding affinity of the peptide in the MHC
binding groove displaces the complex from Tapasin, a protein that
anchors MHC-I in the ER. Released, MHC-I/peptide travels through
the trans-golgi pathway to be presented at the surface of the cell
Figure 1. Endogenous pathway of antigen processing. 1) HIV in-
tegrates its genome into host 2) Transcription of HIV genes and translation
of protein occur 3) Ubiquitination and degradation of HIV protein in the
proteasome 4) Peptide products are further degraded in aminopeptidase,
TOP, and TPPII 5) Optimal epitope is transported into the ER through
TAP 6) and is subjected into further degradation in ER by ERAP, but can
be loaded onto MHC-I heavy chain/Tapasin complex 7) Tapasin disso-
ciates from completely assembled MHC-I/peptide complex 8) Complex
moves through Golgi to 9) cell surface. 10) T-cell receptor (TCR) of CTL
and co-receptor, CD8, recognize MHC-1/peptide complex.
www.thurj.org
RESEARCH
(Groothuis et al., 2005). CTLs primed by DCs recognize the epitope
presented as well as the MHC-I molecule (Frahm et al., 2007; Fig. 1).
If the CTL can recognize a non-self peptide, like an HIV-1 epitope, it
Immunology
kills the infected cell or produces other antiviral responses.
The exogenous pathway
Professional antigen presenting cells (APCs), like DCs, use this
pathway during the initial activation of the immune response to ex-
tracellular pathogens. Here, MHC class II molecules on APCs present
HIV-1 epitopes to CD4+ T-cells after cathepsins peptidase degrada-
tion (Pajot et al., 2007).
Antigen processing in PBMC subsets
It is generally assumed that antigen processing across cell types is
similar, though one can deduce that differences in presented peptides
can lead to variations in the immune response. For example, cellular
production of more antigenic peptides should lead to stronger im-
mune responses. Despite the multi-layered comprehension of antigen
processing, the process is not well understood in PBMC subsets in-
fected by HIV. Since HIV-1 targets certain PBMC cell subsets, such
as CD4+ T-lymphocytes and monocytes, it is necessary to understand
both the type of immune response that these subsets elicit and the
processes by which they are generated.
Although direct comparisons of antigen processing in CD4+ T-
cells and monocytes have not been previously reported, some work
has been published about differences in antigen processing between
other cell types. First, mouse fibroblast and dendritic cell lines in-
fected with lymphocytic choriomeningtitis virus (LCMV) presented
different viral peptides to CTLs, resulting in non-identical cell type-
specific immune responses (Butz and Bevan, 1998). The presented
peptides varied in their antigenicity; thus immune responses to DCs
and fibroblasts varied, showing the possibility of functional dif-
ferences in antigen processing among cell types. Another study in
mice found that dendritic and non-dendritic cells presented different
epitopes to CTLs during primary and secondary infection of influ-
enza (Crowe et al., 2003). This differential presentation affected the
immune response through the generation of distinct memory CD8+
T-cells for each cell type. Both of the aforementioned suggest cell-
type-specific variations in antigen processing that affect the immune
response, making this of high interest to HIV research since HIV-1
has tropism for select cell types.
When this is applied to immune responses to HIV-1, where in-
fection is subset-specific and depletion of lymphocytes is skewed to-
wards CD4+ T-cells, one realizes the need to identify and describe
such differences. Furthermore, this question about the differences
in antigen processing between the leukocyte subsets is applicable to
vaccine research because of the increasing promise of T-cell vaccines
(Johnston and Fauci, 2007). These vaccines attempt to elicit memory
T-cell responses, especially CD8+ T-cell responses to kill infected
cells as soon as a recipient of the virus is infected with the virus. Also,
T-cell vaccines that focus on improving the host’s immune response
during acute and/or chronic infection would advance the epidemio-
logical control of the HIV pandemic. A necessary prelude to creating
such a vaccine is ensuring that infected cells, CD4+ T-cells and mono-
cytes, will present peptides that CTLs can recognize.
A thorough understanding of HIV pathogenesis will require
knowledge about the immune response that HIV-infectible cells, such
as CD4+ T-cells and monocytes, can elicit. First, the two tropisms of
HIV-1 strains are for CD4+ T-cell lines and for monocyte lineage cells
(Weiss, 2008). For instance, CD16+ monocytes, which are precursors
to DCs and macrophages, have increased permissivity to HIV-1 in-
fection with HIV-seropositive individuals having elevated numbers
of this cell population (Ellery et al., 2007). Besides infectibility, both
CD4+ T-cells and monocyte cell lines can serve as latent reservoirs of
19
 RESEARCH
HIV. The creation of reservoirs begins during primary infection in
resting CD4+ T-cells, and there is evidence that monocyte-derived
macrophages in the gastrointestinal tract can serve as reservoirs for
Immunology
the virus during chronic infection (Chun et al., 1998; Smith et al.,
2003).
As important as CD4+ T-cells and monocytes are during HIV in-
fection, there is little known about the differences in their antigen
processing that can potentially affect the immune response. It is
known that gene expression between the PBMCs and T-lymphocyte
subsets varies in HIV-seropositive individuals (McLaren et al., 2004).
This introduces the possibility of variations in the expression of an-
tigen processing genes and in the activity of their protein products,
leading to functional differences. Such variations between subsets
targeted by the virus may affect the kinetics, the amount, or the iden-
tity of peptides presented to CTLs and may alter the capacity of CTLs
to kill infected CD4+ T-cells or monocytes.
Materials and Methods
Blood processing
Blood was drawn from healthy and HIV-infected donors (Massa-
chusetts General Hospital, Boston, MA). PBMCs were isolated from
blood using Ficoll-Hypaque (Sigma, St. Louis, MO) density gradient
separation after a 30-minute spin at 1500rpm at room temperature.
Cells were washed three times in Hank’s Balanced Salt Solution
with 10mM HEPES and 1% PSG. After each wash, cells were spun at
1500rpm for 10 minutes at room temperature.
Isolation of PBMC subsets
CD4+ T-lymphocytes and monocytes subsets were isolated from
PBMC and magnetically immunosorted using the EasySep® Human
CD4+ T-cell Enrichment Kit and EasySep® Monocyte Enrichment Kit
according to the manufacturer’s instructions (StemCell, Vancouver,
Canada). Enriched cells were washed three times in Dulbecco’s Phos-
phate Buffered Saline (Sigma) at 1500rpm for 10 minutes at room
temperature. Dry cell pellets were kept at –80°C until cytosol extrac-
tion.
Cytosolic extracts
Cell pellets were resuspended in a Digitonin buffer (50mM HEP-
ES, 50mM KAc, 5mM MgCl2, 1mM DTT, 10% glycerol, 1mM ATP,
0.5mM EDTA, 0.0125% Digitonin) for 5 minutes to allow sufficient
detergent permeabilization and then spun at 20,000rcf for 15 min-
utes. Protein concentration in both methods was measured with
the DC Protein Assay Kit from Bio Rad Laboratories (Hercules, CA).
Whole cell extracts were kept at –80˚C until use.
Actin normalization
To standardize the amount of extract used in experiments, actin
in each sample was compared to a sample known to contain 3µg cy-
tosol. The normalized amount of each sample was the volume of cell
extract with an actin content that matched 3µg of actin in the known
sample after Western Blot analysis.
Western Blot Analysis
All samples were subjected to Western Blot analysis. First, whole
cell extracts were mixed with laemmli buffer and heated at 85°C for
10 minutes. Lysates were run through a 12% SDS-PAGE gel at 100V
through stacking gel and 125V through running gel. The gel was
transferred to PVDF transfer membrane (GE healthcare) at 25mA
overnight. The membrane was blocked at room temperature for at
least 30 minutes in 5% milk and 1% NP40. Primary anti-beta actin
antibody (Abcam, Cambridge, MA) was diluted to 1/20,000 in 5%
milk and 0.1% NP40, added to the membrane, and incubated for 1
hour at room temperature. Following the primary, the membrane
was washed 5 times with 0.1% NP40 every 10 minutes. Secondary
20
Volume 2 Issue 2 | Fall 2009
anti-mouse and anti-rabbit antibody coupled to horseradish per-
oxidase (GE Healthcare) was diluted to 1/6000 in 5% milk and 0.1%
NP40 and incubated for 40-45 minutes in room temperature with the
membrane. Membrane was washed again as stated above. For imag-
ing, ECL Plus Western Blotting Detection Reagents and Amersham
Hyperfilm ECL were used following manufacturer’s instructions (GE
Healthcare).
Peptidase inhibitors and substrates
Caspase-like, chymotrypsin-like, and trypsin-like active sites of
the proteasome were inhibited by the proteasome inhibitor MG132
using a 50mM stock solution in DMSO (Sigma-Aldrich, St. Louis,
MO). Aminopeptidase activity was inhibited by Bestatin hydro-
chloride using a 12mM stock solution in DMSO (Sigma-Aldrich, St.
Louis, MO). Cpp-AAF-pAb, the TOP inhibitor, was diluted in DMSO
for 1mM stock solution (Bachem, Torrance, CA). The TPPII inhibitor
Butabindide oxalate was diluted with DMSO for a 10mM stock solu-
tion (TOCRIS Bioscience, Northpoint, UK). TPPII, chymotrypsin-
like, and trypsin-like activities were measured with H-Ala-Ala-Phe-
Amc, Suc-LLVY-Amc, and Boc-LRR-Amc [where Amc represents
7-amido-4-methyl-coumarin] respectively and were resuspended
in DMSO to produce stock solutions of 10mM, 50mM, and 100mM
(Biochem Bioscience, Torrance, CA). The aminopeptidase substrate
Leu-AMC and the proteasome caspase substrate ZLLE-Amc were
obtained from Calbiochem (San Diego, CA) to be resuspended in
DMSO for respective stock solutions of 50mM and 50mM. The TOP
substrate Mcc-PLGPK-Dnp was resuspended in DMSO for a stock so-
lution of 10mM. All inhibitors and substrates were stored at –20°C.
Antigen Processing Peptidase Activity Assays
For proteasome peptidase activities, 3µg of whole cell extract
was incubated in a buffer containing 20mM HEPES, 50mM KAc,
5mM MgCl2, 1mM DTT, and 1mM ATP at room temperature for
30 minutes. To ensure the specificity of the assay, 1μM MG132 was
incubated with the extract. After the incubation, substrates for the
different proteasome active sites were added and fluorescence was
immediately measured every 5 minutes over 1 hour at 37°C with a
Victor-3 plate reader (Perkin Elmer, Boston, MA). 75μM ZLLE-Amc
was used to measure caspase-like activity, 100μM Suc-LLVY-Amc
was used for chymotrypsin-like activity, and 25μM Boc-LRR-Amc
was used for trypsin-like active site activity. Aminopeptidase, TPPII,
and TOP fluorescence assays were conducted in similar conditions,
excluding DTT and ATP in the buffer. Specificity was determined
by incubation with 120μM Bestatin, 10μM Cpp-AAF-pAb, or 1μM
Butabindide for aminopeptidase, TOP, and TPPII activities respec-
tively. Following the 30 minute incubation, respective substrates
were added: 50μM Leu-Amc, 20μM Mcc-PLGPK-Dnp, and 100μM
H-Ala-Ala-Phe-Amc for aminopeptidase, TOP, and TPPII. Fluores-
cence was read as stated above. Excitation and emission wavelengths
for trypsin-like, aminopeptidase, and TOP fluorescent products were
345nm and 405nm, respectively. Those wavelengths for caspase-like,
chymotrypsin-like, and TPPII fluorescent products were 380nm and
460nm, respectively.
Synthetic peptides and RP-HPLC peptide analysis
Peptides were made by the AAPPPTEC Apex 396 multiple peptide
synthesizer at the MGH peptide core (Massachusetts General Hospi-
tal, Boston MA). Peptides were then purified by reverse-phase high
pressure liquid chromatography (RP-HPLC) and their sequences
were verified by mass spectrometry, which showed greater than 95%
purity (Partners Proteomics, Cambridge, MA). Eluted peptides pro-
duced defined peaks on the RP-HPLC where the area under the peak
was proportional to the amount of peptide in the analyzed sample.
Defined peptide peaks were calibrated using a 4.6x50mm 3mm C18
column (Waters, Milford, MA).
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Intracellular peptide degradation assays
PBMC, CD4+ T, and monocyte whole cell extracts from the same
healthy donor were resuspended in buffer containing 20mM HEPES,
137mM KAc, 1mM MgCl2, and 1mM ATP in nano-pure H2O. Subse-
quently, peptide was added to the solution, at which point the incuba-
tion at 37°C began. At 0, 10, and 30 minutes, aliquots of the reaction
containing 30μg whole cell extracts and 6nmol peptide were stopped
with 0.3% Trifluoroacetic acid (TFA) (Sigma Aldrich, St. Louis, MO).
6nmol of pure peptide in same buffer and TFA conditions was used
to identify the elution time and amount of the undigested peptide
through RP-HPLC. All peptides were eluted using a gradient solution
of two buffers. The first was 0.05% TFA in nanopure H2O, while the
second buffer varied depending on the peptide. For peptides 5RK3,
A3-RK9, B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-K11, the second
buffer either contained 50% acetonitrile (AcN) and 0.01% TFA or
100% methanol and 0.05% TFA. The second elution buffer for B7-FL9,
B7-TL10, and B7-FR10 contained either 100% AcN and 0.03% TFA
or 100% methanol and 0.05% TFA. Integrated peaks from digested
peptides were compared to the undigested peptide. Identities of some
digested peaks were verified by mass spectrometry.
Antigenicity of produced peptides
Peptide products from the 17-mer (gag) intracellular degradation
assay were purified with 10% trichloroacetic acid precipitation and
diluted in RPMI-1640 without serum. Subsequently, pH was adjusted
to 7.4. HLA-A3+ B cells derived from HeLa cells served as target cells
and were labeled with 51Cr and pulsed with 0.5ug/ml of the peptide
products for 30 minutes at 37°C without serum. CTL clones specific
for RK9 were incubated for 4 hours with the B cells at a 4:1 effector-
target ratio. Cell lysis was defined as [(51Cr release from B cells incu-
bated with digested products - spontaneous release) / (total release –
spontaneous release)]. Cell lysis values were also compared to that of
HLA-A3+ B cells that were incubated with undigested 17-mer peptide
at a 0.5µg/ml concentration.
Statistical analysis
Maximum slope in the fluorescence assays was calculated with
Workout 2.0 using a liner kinetic fit of the fluorescence at different
time intervals (Perkin Elmer, Boston, MA). The differences in maxi-
mum slopes and fluorescence levels between CD4+ T-lymphocytes
and monocytes were analyzed using the non-parametric Mann-
Whitney test with GraphPad Prism 5 (La Jolla, CA). This test was
used since it does not assume a normal distribution for the data, yet
it assumes that both samples are independent. The half-lives of pep-
tides used were determined using a non-linear regression of an one
phase exponential decay with the same software.
Figure 2. Intracellular fluorescence-based hydrolytic activity
assay. Cell extracts were incubated with specific fluorogenic substrates at
37°C for 1 hour. Cleavage of those substrates produced fluorescence. Pep-
tidase activity is proportional to fluorescence. Sample proteasome-trypsin
assay shows fluorescence level over time. Slope and 1-hour fluorescence
are indicated on graph.
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RESEARCH
Results
Fluorogenic assays of antigen processing associated peptidases
Immunology
As protein degradation of HIV-1 proteins is crucial to the even-
tual presentation of viral peptides to CTLs, I sought to characterize
the activity of peptidases associated with antigen processing—pro-
teasome-caspase-like, -chymotrypsin-like, and -trypsin-like; amino-
peptidase; TOP; and TPPII—in CD4+ T-cells and monocytes. To do
this, I utilized an in vitro fluorescence-based hydrolytic assay already
developed by others in the group. Here, I incubated cytosolic extracts
from peripheral blood mononuclear cells (PBMC), CD4+ T-cells, and
monocytes with substrates that fluoresce upon cleavage by specific
peptidases (Figure 2). The measured fluorescence of the sample was
directly proportional to the activity of the peptidase over time. Af-
ter a 30-minute pre-incubation with peptidase-specific inhibitors,
I found that the average specificity of substrates to their respective
peptidases in PBMC, CD4+ T, and monocyte subsets was between
80% and 95% (Sup. Table 1). I must note that cleavage of Leu-Amc in
monocytes was about 50% specific to aminopeptidases, which indi-
cated that another peptidase along with aminopeptidase cleaved Leu-
Amc. However, fluorescence from cleavage in monocytes was much
greater than in other cell extracts that we considered the discrepan-
cies to be negligible (data not shown).
CD4+ T-cells and monocytes have different peptidase activity
levels
After ensuring the specificity of the fluorescence assay, I sought
to characterize the activity of the proteasome, aminopeptidase, TOP,
and TPPII in two PBMC subsets targeted by HIV: CD4+ T-cells and
monocytes. Prior research indicated that activities of the antigen
processing machinery varied according to cell type (Butz and Bevan,
1998). Therefore, we hypothesized that the proteasome, aminopep-
tidase, TOP, and TPPII would show differences in activity between
CD4+ T-cells and monocytes. Using blood from 8 healthy donors, I
purified the PBMC and immunosorted CD4+ T-cells and monocytes.
With equal amounts of cell extracts normalized through western
blots against actin and GAPDH, I compared peptidase activities of
whole PBMC, CD4+ T-cells, and monocytes by using the fluorescence
emitted by the sample at 1 hour after the 37°C incubation. All assays
Figure 3. Different activity levels of peptidases associated with
antigen processing between CD4+ T-cells and monocytes. Pro-
teasome caspase-like and trypsin-like, aminopeptidase, TOP, and TPPII
activities were measured in whole cell extracts from PBMC (•), immuno-
sorted CD4+ T-cells (■) and monocytes (▶) after a 1 hour incubation with
peptidase-specific substrates at 37°C. Fluorescence was compared using
a non-parametric Mann-Whitney statistical test. Significant differences
between CD4+ T-cells and monocytes were found. *p<0.05, **p<0.01,
***p<0.001. n=8.
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Immunology
RESEARCH
Figure 4. Faster peptidase kinetics in monocytes. The degradation
of fluorogenic substrates specific for either the proteasome: caspase-like or
trypsin-like active sites, aminopeptidase, TOP, or TPPII were measured in
whole cell extracts of PBMC (•), CD4+ T-cells (■), and monocytes (▶) over
1 hour at 37°C. The maximum slopes of degradation in CD4+ T-cells and
monocytes were compared using non-parametric Mann-Whitney statisti-
cal analysis. Monocytes showed significantly higher kinetics than CD4+
T-cells. *p<0.05, **p<0.01, ***p<0.001. n=8.
were repeated three times, and the arithmetic mean of all trials was
used as the final value for comparison. A representative sample shows
the typical fluorescence pattern with monocytes having a higher
fluorescence at the 1-hour time point than CD4+ T-cells (Figure 2).
Overall, I found that proteasome-caspase-like, proteasome-trypsin-
like, aminopeptidase, TOP, and TPPII had significantly higher ac-
tivity in monocytes than in CD4+ T-cells (Figure 3). In proteasome-
chymotrypsin-like activities, I did not find a significant difference,
though other members of my lab performed a similar assay with 14
samples and found significant differences between CD4+ T-cells and
monocytes (data not shown).
Faster kinetics in peptidases associated with antigen processing
in monocytes
In addition to the overall level of peptidase activity, the speed at
which the peptidase cleaves proteins is crucial during viral infection
since the virus depends on the slow recognition of an infected cell for
its replication. We investigated whether observed variations in pep-
tidase activity levels between CD4+ T-cells and monocytes occurred
with kinetic differences. Therefore, I measured the kinetics of pepti-
dase degradation for the proteasome’s caspase-like, chymotrypsin-
like, and trypsin-like active sites and for aminopeptidase, TOP, and
TPPII. Like in the previous experiment, whole cell extracts of PBMC
and immunosorted CD4+ T-cell and monocytes from 8 healthy do-
nors were incubated with fluorogenic substrates for 1 hour. A repre-
Figure 6. Intracellular degradation of 5RK3 to produce RK9.
Whole cell extracts of PBMC (+), CD4+ T-cell (■), and monocytes (◊) were
incubated with 5RK3, an HIV-1 Gag p-17 fragment, at 37°C. At 2, 10, 30,
60, 120 minutes, samples were collected and eluted through RP-HPLC for
analysis and quantification. A) Monocytes showed faster degradation of
5RK3 than CD4+ T-cells. B) Monocytes produced the HLA-A3 restricted
optimal epitope, RK9, faster and at sustained levels for over 2 hours. Iden-
tities of 5R3 and RK9 were confirmed by mass spectrometry.
22
Volume 2 Issue 2 | Fall 2009
Figure 5. Intracellular epitope degradation and 51Cr release as-
say. A) Intracellular peptidases in whole cell extracts of PBMC, CD4+ T-
cells, and monocytes degrade 5RK3 to smaller products when incubated in
37°C over 2 hours. Peptides were eluted in RP-HPLC and produced unique
peaks where the integral under the peak is proportional to the amount of
that peptide. B) HLA-A3+ B cells were pulsed with peptide degradation
products and used as targets in the 51Cr release assay. CTLs specific for the
HLA-A3+ -restricted epitope RK9 (orange) were used as effectors.
sentative sample shows the observed differences in slope over time
(Figure 2). I used the average of the maximum slope from 3 experi-
ments as my comparison values. Peptidases in monocytes have sig-
nificantly faster degradation capacities than those in CD4+ T-cells
(Figure 4). Again, the proteasome chymotrypsin-like subunit failed
to show a statistically significant difference between the two subsets,
but other members of the group observed differences when using
a larger sample size (data not shown). These differences in antigen
processing activities and kinetics imply that monocytes are capable
of processing intracellular peptides to a greater extent than CD4+ T-
cells, which may have downstream effects on the recognition of the
infected cell.
Figure 7. Peptide products from intracellular 5RK3 digestion.
Whole cell extracts of PBMC, CD4+ T-cell, and monocytes were used to
digest an HIV-1 Gag p17 fragment (RWEKIRLRPGGKKKYKL aa 15-31),
called 5RK3 (uppermost rectangle). It contains an HLA-A3 restricted
epitope, RK9 (black text); N-terminal flanking regions are to the left; C-
terminal flanking regions are to the right. Degradation products at 2 and
60 minutes that were found through mass spectrometry are listed. Prod-
ucts with dotted outlines are unique to the indicated cell type. Shaded
products are previously described antigenic epitopes. Monocytes showed
shorter and more antigenic degradation products than CD4+ T-cells.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Intracellular degradation of longer peptides to produce optimal
HIV-1 epitopes
After ascertaining significant differences in activity and kinetics
of antigen processing peptidases between CD4+ T-cells and mono-
cytes, we wanted to know if those differences affect HIV-1 epitope
processing. To do this, I utilized an in vitro intracellular degradation
system where I incubated whole cell extracts of PBMC, immunosort-
ed CD4+ T-cells, or monocytes with a 17-amino-acid sequence from
HIV-1 Gag p17 (RWEKIRLRPGGKKKYKL, aa 15-31), called 5RK3
in this experiment, for 0 to 120 minutes (Figure 5a). When intracellu-
lar peptidases degrade 5RK3, one can visualize the peptide products
through RP-HPLC where one peak indicates a distinct peptide, and
the integral below the peak is proportional to the amount of peptide.
At different time points during the incubation, one can examine the
quantity of 5RK3 and its degradation products. Members of the lab
previously showed this system to be a good approximation of the en-
dogenous processing of intracellular proteins (Le Gall et al., 2007).
Faster degradation of 5RK3 fragment and sustained levels of
RK9, a peptide product, in monocytes
Using the in vitro intracellular degradation assay described in the
earlier section, I measured the degradation of 5RK3 and the genera-
tion of its optimal epitope RK9 (RLRPGGKKK), which requires a
multistep cleavage of the 5 N-terminal residues (RWEKI) and 3 C-
terminal residues (YKL) of 5RK3.
I studied the production of RK9 since it is associated with delayed
progression to AIDS through HLA-A3 restricted CTL responses in
some HIV-infected patients (Altfeld et al., 2006). Furthermore, I
could infer that the observed differences in peptidase activity and
kinetics affected 5RK3 degradation since this process requires the
proteasome, aminopeptidases, and TPPII (Le Gall et al., 2007). I
found that the degradation of 5RK3 was faster in monocytes than in
CD4+ T-cells, and monocytes degraded all the 5RK3 by the second
minute of the incubation (Figure 6a). As for the production of RK9,
monocytes not only produced it with faster kinetics, but they also
maintained higher levels of RK9 over time compared to CD4+ T-cells
(Figure 6b).
Antigenic degradation products from monocytes
Besides the degradation of the original peptide, and the produc-
tion of RK9, we wanted to understand the differences among peptide
products of the 2 PBMC subsets. No study had shown the potential
differences in peptide products from the antigen processing machin-
ery. Such a difference could eventually affect CTL recognition of
the infected cell subset. The design of the intracellular degradation
Figure 8. Antigenicity of peptide products from intracellular
5RK3 digestion. Extracts from 2, 10, 30, 60, 120 minutes in the 5RK3
degradation in PBMC, CD4+ T-cells, and monocytes were purified. HLA-
A3+ B cells were pulsed with these products to be used as targets in 51Cr
release assay. CTLs specific for HLA-A3+ restricted epitope RK9 were used
as effectors. For each extract from all time points during the degradation,
percent specific lysis was measured. Monocyte degradation products (▶)
elicited greater CTL killing, and thus, were more antigenic than that of
CD4+ T-cells (■).
www.thurj.org
RESEARCH
assay allowed me to attribute any observed differences in the prod-
ucts between CD4+ T-cells and monocytes to the antigen processing
machinery. We sent the products for mass spectrometry analysis to
Immunology
identify the peptide sequences. I found that peptides produced from
5RK3 in monocytes were shorter than those from CD4+ T-cells, pre-
sumably allowing for a better fit on MHC-I (Figure 7). In addition,
monocyte degradation products contained already identified optimal
HIV-1 epitopes: HLA-B27 restricted IK9 (IRLPPGGKK) and HLA-
A3 restricted KK9 (KIRLPPGGK), RK9 (RLRPGGKKK), and RY10
(RLRPGGKKKY) (Korber et al., 2008; Fig. 7). This alluded to differ-
ences in the CTL recognition and antigenicity of peptide products
between the two subsets. My supervisor performed a 51Cr release as-
say on the peptide products from the 5RK3 intracellular degradation
assay to determine their antigenicity. She purified the small peptides
from the incubated extracts and pulsed identical HLA-matched B
cells with them (Figure 5b). Those B cells served as the targets in the
release assay, and CTLs specific for HLA-A3 RK9 were the effectors.
We found that products from monocytes were more antigenic than
those of CD4+ T-cells (Figure 8). In fact, we achieved a maximum
specific lysis of 51% from monocyte degradation products whereas
those from CD4+ T-cells could not generate greater than 3% lysis.
Other members of the lab observed similar results after replicating
the assay twice (data not shown).
Intracellular stability of HIV-1 epitopes
The previous experiments showed how differences in peptidase
activity between CD4+ T-cells and monocytes could affect the pro-
duction of HIV-1 epitopes. However, antigen processing is a balance
between both the production and degradation of epitopes since intra-
cellular peptidases are constitutively active. Differences in the deg-
radation of optimal epitopes between CD4+ T-cells and monocytes
could affect epitope presentation and CTL recognition of optimal
epitopes. Little is known about the intracellular stability of short
peptides, though aminopeptidases have been implicated in regularly
cleaving short and intermediate peptides in the cytosol (Smyth and
O’Cuinn, 1994). Thus, I examined whether the degradation of HIV-
epitopes, which are short peptides, was the same in both CD4+ T-
cells and monocytes. Other members of my lab had characterized the
half-lives of many of the known HIV epitopes in PBMC and found
that although some epitopes were highly unstable, others resisted
degradation over time (data not shown). We speculated that HIV
epitopes that were stable in PBMC could have varying levels of stabil-
ity between CD4+ T-cells and monocytes since we already saw cell
Figure 9. Intracellular degradation of optimal HIV-1 epitopes. A)
8 optimal HIV-1 epitopes (B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-
KK11, B7-FL9, B7-TL10, and B7-FR10) were incubated in whole cell extract
of PBMC and immunosorted CD4+ T-cells and monocytes at 37°C for 30
minutes. B) At 2, 10, and 30 minutes, portions of the reaction were stopped
and eluted through RP-HPLC. Peptides produce unique peaks where the
area under the peaks represents the amount of peptide in the eluate.
23
 RESEARCH
Immunology
Figure 10. Shorter half-life of HIV-1 epitopes in monocytes. 8
optimal HIV-1 epitopes (B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-
KK11, B7-FL9, B7-TL10, and B7-FR10) were subjected to intracellular
degradation in whole cell extracts of PBMC (circle) and immunosorted
CD4+ T-cells (square) and monocytes (triangle). Peptide products at 2, 10
and 30 minutes were analyzed and quantified using RP-HPLC. One-phase
exponential decay was used to estimate the half-lives of the epitopes. Non-
parametric Mann-Whitney test was used for comparison of the half-lives.
Optimal epitopes showed significantly shorter half-lives in monocytes
than in CD4+ T-cells. **p<0.01.
type differences in epitope production. To measure the intracellular
half-life of optimal HIV epitopes, I incubated them with whole cell
extracts of PBMC, immunosorted CD4+ T-cells, or monocytes over
30 minutes (Figure 9). At 2, 10, and 30 minutes, I eluted the extract-
peptide mixture through RP-HPLC. Like the prior RP-HPLC assay,
each peptide produced a unique peak where its integral reflects the
amount of peptide present in the eluate allowing me to quantify the
24
Volume 2 Issue 2 | Fall 2009
percent left of the original epitope. By examining the proportion of
peptide remaining at each time-point, I could map the epitope degra-
dation to a non-linear regression of exponential decay that provided
me with parameters to calculate the half-life of each epitope. From
the existing data in the group, I chose epitopes that were relatively
stable overtime in PBMC, in order to measure any notable deviations
in stability in CD4+ T-cells and monocytes. Using 8 epitopes (B57-
YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-KK11, B7-FL9, B7-TL10, and
B7-FR10), I found that the half-life of those optimal HIV-1 epitopes
was significantly shorter in monocytes than in CD4+ T-cells (Figure
10). To ensure that my results were replicable, I repeated each degra-
dation assay 3 times and averaged all values.
Different intracellular degradation of HIV-1 optimal epitopes in
CD4+ T-cells and monocytes
The calculated differences in half-life of HIV optimal epitopes
were not the only interesting findings revealed in our comparison of
intracellular epitope degradation between CD4+ T-cells and mono-
cytes. Apart from understanding the overall pattern of optimal
epitope degradation in PBMC, we wanted to know if the kinetics of
degradation was similar across the PBMC subsets that HIV-1 infects.
In the same assay as above, I qualitatively assessed the kinetics of deg-
radation for each optimal epitope (B57-YY9, Cw3-RL9, A33-ER8, B7-
HA9, A3-KK11, B7-FL9, B7-TL10, and B7-FR10) between the subsets.
I found that there was no consistent pattern of degradation among
epitopes in both cell subsets, and these differences existed regardless
of the epitopes protein’s origin (Figure 11). This suggested functional
variations in subset-specific degradation of HIV epitopes.
Discussion
HIV-1 infection depends on CD4+ T-cells and monocytes
as vessels for proliferation and latency (Pierson et al., 2000).
CD4+ T-cells undergo a more severe depletion than mono-
cytes during infection (Pedersen et al., 1989). To delay the
onset of immunodeficiency following HIV infection, the
host must mount an HIV-specific immune response. One
study showed that the presence of CTLs that recognized the
HIV-1 envelope glycoprotein allowed the patient to control
viral levels during primary infection since CTL recognition
of infected cells can stimulate antiviral responses (Borrow
et al., 1994). This recognition event requires the intracellu-
lar degradation of viral proteins and their assembly on an
MHC-I for cell surface presentation.
Although CD4+ T-cells and monocytes are critical in
HIV infection, antigen processing and presentation of HIV-1
epitopes have not been studied in these two cell types. More-
over, the type and efficacy of the immune response that those
infected cell subsets can induce is unknown. Therefore, this
study aimed to understand the antigen processing machin-
ery in CD4+ T-cells and monocytes in the context of HIV-1
epitope production, degradation, and antigenicity. I focused
Figure 11. Various degradation kinetics of HIV-1 op-
timal epitopes in CD4+ T-cells and monocytes. Whole
cell extracts of PBMC (+), immunosorted CD4+ T-cells (■), and
monocytes (◊) digested various HIV-1 optimal epitopes (B57-
YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-KK11, B7-TL10, and B7-
FR10) over 30 minutes at 37°C. RP-HPLC was used to analyze
the extract-peptide mixture to determine the amount of peptide
left. Extensive variation in degradation kinetics was seen among
epitopes from the same protein as well as across all epitopes.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
on identifying the levels and kinetics of peptidases associated with
the production and degradation of these peptide fragments and found
that there were indeed differences in activity level and kinetics, which
resulted in variations in the processing of HIV-1 epitopes. Observed
heterogeneity in the identity and production time course of peptide
products might have important consequences for CTL recognition of
infected PBMC subsets and the ensuing immune response.
Using the intracellular fluorescence-based hydrolytic assay for
peptidase activity, I showed how proteasome-caspase-like and
trypsin-like, aminopeptidase, TOP, and TPPII activity levels and
speed in CD4+ T-cells and monocytes differ for both healthy and
HIV infected donors. We do not know whether these differences
stem from variations in transcription or translation. Moreover, we do
not understand what differences in intracellular peptidase quantity
lead to observable differences in activity. Therefore, I cannot know if
variations in activity come from increased expression of the proteins
or from increased activity of each individual peptidase in mono-
cytes. Another potential reason for the increase in peptidase activity
in monocytes might be a contamination in the cytosol of cathepsins,
endosomal peptidases that function in the exogenous pathway of an-
tigen presentation. I corrected for this by stabilizing the pH of the
whole cell extracts at 7.4, which considerably inhibits the function of
cathepsins (Turk et al., 1999). Despite the uncertainty in the source
of these functional differences between CD4+ T-cells and monocytes,
the effects of these variations are of primary interest because we must
fully understand the consequences for the immune response.
One limitation of this study was that it did not measure differ-
ences in non-cytosolic peptidases, such as ERAP-1 and ERAP-2, be-
tween the two PBMC subsets. Much of the literature about antigen
processing of epitopes implicates ERAPs and not cytosolic peptidases
(York et al., 2002). York and his group showed that purified ERAP-1
could trim all peptides that were longer than 10 residues and half of
the 9-residue peptides. Though their experiment was not performed
under the typical intracellular environment of the cell, it suggested
that ERAP-1 was important in determining the loaded peptides for
MHC-I presentation. My data on intracellular epitope processing
does not conflict with the existing research, but it adds to the litera-
ture by showing that cytosolic peptidases can produce optimal HIV-1
epitopes from longer peptides. However, it is still important to study
the potential differences in ERAP activity between the PBMC subsets
as those peptidases can make the final peptide trimmings in the ER
that may affect its loading on the MHC-I and CTL recognition.
Even with the exclusion of non-cytosolic peptidases, my study
revealed differences among intracellular peptidases. For example,
aminopeptidase showed the greatest fold differences in average activ-
ity values between CD4+ T-cells and monocytes. Compared to the
proteasome, TOP, and TPPII, whose fold differences in the average
activities between the subsets fell between 1.5 and 2.1, aminopepti-
dase activity in monocytes was about 3.5 times greater than that of
CD4+ T-cells. This suggests that each peptidase can have unique dif-
ferences in activity in different subsets. Also supporting this theory
is the inconclusive result on the subset-specific differences in the pro-
teasome-chymotrypsin active site. Although average activity levels of
this active site were greater in monocytes, there was no statistically
significant difference in values between the two cell types (data not
shown). Other active sites within the proteasome showed significant
subset-specific differences with 8 samples in the cohort, indicating
non-uniform subset-specific variations in the proteasome active sites
and perhaps, in most intracellular peptidases.
In addition to studying the differences in intracellular peptidase
activity between the CD4+ T-cells and monocytes, it is important to
study the entire antigen processing pathway because the presentation
www.thurj.org
RESEARCH
of the epitope is dependent on a multi-step process where each step
can affect the identity of the final presented product. This process
incorporates the kinetics of peptide production, degradation, trans-
Immunology
port, and MHC-I binding, which must be completed in a timely man-
ner before the proliferating virions can escape. Here, I studied one
of the earlier steps, intracellular epitope processing, which did not
include epitope processing in the ER, peptide transport, or MHC-I
binding kinetics. Products from intracellular processing need trans-
port into the ER by TAP, which displays binding preferences for cer-
tain peptide sequences to transport into the ER (Procko and Gaudet,
2009). Thus, it is possible to infer that TAP might also influence the
final product presented by MHC-I. Therefore, further experiments
to discern the differences in the entire antigen processing pathway
between CD4+ T-cells and monocytes must be completed.
Moreover, my research only focused on 2 subsets that HIV infects:
CD4+ T-cells and monocytes, and did not include the entire repertoire
of HIV-infectible PBMC subsets, such as macrophages, natural killer
(NK) cells, and dendritic cells (DCs), which all contribute to infec-
tion (Weiss, 2008; Valentin et al., 2002). Macrophages are monocyte-
derived cells that can store dormant virus in the gastrointestinal tract
during chronic infection (Smith et al., 2003). Reduced levels of NK
cells in infected patients are associated with worse prognoses (Ullum
et al., 1995). DCs activate CTLs in cell-mediated immunity, making
them especially important in HIV pathogenesis (Zarling et al., 1999).
Although most of the infected cells in HIV-individuals are CD4+ T-
cells, it is still important to understand the antigen processing capa-
bilities of all PBMC subsets that contribute to this disease in order to
understand the entire scope of presented HIV-1 peptides.
Apart from exposing peptidase activity differences between the
subsets, my results introduce the concept of a balance between pro-
duction and degradation of intracellular peptides within the infected
cell. The observations that monocytes both produce and degrade
optimal HIV-1 epitopes faster than CD4+ T-cells can be seemingly
contradictory if one quickly attributes faster degradation of epitopes
as a negative factor in antigen processing. However, one must remain
aware of the balance between production, degradation, and transport
of epitopes in the cytosol compartment. If monocytes have a more
efficient transport system into the ER, then the increased degrada-
tion of optimal epitopes in monocytes might be negligible. Similarly,
slower degradation of HIV-1 epitopes in CD4+ T-cells might allot
more time for TAP binding and transport into the ER. With my re-
sults, I have reiterated the importance of kinetics in the entire antigen
processing pathway and established the value of a balance between
intracellular production and degradation of viral peptides destined
to be presented to CTLs.
Additionally, I have shown that these observations of differential
epitope processing might also be applicable to HIV-infected individ-
uals. Though I did not perform the intracellular degradation of an
HIV epitope on whole cell extracts from HIV-infected donors, the
significant difference in peptidase activities between CD4+ T-cells and
monocytes of HIV-infected samples suggests similarities in epitope
production (Sup. Fig. C and D). However, the estimated frequency of
infected CD4+ T-cells and monocytes waivers between 0.01% and 1%
in an HIV-positive individual; therefore, the differences in peptidase
activity between the subsets that I detected might stem from the un-
infected cells (Poznansky et al., 1991; Smith et al., 2003). Although
HIV epitope processing in infected cells remains unclear, a larger
study that compares intracellular peptidase activity and epitope pro-
cessing in HIV-infected PBMC will elucidate unknown patterns.
Furthermore, since HIV-1 has two main tropisms for PBMC cell
subsets—T-cells and monocyte-derived cells—one can speculate that
the untimely recognition of infected CD4+ T-cells may skew the viral
25
 RESEARCH
population in an infected patient to the T-cell tropic strains. Recently,
the prevalence of HIV-1 viral strains that preferentially infect CD4+
T-cells was found to be significantly greater in PBMC (Verhofstede et
Immunology
al., 2009). With increased viral levels of T-cell-tropic strains, greater
numbers of CD4+ T-cells might be infected, thus contributing to the
drastic reduction of CD4+ T-cells during HIV infection. Already, the
period following infection before the CTL response has been identi-
fied as crucial for the virus to establish reservoirs in T-cells (Daven-
port et al., 2004). This study complements my findings to suggest that
kinetics of antigen processing and presentation and CTL recognition
are important for disease progression. Though it might be imprecise
to generalize laboratory findings to the disease progression, my find-
ings provide a different way of examining the dissimilar fates of CD4+
T-cells and monocytes during HIV infection.
This research is applicable to other viral infections, especially ones
with tropism towards specific cell subsets. For the cell-mediated im-
mune response to be effective, the infected cell must present a viral
peptide that DC-primed CTLs can recognize. Since uninfected DCs
can undergo cross-presentation, where extracellular peptides enter
the endogenous pathway, it is important to ensure that the peptides
presented by the infected cells are the same ones that the DCs can
process for CTL priming. For example, one study on the DC-tropic
human cytomegalovirus (HCMV) strain found that whole PBMC in-
fected with HCMV could elicit more responses than infected DCs
(Gerna et al., 2005). Here, differences in viral peptide presented might
be the cause for differences in the elicited immune response. Further
research on cell subsets infected by viruses with particular tropisms
must be completed to understand the extent of cell-type-specific dif-
ferences in antigen processing.
My results also pertain to the current literature on immunodomi-
nance of MHC-I restricted epitopes. For this purpose, I will define
MHC-I immunodominance as the phenomenon where CTLs against
a certain viral epitope are more prevalent in an infected individu-
al. Mathematical models based on patient observations show that a
dominant CTL response usually arises when a pathogen is homoge-
neous; however, this does not explain the molecular reasons for over-
representation of that particular viral peptide (Nowak et al., 1995). A
recent study on intracellular processing found that flanking sequenc-
es near the epitope contributed to the processing of those epitopes
and strongly determined the intracellular peptide products (Le Gall
et al., 2007). If cell subsets exhibit different kinetics in the production
and identity of presented HIV-1 epitopes, then the immunodominant
epitope might not be presented by one of the subsets. This would cre-
ate dire consequences for the PBMC subset that does not present this
dominant epitope, and clearance of infected cells in that subset might
be compromised.
Besides understanding the immune response to infect CD4+ T-
cells or monocytes, my findings also add to the existing literature on
HIV vaccines. Unfortunately, the search for an HIV vaccine has not
been very successful due to the complexity of HIV and its interac-
tions with the immune system (Johnston and Fauci, 2007). Vaccines
function by delivering antigen to a host; the antigen activates the im-
mune system and induces the creation of antibody responses and an-
tigen-specific memory T-cells, which persist in the host. Upon infec-
tion with that same pathogen, infected cells present foreign peptides
that optimally trigger proliferation of those memory immune cells to
perform their effector functions to curb the disease. An efficacious
vaccine necessitates memory T-cells recognizing the viral peptides
presented by infected cells. Since HIV infects various cell types that
potentially have differences in their antigen processing machinery,
a great candidate for a vaccine should contain a viral peptide that
all cell types can process efficiently and present to CTLs. Thus, the
26
Volume 2 Issue 2 | Fall 2009
consequences of subset-specific differences in antigen processing and
presentation are broad reaching. The identity of the peptide present-
ed might affect memory T-cell activation and CTL recognition of the
infected cell.
Even without observations on other components of the antigen
processing pathway or on other cells infected by HIV, my results dem-
onstrate that differences in intracellular processing might affect the
production, degradation, identity, and antigenicity of HIV-1 epitopes
in two PBMC subsets. Monocytes showed increased activity in anti-
gen processing peptidases, which might lead to a different repertoire
of presented HIV-1 epitopes than CD4+ T-cells. The rapid production
and larger amounts of more antigenic peptides in monocytes may
increase the probability of transport into the ER for those peptides. If
infected monocytes present more antigenic epitopes, then these cells
might elicit a better immune response than infected CD4+ T-cells.
Accordingly, it is important to further research on subset-specific
differences in antigen processing not only to better understand the
host’s immune response to infection but also to develop highly effec-
tive clinical interventions, such as vaccines.
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Acknowledgements
I would like to thank Sylvie Le Gall for giving me the opportunity to conduct
research and for being a great mentor to me. Her dedication to the scientific pro-
cess is a quality that I will always cherish. Furthermore, my experiments would
have been impossible without the amazing supervision by Estibaliz (Esti) Lazaro.
She helped me grow as both a researcher and as a young adult. Furthermore, I
truly appreciate working in a world-class laboratory whose director, Bruce Walk-
er, gave me encouragement and support during my time there. This project was
supported in part by Howard Hughes Medical Institute (HHMI), the National
Institutes of Health (NIH), and Bill and Melinda Gates Foundation.
I would also like to individually thank all members of the Le Gall lab, past and
present: Sasha Blue Godfrey, Jeremy Ho, Christopher Kerrigan, Mei Zhang, Ser-
gio Martinez, Paul Bourgine, Shao Chong Zhang, and Jonathan Chow for sharing
their projects with me, assisting me with portions of my research, and staying
positive in times of stress. Professor Losick’s HHMI program also supported me
immensely in my research.
To end, my family and friends have encouraged me and kept me sane through-
out this project. I cannot imagine completing this process without them. They
always believed and still believe in my commitment to understanding and com-
bating this destructive virus.
27
 RESEARCH Volume 2 Issue 2 | Fall 2009

Victory by association:
Using electronic prediction markets to
measure coattail effects
Stephen Travis May
Harvard College ‘09, travis.may@post.harvard.edu
Economics

In this paper, we study the magnitude of coattail effects in the 2008 election, or the impact of the presidential elec-
tion on congressional elections. We utilize data from electronic prediction markets to measure these effects. In order
to eliminate endogeneity biases in our analysis, we measure coattails by using the occurrence of a major event as an
instrument. We select days in which major events occurred (such as presidential debates) that affected the presidential
election without any direct effect on local elections. Since the shifts that occur on these days in congressional markets
are due exclusively to coattails, we then measure the impact the exogenous events had on the congressional elections.
We find strong coattail effects in House elections and insignificant effects in the overall Senate race. We then apply
our methodology to the Minnesota Senate election, where we find strikingly strong coattail effects. We discuss these
results in the context of our simple model of voting preferences.

Introduction
As the cornerstone of democracy, elections play a critical role in
the United States political system. Every four years, star candidates
rise and fall, scandals emerge and disappear, and long campaigns are
fiercely fought before settling on a winner. Pundits devise dozens of
stories that “explain” what determined the winner, and television and
radio talk shows will debate these theories for months. But to the
academic empiricist, understanding an election is problematic. To
understand the determinants of a specific election, the sample size is
only one event—so how can we know whether John McCain would
have fared better with a different vice-presidential candidate? To un-
derstand election determinants, political economists like Ray Fair
have pooled the data of many elections and constructed models of
very broad trends; yet because of the small sample size and the com-
plexity of the variables impacting elections, these models yield only
a few answers about what shapes elections in general, and much less
about what shapes specific elections.
In this paper, we propose the use of a new tool for understand-
ing elections: electronic prediction markets. These markets have been
studied at length, with a broad theoretical and empirical consensus
that they yield reasonably unbiased, efficient estimates of the prob-
ability that an event occurs. Instead of focusing on their efficiency,
as most papers about electronic prediction markets have, we will in-
stead assume that they are efficient. In this paper, we will begin to an-
swer the following question: given that electronic prediction markets
are efficient, what can they tell us about political institutions? We will
provide a case study of the potential utility of this methodology in the
example of coattail effects. While this paper yields interesting results,
our focus is primarily methodological and aims to understand how
the existence of a continuous metric of election probabilities can be
useful for causal analysis.
Background on electronic prediction markets
Electronic prediction markets are futures exchanges in which the
value of an asset is tied to the outcome of a particular event. In a
vote share market, traders bid in a continuous double auction for the
28
optimal price that they will pay for a contract that pays $1 for every
percentage point that a particular party receives. For example, in the
2000 election, George Bush received 47.9% of the vote, and thus the
Republican contract paid off $47.90 at the end of the election. In a
winner-take-all (WTA) election, traders bid on contracts for which
candidate will win the election and receive $100 per share of the win-
ning candidate held.
In a system with no transaction costs and risk neutrality, Wolfers
and Zitzwitz (2004) show that the market price of the contract should
equal the median market participant’s expected election outcome
weighted by trading volume. Furthermore, Berg et al. (2001) show
that the market price is a remarkably accurate short-term predictor
of the real election outcome, consistently outperforming polls as a
last-day prediction tool. In a follow-up study, Berg et al. (2003) find
that electronic markets have exceptional long-term predictive abili-
ties, and greatly outperform both polls and forecasting models.
The intuition behind these results is clear: traders are forced to
“put their money where their mouth is,” and they are thus incentiv-
ized to be accurate. Since projection models and poll results are gen-
erally publically available, that information can be incorporated into
the market price. Thus the market prediction should be at least as
accurate as any type of publicly-known prediction mechanism, if not
more so. Interestingly, this assumption does not depend on traders
being a random sample of the population, as the market can incorpo-
rate information without all voters participating. As Berg et al. (2001)
point out, market participants are far from a random sample; they
tend to be wealthy, young, and highly-educated. Furthermore, the av-
erage trader in the market can be biased and trade based on his own
bias; empirically, there is a small set of “marginal traders” who are
the ultimate price-makers and hunt for arbitrage opportunities—and
their probabilistic assumptions are quite accurate.
With this consensus of research demonstrating that prediction
markets are accurate estimators, we intend to utilize the market re-
sults in this paper as a continuous set of election results­—changing
over the course of the election only as the expected outcome changes.
Thus, we have a much more complete data set, with a continuous set
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
of data points that can be evaluated in order to measure the impor-
tance of contextual events.
Background on coattail effects
In the paper, we apply electronic prediction markets to understand
the interplay between national and local elections. While resting par-
tially on local issues and candidate personalities, local elections are
often a referendum on national political figures. Local elections are
heavily correlated with national results, and many elections—such
as the 2008 election—have the same party sweep to victory in the
House, the Senate, and the presidency.
Some of this correlation is due to contextual shifts, such as macro-
economic changes or wars abroad. Other sources of correlation may
be due to a demographic transition, leading to partisan realignment.
A third part of the correlation comes from the presence of coattail
effects, or the effect that a top-level candidate has on a local candi-
date. A popular presidential candidate can register new voters, drive
partisan turnout and party-line voting, and change voter preferences
on issues, leading to coattail effects that change the outcome of con-
gressional elections.
Despite the intuitive nature of coattail effects, attempts at effective-
ly quantifying these effects have generally floundered. Since elections
are correlated for a wide variety of reasons, measuring basic correla-
tion between elections is insufficient to establish coattail effects. The
methodological obstacles to measuring coattail effects were pointed
out by Miller (1955), who noted the simultaneity of decisions taking
place in elections. Mondak (1990) asserted that “a growing consensus
holds that the presidential vote does exert significant influence on
congressional elections,” though he added that these analyses were
set back by the “long–recognized difficulties associated with measur-
ing political coattails.”
Two primary obstacles exist in measuring coattail effects. First,
there is a highly limited sample size: there is only one presidential
election every four years from which to draw data, and there are
substantial contextual changes that occur over that time frame. To
overcome the limited datasets, we use electronic prediction markets
to assess the status of elections on a continual basis. A second ob-
stacle is one of endogeneity: much of the correlation between candi-
dates is due to agreement on issues and shifts in economic context,
rather than coattail effects? In this paper, we measure coattail effects
within the 2008 election by using exogenous changes in the presi-
dential election (that do not directly affect congressional elections) as
an instrument. We find substantial coattail effects in the House and
insignificant effects in the Senate.
The paper is organized as follows: first, we describe a theoretical
model for the existence of coattail effects; then, we propose an em-
pirical methodology based on the electronic prediction market data-
set; next, we analyze the results of this methodology; and finally, we
provide concluding remarks and suggest areas for future research.
Modeling Coattail Effects
Despite the difficulties in measurement, there are intuitive rea-
sons why coattail effects are likely to exist. First, voter registration
and turnout are often driven by grassroots campaigns and excite-
ment for a top-level candidate. If a presidential candidate is able to
register millions of first-time voters, those voters are likely to vote in
lower-level elections for the same party. Similarly, excitement about
a top-level candidate can help drive party-line voting in some states,
as voters that are ambivalent about lower-level elections choose out
of simplicity and expediency to vote for a party generally rather than
evaluating individual candidates. While these effects can help shift
votes towards a popular presidential candidate’s party, a more subtle
www.thurj.org
RESEARCH
source of coattail effects shapes the median voter’s preferences direct-
ly in lower-level elections. The median voter, typically a swing voter
who was planning to vote and does not make a straight party-line
vote, may change her lower-level preferences based on a change in
her perception of a top-level candidate; her preference for a particu-
lar presidential candidate informs her decision of which lower-level
candidate to support.
To gain a better understanding of the impact of coattail effects on
the election, we will consider a congressional election that is simulta-
neous with a presidential election. Define candidate C as a congres-
sional candidate in the same party as presidential candidate J. The
decision of whether to vote for candidate C can be modeled as:
Economics
X iC = 1 if Udif_C (Equation 1)
X iC = 0 if –Ū < Udif_C < Ū
X iC = –1 if Udif_C < –Ū
Where Udif_C = E[UiC]–E[UiC2]
X iC: vote cast by voter i for candidate C. Not voting counts as 0 votes,
and voting against counts as a negative vote for the candidate.
UiC: utility voter i receives in the state of the world where candidate
C wins
UiC2: utility voter i receives in the state of the world where candidate
C’s opponent wins
Ū: absolute utility difference threshold that causes voter i to cast a
vote.
Additionally, we can further expand these variables as:
E(UiC) = E(F(ii, v iC)) (Equation 2)
ii = i(c, ti)
E(v iC) = v(c, riC, pC, siC)
UiC: utility voter i receives in the state of the world where candidate
C wins
v iC: vector of expected agreement between voter i and candidate C’s
decisions
ii: vector of weights of importance that a voter gives to particular is-
sues
c: context of election
ti: voter i’s tastes
pC: personal characteristics of candidate C
riC: historical correlation between voter i and candidate C’s known
beliefs
siC: signals of future agreement between voter i and candidate C
Under this model, a voter’s tastes (ti) and the election’s context (c)
are the same in both the presidential and congressional elections, and
are not affected by either candidate. Similarly, the personal charac-
teristics of a candidate (pC) and the agreement between a voter and a
candidate’s revealed beliefs (riC) are specific to a particular candidate,
and we assume that they are not affected across elections. Instead,
we model coattails as an informational phenomenon, with signaled
beliefs (siC) as the source of coattail effects between the elections.
Intuitively, the model suggests that coattail effects occur as voters
use presidential candidates as partial proxies for local, less-known
candidates. Beyond any information known about the candidate per-
sonally, the candidate’s party affiliation is also visible to a voter­—a
­
strong signal of the candidate’s future decisions if elected. Mondak
(1990) finds that voters without much knowledge of a political race
may use their views of the presidential candidate in the same party as
a factor in their votes. Given the time cost of information collection
29
 RESEARCH
and the voter’s scarce budget of time, a typical voter spends only part
of her time considering the election, and uses the presidential candi-
date as a proxy for the views of the local candidate. We formalize this
view of election signaling as a simplified model:
siC = s(v ij, w ic) (Equation 3)
siC: signals of future agreement between voter i and candidate C
v ij: vector of expected agreement between voter i and presidential can-
didate j’s decisions
w iC: relative strength of presidential views as a proxy for congressio-
nal candidate’s views
The extent that the voter expects to agree with the presidential
Economics
candidate’s political decisions (v ij) affects the voter’s expectation of
how much she expects to agree with the lower-level candidate (v ic).
The importance of the presidential candidate as a proxy is given a
weight (w ic) that shows the extent to which the voter relies on the
presidential candidate as a proxy (which in turn is affected by the
perceived correlation between the presidential candidate and the lo-
cal candidate as well as the relative amount known about the views
of each). Assuming a candidate’s views are considered to be positively
correlated with the presidential candidate of the same party, then
∂sic /∂v ij > 0, implying that ∂Uic/ ∂v ij > 0. Thus, the probability that
the voter votes for candidate c increases with the voter’s expected de-
gree of agreement with the presidential candidate (v ij). Furthermore,
the magnitude of impact of coattails on voter choice increases with
weight, so ∂2Uic/ ∂v ij∂w ic > 0.
This model outlines a mechanism for why, ceteris paribus, a candi-
date’s popularity might be affected by a change in political popular-
ity of the presidential candidate from the same party. Since a voter
often knows more about a presidential candidate than about a local
candidate, she uses the presidential candidate as a proxy for the local
one – resulting in coattail effects.
The problem with traditional measurement approaches
For an observer trying to estimate the impact of coattail effects,
many of the variables from Equations 1 and 3 are not directly observ-
able; instead, what is observable is the median voter’s decision on the
presidential election, X ij and their decision in the lower-level election
X ic. Previous attempts at measurement, such as the C-Correlation ap-
proach, have effectively regressed lower-level election results directly
on presidential election results, or X ic on X ij. Equation 3, however,
demonstrates why this is problematic. The unobserved effect of con-
text is a factor in both X ic and X ij, causing correlation without coat-
Figure 1. House and presidential election correlation. Figure 1
shows weekly changes in prediction market prices for Democrats winning
the House (on the y-axis) against weekly changes in price for Obama win-
ning the election (on the x-axis). There is a general positive correlation
between these two probabilities.
30
Volume 2 Issue 2 | Fall 2009
tails.
To give a concrete example of this problem, in the 2008 election,
negative events in the economy tended to shift voters’ views in ways
that were favorable to Democrats in general. There was substantial
correlation between local elections and the presidential election, but
much of this relationship was due to the favorable context for Demo-
cratic policies that were shared among candidates. The correlation
in such a case could not be attributed to coattails, since it was eco-
nomic changes—rather than changes in the presidential election—
that shaped the association between the two elections.
A New Empirical Methodology
Despite the intuitive theoretical justifications for coattail effects,
attempts to measure the effects empirically have been hindered by
both causality questions and a limited dataset. To overcome these
obstacles, we propose an instrumental variable approach that uses
data from electronic prediction markets. There are three steps to our
methodology: first, we will broaden the dataset by turning to elec-
tronic prediction markets; second, we will select specific events where
the presidential elections were exogenously affected without any di-
rect effect on congressional elections; and finally, we will analyze the
impact of these events on the congressional elections.
Step 1: Broadening the dataset
In order to broaden the dataset, we use electronic prediction
market data for the 2008 election from the Iowa Electronic Markets.
We track daily closing prices for Democrats in the winner-take-all
(WTA) market for the presidential election and the seat gain WTA
market for the House and Senate elections from August 26, 2008
(the date the congressional markets opened) until November 4, 2008
(Election Day). In these markets, House and Senate prices are ag-
gregated total probabilities of the Democrats winning seats in each
respective chamber rather than looking specifically at particular dis-
tricts. Figures 1 and 2 show the association between weekly changes
in presidential markets and changes in the House and Senate races,
respectively. As expected, the figures demonstrate a clear correlation,
from which we intend to isolate the coattail effects.
Due to light trading and large bid-ask spreads, we use weekly
price changes for the congressional prices instead of daily changes.
However, if we were to compare weekly prices for each, we would un-
intentionally include correlation that does not result from coattails
by expanding our time horizon too greatly (for example, economic
changes over that time period may affect both if the time horizon is
Figure 2. Senate and presidential election correlation. Figure 2
shows weekly changes in prediction market prices for Democrats winning
the Senate (on the y-axis) against weekly changes in price for Obama win-
ning the election (on the x-axis). There is a general positive correlation
between these two probabilities.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

too long). Thus, throughout this paper, we are generally comparing
weekly congressional shifts with daily presidential shifts, assuming
that any congressional price change that occurred in the preceding
six days is in a random direction. We also remove from the dataset
all periods in which there was no trading volume. Finally, we restrict
the dataset to periods on which presidential candidate Barack Obama
gained vote share as a proxy for an election event being positive. Sum-
mary statistics are shown in Table 1.
stantial effect of major presidential events on the congressional mar-
kets that increases average gains by a factor of eight in the Senate and
a factor of four in the House.
To quantify this effect, we run a two-stage least squares regression
using the presence of a major event as an instrument. As our first
step, we regress changes in the probability of Obama winning on the
presence of a major event on days where Obama gained popularity in
the election:

Variable Obs Mean Std. Min. Max. Change_DemPres_WTA = α0 + α1 × MajorEvent + u (Equation 4)

Dev. Change_DemPres_WTA: daily price change for prediction market
outcome of Obama winning presidency
Presidential WTA Price 46 0.693 0.106 0.553 0.903 Major_Event: binary variable equal to one on days with major presi-

Economics
dential events
Pres. Daily Change 45 0.003 0.023 -0.09 0.054 u: error term

Senate WTA Price 30 0.923 0.045 0.806 0.993 We can now plug this result in to measure coattail effects. Equa-
tion 4 shows that the probability that a particular local candidate
Senate Daily Change 29 0.603 0.044 -0.149 0.059 wins the median voter’s support is a function of candidate-specific
effects (including the candidate likability and the agreement between
House WTA Price 30 0.881 0.065 0.764 0.974 the voter and the candidate’s revealed beliefs), time-specific contex-
tual effects, and changes in the popularity of the party’s presiden-
House Daily Change 29 0.002 0.054 -0.133 0.069 tial candidate. In a naïve model regressing change in congressional
Table 1. Summary statistics. Table 1 provides summary statistics for probability on change in presidential probability directly, the er-
our dataset. The Winner-Take-All (WTA) Price is the market probability ror term would include both the candidate-specific effects and the
that the Democrats win a particular election. time-specific contextual effects and would thus be correlated with
the regressor. To overcome this problem with the naïve model, we
Step 2: Choosing events instead use the predicted effect from Equation 4 as a regressor. Since
In order to construct an instrument, we establish a binary variable we constructed MajorEvents to be uncorrelated with macroeconomic
dependent on whether or not there is a major shift that affects the contextual changes and based exclusively on exogenous events in the
presidential election without directly impacting lower-level elections. campaign, its expected correlation with the error term is 0. Aggre-
While some factors in the presidential election (such as economic gating local elections into a metric of all ongoing congressional elec-
context) are also major direct factors in lower-level elections, certain tions, this instrument enhances our model for a change in probability
events only affect the presidential election directly. For example, a of the Democrats winning the election:
presidential debate—designed to influence public opinion about only
the presidential candidates—would impact congressional outcomes Change_DemCongress_WTA =
(Equation 5)
exclusively through coattail effects. After analyzing major events in β0 + β1 × Predicted_Change_DemPres_WTA + ε
the election,1 we select seven such events to set our binary variable Where Predicted_Change_DemPres_WTA = α0 + α1 × MajorEvent
equal to one: (with α0 and α1 from Equation 4)
• 8/23/08 – Obama selects Biden for VP Change_DemCongress_WTA: weekly price change for prediction
• 8/28/08 – Obama Acceptance Speech market outcome of Democrats winning seats in Congress
• 8/29/08 – McCain selects Palin for VP
• 9/27/08 – Day after 1st debate

• 10/3/08 – Day after VP debate  
• 10/8/08 – Day after 2nd debate 
    
• 10/16/08 – Day after 3rd debate 
 

To confirm that these events had a substantial effect on the presi- 
dential election, we compared prediction market price changes on
major event days in which Obama’s market price for winning the 
presidency improved. As Figure 3 shows, the average gain in the 
presidential markets nearly doubles on the dates with major events. 
Step 3: Measuring impact

Now that we have measured the impact of these events on the
presidential elections, we turn to the congressional elections to gauge 
the impact that these major, exogenous events from the presidential 
election have on the congressional markets. Figure 3 shows a sub-   
1
While a number of resources were used to select the largest events of the elec-
Figure 3. Average gains in election markets. Figure 3 displays the
tion, the Pew Research Center’s “Top 25 Events of 2008 Election” was particularly
useful. In choosing events, we selected events that were clearly exogenous to the
average daily gains for the Obama WTA contract and the average weekly
election and were confined to a discrete time period. While there were several gains in House and Senate markets on days where the closing price in-
other significant events in the Presidential election (such as Obama travelling to creased. Days with major events showed substantially larger average gains
Europe), we specifically selected for events with measurable short-term effects. in all markets.
www.thurj.org 31
 RESEARCH
Change_DemPres_WTA: daily price change for prediction market
outcome of Obama winning presidency
ε: error term
Through the two-stage least squares outlined in Equation 4 and
Equation 5, we observe the effect of an exogenous shift in presidential
preferences on Congressional prediction markets, yielding an unbi-
ased estimate of coattail effects as β1.
Results and Discussion
General results and discussion
Our results from the two-stage regression in Equations 4 and 5
Economics
are reflected in Table 2. The results display strong, significant coattail
effects in the House election but weaker, insignificant results in the
Senate. A 1% increase in Obama’s likelihood of winning is associated
with a 2.1% increase in the Democrats winning the House, but only
a 0.76% change in probability in the Senate. The coattail effect in the
House election is quite strong, as an event occurring in the presiden-
tial election has a larger effect on candidates in the House than on the
presidential candidates themselves.
The general model of coattail effects in Equation 3 helps explain
the difference in magnitude of the effects in the House and Senate.
One of the model’s core results is that a voter’s perception of the presi-
dential candidate matters in congressional elections only to the ex-
tent that the presidential candidate’s views are a useful proxy for the
congressional candidate’s views, or the magnitude of the variable w ic.
In an election where the median voter knows less about the candi-
date, coattail effects are expected to be stronger, since the party affili-
ation of the candidate carries more meaning. Typically, Senate candi-
dates are better known than their House counterparts; since senators
serve longer incumbencies, wield more personal power, spend more
on their campaigns, and have broader constituencies, they generally
capture a larger share of news coverage and political buzz. Thus, the
median voter often knows more about the candidates in the Senate
election than the candidates in the House election, and presidential
views are more necessary as a proxy in the House election.
Amplification
One of the more surprising results of this analysis is the amplifi-
cation of effects across elections. A one percent change in the prob-
ability of Obama winning is transformed into a greater than one per-
cent change in the probability of Democrats winning in the House
election. We propose two hypotheses for why amplification exists:
geographic influences and the self-fulfillment of predicted success.
In these markets, changes in voting trends in different states are not
equally important, as a vote shift in a non-swing state is not as mean-
Variable House Senate
Predicted_Change_Pres_WTA_Price 2.102 ** 0.764
[0.901] [0.587]
_const -0.020 -0.005
[0.021] [0.015]
Observations 29 29
Table 2. 2-stage least squares results. This table shows the results of
Equation 5, demonstrating the predicted effect of exogenous changes in
presidential market probabilities on House and Senate probabilities. The
House demonstrates strong, significant coattail effects, while the effects in
the Senate are minimal. Heteroskedasticity-adjusted standard errors are
displayed in brackets. ** significant at 5%
32
Volume 2 Issue 2 | Fall 2009
ingful as a vote in a swing state. The presidential swing states may
vary from congressional swing states. Thus, a presidential announce-
ment that “rallies the base” may have an amplified effect in states with
close congressional elections but that are heavily leaning towards a
particular party already in the presidential election.
An alternative hypothesis for amplification is that success is self-
fulfilling, as the perception of being likely to win an election can in-
crease the actual likelihood of winning. Campaign contributors, key
politician endorsers, and even news editorial boards are often eager
to support the winning side of an election and reap potential benefits
of early support. A company’s executives may choose to donate to
a campaign they expect will win in order to gain potential political
goodwill, and they must base their decisions on probability estimates
made weeks or months before an election. Since these acts of sup-
port may directly shift the outcome of an election (and likely have
larger effects in elections where candidates are lesser known), success
may have inertia that amplifies a small probability change into strong
gains.
Case study: Minnesota
Turning from the general application of our model across all con-
gressional and Senate elections in 2008, we now apply our model to
one specific election: the 2008 Minnesota Senate election. Because of
the perceived closeness of Minnesota’s election, the Iowa Electronic
Markets introduced a market with reasonable trading volume that
measured the vote share in state’s senatorial election. Table 3 shows
the results of applying the instrumental variable regression from
Equation 5 on Minnesota’s results. A 1% change in likelihood of
Obama’s victory is associated with a 5.865% increase in the expected
vote share for Franken (the Democratic candidate in this election).
The magnitude of the coattail effect is strikingly large, especially
for a Senate race. The difference in effect between this election and
other Senate elections is likely due to the extreme closeness of this
race – a race that was ultimately decided by just a few hundred heav-
ily-contested ballots. Thus the impact of any perceived political shift
would be heavily amplified in this ultra-close election. Furthermore,
Minnesota was not a heavy swing state in the presidential election,
and ultimately voted by a substantial margin for Obama. Thus, events
in the presidential election that resounded with liberal voters may
have had a stronger effect on the Senate election outcome than the
general election probabilities.
Limitations and further research
There are several limitations to these conclusions, and several
questions are raised for further research. First, while this methodolo-
gy could be generalized, we only used data from the 2008 elections in
this analysis due to the limited history of electronic prediction mar-
kets for Congressional elections. As more elections occur and the use
Variable Coefficient
Predicted_Change_Pres_WTA_Price 5.865 ***
[1.349]
_const -0.057
[0.035]
Observations 39
Table 3. 2-stage least squares results in Minnesota. This table
shows the results of Equation 5, demonstrating the predicted effect of ex-
ogenous changes in presidential market probabilities on the Minnesota
Senate election. The election demonstrates strong, significant coattail ef-
fects, while the effects in the Senate are minimal. Heteroskedasticity-ad-
justed standard errors are displayed in brackets. *** significant at 1%
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
of electronic prediction markets expands, a broader analysis could
be performed on the impact of coattail effects on elections and the
determinants of the magnitude of the effects. In particular, the 2008
election was unique in recent elections because of the relative cer-
tainty of the eventual outcome for Senate and House control, as the
probabilities that Democrats would control each chamber were over
90% for much of the period evaluated. Thus, an analysis performed
in a year when both the presidential and congressional elections are
expected to be close may yield different results.
As prediction markets continue to mature, another potential ex-
pansion would be to observe coattail effects on a state-by-state level.
While state-by-state election details from electronic prediction mar-
kets were limited in the 2008 election, further expansion in market
availability and trading volume would enable a study on state-specific
coattail effects and determinants of the magnitude of coattails.
Finally, several assumptions were made in choosing to use elec-
tronic prediction market prices as proxies for probabilities of actual
outcomes. First, we assumed that they are unbiased, reasonably accu-
rate estimators, claims that are generally supported by both empiri-
cal and theoretical literature on prediction markets. A more subtle
assumption is that changes in prediction market prices are due to
events impacting the election directly, rather than merely changing
perceptions of the candidates themselves by market participants. For
example, an eloquent speech by a candidate would not only boost
his chances in the election directly, it might also signal to predic-
tion market participants that he will be similarly eloquent in the
future—and traders will factor these future events into the market
price. This effect would likely be particularly magnified early in an
election, since market participants know less about the strategic and
tactical competence of particular candidates. In order to mitigate this
effect, we selected events for this study that occurred relatively late in
the election and would likely have a primary effect of directly alter-
ing the election, such as the selection of a vice-presidential candidate.
Furthermore, even if the market changes its perception of a candidate
and incorporates information about future expected events because
of one of the events we selected, as long as those future events also
generate coattails, they will also be incorporated into congressional
prediction markets, leaving our estimate unbiased.
Concluding Remarks
Our results provide an intriguing insight into one of the major
determinants of United States elections outcomes. Using electronic
prediction market trading data, we find that coattail effects have
a major impact on House elections, but a more limited impact on
Senate elections. By using major exogenous shifts in the presidential
election as an instrument, we isolate the coattail effects in the elec-
tion and overcome causality concerns intrinsic to most past studies
of empirical effects.
While this result generally confirms our intuitive expectations
about coattail effects, the methodology provides an intriguing new
way to study major factors shaping elections. Similar to coattail ef-
fects, many factors that intuitively have large effects on elections are
difficult to show empirically, since elections occur only once every
few years and substantial differences in context occur from election
to election that complicate any attempts to hold multiple factors con-
stant. By using market prices of a particular outcome as a proxy for
the status of an election, electronic prediction markets promise to
www.thurj.org
RESEARCH
widely expand the data set available for analyzing election phenom-
ena. Nonetheless, most literature on electronic prediction markets to
date has been either theoretical in nature or an empirical measure-
ment of the market’s predictive ability. By assuming the markets are
in fact reasonably accurate (which most analysis supports), we use
prediction markets as robust, real-time indicators of the status of an
election. Our results indicate that – beyond their predictive value –
prediction markets can also be a useful tool in isolating empirical
effects in presidential elections and uncovering the determinants of
political success.
References
1. Berg, J. et al., “Results from a Dozen Years of Election Futures Markets
Economics
Research.” Working Paper (2001).
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3. Erikson, Robert et al. “Was the 2000 Election Predictable?” Political Sci-
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Perspective.” American Journal of Political Science, Vol. 28, No. 1 (Feb.,
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paign Spending on Election Outcomes in the U.S. House.” The Journal of
Political Economy. Vol. 102, No. 4 (Aug., 1994), pp. 777-798.
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Methodology.” The Public Opinion Quarterly, Vol. 19, No. 4 (Winter
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sults/president/map.html. Accessed February 2009.
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wide Presidential Votes, 1988-96.” The American Political Science Review,
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grow; Over $28 million spent from September 28-October 4.” Univer-
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33
 RESEARCH Volume 2 Issue 2 | Fall 2009

Element of survival: Isolating the causal

effect of access to iodized salt on child
health in India
Shubha Bhat
Harvard University ‘09, shubhat@gmail.com

Despite India’s longstanding efforts to combat Iodine Deficiency Disorders through a Universal Salt Iodization pro-
gram, only 51% of households were using iodized salt in 2005. In order to justify efforts to actively expand the pro-
gram, it is crucial to establish a causal link between access to iodized salt and child health. This study examined
household salt iodine concentration, anthropometric outcomes, and birth histories of over 18,000 children from the
1998 India National Family Health Survey. To isolate causality, two-stage least squares (2SLS) regressions were used
with state-fixed-effects. Innovative district-level instruments were constructed using Geographic Information Sys-
tems to predict salt iodine concentration in households and targeting efforts of the government. The 2SLS estimate
and Medicine
Public Health

revealed that increasing the iodine level in salt led to a 1.168 standard deviation increase in height-for-age (p<0.05),
a 15.9% decreased likelihood of having below-average birth weight (p<0.05), a 18.6% increased likelihood of having
above-average birth weight (p<0.05), and a 2.8% increase in child survival (p<0.10).

Introduction
Iodine deficiency disorders (IDD) are one of the most common
causes of preventable mental retardation (X. Y. Cao et al., 1994). A
meta-analysis of 18 studies of 2214 subjects comparing the perfor-
mance of iodine-deficient children with that of iodine-sufficient peers
on a standardized intelligence tests, concluded that iodine deficiency
lowered the mean intelligence quotient by 13.5 points, which indi-
cates a staggering public health problem (Bleichrodt and Born 1994).
This shortcoming affects a child’s ability to learn, and later in life, to
earn. In this way, the negative effects iodine deficiency on both men-
tal and physical health can significantly impede worker productivity
and the economy at large. Clearly, eliminating iodine deficiency has a
significant impact on the world’s poor.
To combat IDD, the WHO and UNICEF recommended in 1994
the use of iodized salt as a safe, cost-effective and sustainable strategy
to ensure sufficient intake of iodine by all individuals. However, de-
spite pushing a Universal Salt Iodization program, progress has been
limited. In India, which is a major salt-producing country, only 51%
of households were using iodized salt in 2005. This is a particular
problem since out of 38 million newborns in developing countries
every year that remain unprotected from the lifelong consequences
of IDD-related brain damage, a large percentage live in South Asia,
as shown in Figure 1 (UNICEF 2008).
However, to justify efforts that the Indian government is making
to improve the system, it is important to understand what effect the
lack of iodized salt coverage has had on public health. Specifically, it
is necessary to determine whether IDD affects the physical as well as
mental capacities of children in India. Thus, identifying the causal
link between iodine deficiency and child survival and growth will
more effectively direct the appropriate resources toward its correc-
tion. In doing so, this study also aims to contribute to the method-
ological literature on measuring intervention impacts.
Figure 1. Children unprotected from IDD (2000-2006).
Methodology
Datasets
The primary dataset used in this study was from India’s second
National Family and Health Survey (NFHS-2), which was conducted
from 1998-1999 by the International Institute for Population Sciences
(IIPS) in Mumbai (IIPS and Macro 2000).1 This dataset was ideal
for two reasons. First, it captured key socioeconomic, cultural, and
health information about over 91,880 households, 90,303 women and
33,132 children in India. Second, it provided district-level identifiers
for each household, which was not available in the most recent 2005
NFHS-3 because of privacy issues. This was crucial because it allowed
for the use of within-state variation by district in order to estimate
causality, which is particularly helpful for assisting policymakers
in planning and implementing strategies for improving population
health and nutrition programs.
Six secondary datasets were superimposed on a map of India and
linked to the NFHS-2 through district-level household identifiers to
create a series of district-level control and instrumental variables.
Measurements were made using the ArcGIS9 (ArcMap Version 9.3)
1
At the national level, the overall sample weight for each household or woman
is the product of the design weight for each state (after adjustment for non-
response) and the state weight. I use these sample weights in my main results.

34 The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009
software. First, MLInfoMap: 1991 Census provided map data on
district borders, area and population. Second, the Global Precipita-
tion and Climatology Center’s Full Data Reanalysis Project provided
information on average annual rainfall for each district. Third, the
Shuttle Radar Topography Mission provided 90-meter by 90-meter
raster images of elevation. Fourth, the Global Self-consistent, Hier-
archical, High-resolution Shoreline (GSHHS) Database Version 1.3
provided information on distances to the closest coast. Fifth, Collins
Bartholomew World Premium 2007 provided information about the
main and secondary railway lines, as well as the road networks in
India. Sixth, the Government of India Salt Department’s 2005-2005
Annual Report provided information on how much salt was trans-
ported to each state by railroad, road, or sea.
Variables
The explanatory variable of interest in this paper was the iodine
level of the salt used in a household. In NFHS-2, interviewers mea-
sured the iodine content of cooking salt in each interviewed house-
hold using a rapid-test kit and recorded the iodine level as 7, 15, or 30
parts per million (ppm) (IIPS and Macro 2000).
Overall, half of households used cooking salt that was iodized at
the recommended level of 15 ppm or more, one-quarter of house-
holds used salt that was not iodized at all, and 21 percent used salt
that was inadequately iodized (less than 15 ppm). The use of iodized
salt varied dramatically from one state to another (as shown in Figure
2). The variations could be due to a number of factors, including the
scale of salt production, transportation requirements, enforcement
efforts, pricing structure, and storage patterns. In particular, salt io-
dization was likely to be more common in states where salt was trans-
ported exclusively by railways, partly because the Salt Department
monitored the iodine content of salt shipped by railways.
The five child health outcomes that this paper examined were
height-for-age, weight-for-height, likelihood of being born small,
likelihood of being born large, and overall child survival. These out-
comes were based on NFHS-2 anthropometric data of 18,521 chil-
dren ages 0-5 years, and NFHS-2 birth history of 21,388 children of
all ages.
Figure 2. Iodine concentration.
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RESEARCH
The first two outcomes, children’s height-for-age and weight-
for-height were evaluated relative to the median height-for-age and
weight-for-height of the 1997 U.S. National Center for Health Sta-
tistics (NCHS) reference population, recommended by the World
Health Organization. Measures greater than two standard deviations
below the reference median considered stunted (height-for-age) or
wasted (weight-for-height). Stunting is a sign of chronic, long-term
under-nutrition, whereas wasting is a sign of acute, short-term un-
der-nutrition. It was therefore hypothesized that access to household
iodine would lead to improved height-for-weight outcomes and have
no effect on weight-for-height outcomes. Such a result would support
the fetal origins of disease hypothesis.
On average, children in India were borderline stunted at 1.91
standard deviations below the reference population. Those children
living in households with salt iodine content of 0, 7, 15 and 30ppm
were 2.06, 2.07, 1.98 and 1.67 standard deviations below the median,
respectively. Thus, there is a clear corresponding trend between io-
dine concentration and height-for-age outcome. On the other hand,
though children were on average 0.78 standard deviations below the
reference median, they were not low enough to be considered wasted.
However, unlike the prediction, there seemed to be a similar rising
trend of weight-for-height (0.96, 0.89, 0.75 and 0.6 standard devia-
and Medicine
Public Health
tions below the reference), which corresponded with rising iodine
concentrations (0, 7, 15, and 30ppm, respectively). Such a pattern pos-
sibly indicates that there are other factors such as standard of living
that may be driving these similar trends.
The third and fourth outcomes, likelihood of being born small
and large, were chosen because small newborns generally face sub-
stantially higher risks of dying than do newborns of normal or large
size. The average birth weight of the small, average and large children
was 2.2 kg, 2.8 kg and 3.4 kg, respectively. According to mothers’ esti-
mates, 25 percent were small, 60 percent were average, and 14 percent
were large. As the salt iodine concentration went up, there was also
a corresponding decrease in fraction of children born small and in-
crease in fraction of children born large.
The fifth and final health outcome examined was childhood sur-
vival, which was calculated as the fraction of children still living over
the total number of children ever born to each woman. In total, the
child survival rate was 91 percent. In this case, when broken down by
iodine concentration, there seemed to be no difference in child sur-
vival rates within households that had 0, 7 or 15ppm. However hav-
ing 30ppm iodized salt corresponded with a 93 percent survival rate.
Estimation Strategy
To test whether trends in health outcomes are driven by iodine
concentration and not other factors ordinary least squares (OLS) and
two-stage least squares (2SLS) regressions were conducted. Below,
the empirical specification for the OLS regressions run in this analy-
sis is presented:
Y = α+β1(I)+β2(M)+β3(H)+β4(D)+β5(S)+ε   ( Equation 1)
In equation 1, Y is the child health outcome of interest. This in-
cludes height-for-age standard deviations from the international
mean, weight-for-height standard deviations from the international
mean, child’s size at birth, and child survival. The α is the constant,
β1 is the coefficient for I, the explanatory variable. Iodine levels of
1, 2, 3 and 4 correspond with iodine concentrations of 0, 7, 15 and
30 parts per million, respectively. β2 is the vector coefficient for M,
the mother-level controls. These include characteristics such as age,
education level, possession of a health card, body-mass-index, and
health status (smoking habits, alcohol consumption, tobacco use, TB,
jaundice, asthma, and malaria). β3 is the vector coefficient for H, the
35
 RESEARCH
Figure 3 Figure 5
Figure 4
and Medicine
Public Health
Figure 3. Railroad.
Figure 4. Road length.
Figure 5. Precipitation.
Figure 6. Elevation.
Figure 7. Coast.
household-level controls. These
include characteristics such as
the standard of living index,
Figure 7 urban environment, and reli-
gion of household head. β4 is
the vector coefficient for D, the
district-level controls. These
include district population
density and area. β5 is the vec-
tor coefficient for S, the state-
fixed effects. Finally, ε is the
error term.
The disadvantage with OLS
is that there are several unob-
servable characteristics that
cannot be controlled for which
may affect both the outcome
and explanatory variable. Al-
though OLS helps identify pat-
terns, it is difficult to use an
OLS estimation to determine
causality. Therefore, a two-stage least squares (2SLS) methodology
is used. The 2SLS technique employs instrumental variables, which
are supposed to be highly correlated with the outcome variable only
through a correlation with the explanatory variable. Below, the em-
pirical specification for the 2SLS regressions run in this analysis is
presented:
Î = α+β 1(Z)+β 2 (M)+β 3 (H)+β 4 (D)+β 5 (S)+ε (Equation 2)
Y = α+β 1(Î)+β 2 (M)+β 3 (H)+β 4 (D)+β 5 (S)+ε (Equation 3)
36
Volume 2 Issue 2 | Fall 2009
Figure 6
Equation 2 corresponds with the first stage of the 2SLS estimate. Î
is the predicted value of household salt iodine. Unlike equation 1, β1
in equation 2 is the vector coefficient for Z, the instruments. These in-
clude both institutional and geographic instruments. The institution-
al instruments include distance to any railroad (Figure 3), distance to
the nearest main railroad, fraction of salt transported to the state by
railroad, and total road length in the district (Figure 4), which pre-
dict access to iodized salt. The geographic instruments include aver-
age district precipitation (Figure 5), elevation (Figure 6), and distance
to the nearest coastline (Figure 7), which predict baseline endemic
iodine deficiency.
Equation 3 corresponds with the second stage of the 2SLS esti-
mate. Here, Y is the child health outcome of interest. β1 is the coef-
ficient for Î, the iodine level predicted by the instruments. As before,
the remaining coefficients correspond with mother-, household-, dis-
trict- and state-level controls.
The 2SLS regressions were also broken down to capture heteroge-
neous treatment effects on male versus female children and on urban
versus rural households to determine whether the effect of iodized
salt had a greater impact on certain groups. Finally, three robustness
checks were carried out to assess how sensitive the results were to the
econometric specification.
Results
When district-, household-, and mother-level observable charac-
teristics were controlled for in the OLS regression (Column 1, Table
5), it was found that increasing the iodine concentration by one level
(i.e. 0 to 7ppm, 7 to 15ppm, or 15 to 30ppm) correlated with a 0.0375
standard deviation increase in height for age (p<0.01). The 2SLS esti-
mate (Column 2, Table 5) revealed that increasing iodine concentra-
tion by one level led to a 1.168 standard deviation increase in height
for age. Looked at another way, increasing the salt iodization con-
centration by two levels, from 0 to 15ppm (the concentration recom-
mended by the WHO) led to a 2.336 standard deviation increase in
height for age. Essentially, such an effect would put children in India
(who on average are at 1.91 standard deviations below the median) at
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Height-for-Age Weight-for-Height
1 2 3 4
OLS 2SLS OLS 2SLS
Iodine 0.0375*** 1.168*** 0.0256** -0.0341
(0.0131) (0.311) (0.0107) (0.190)
n 20328 18521 20328 18521
Table 5. Effect of iodine concentration on child health outcomes. Controls = State fixed effects, population density, district area, standard of
living, urban, hindu, muslim, christian, mother’s characteristics (age, education, has health card, bmi, smoking habits, alcohol and tobacco use, jaundice,
malaria, TB, asthma). Instruments = distance to railroad, distance to main railroad, %salt transported to state by railroad, total road length, annual
precipitation, elevatin, distance to coast (F = 153.22). All regressions are run with sample weights. * p < 0.10, ** p < 0.05, *** P < 0.001; Robust standard
errors in parentheses.
just over normal, on the scale of international growth standards.
Contrary to the prediction, in the OLS estimate (Column 3, Table
5), an increased iodine level correlated with a 0.0265 standard de-
viation increase in weight-for-height outcomes for children (p<0,05).
However, as expected, the significance of this estimate became nil in
the 2SLS (Column 4, Table 5).
Increasing the iodine concentration by one level led to a statisti-
cally significant (p<0.05) but small (0.008 percent) decrease in the
likelihood of being born small in the OLS estimate (Column 5, Table
5). This value became much larger and more significant in the 2SLS.
As shown in Column 6, Table 5, increasing iodine concentration level
led to a 16% drop in the likelihood of being born small (p<0.01). The
correlation between iodine concentration level and likelihood of be-
ing born large was insignificant in the OLS regression (Column 7,
Table 5). However, iodine concentration seemed to have a large and
significant effect in the 2SLS (Column 8, Table 5). An increase in io-
dine level led to an 18.6% increase in likelihood of being born large
(p<0.01).
The OLS and 2SLS estimates (Columns 9 and 10, Table 5) showed
that an increase in salt iodine concentration level led to a 0.22 per-
cent and a 2.8 percent increase in child survival rates, respectively
(p<0.1).
When the sample was broken down by gender and place of resi-
dence, there were significantly greater effects of iodized salt on fe-
male children and rural households.
Discussion
The goal of this study was to establish a causal link between access
to iodized salt and child health outcomes. Showing the direct relation
between iodine deficiency and child survival and growth would help
motivate researchers and policy-makers to identify the barriers that
prevent India from reaching more widespread use of iodized salt. In
addition, illuminating the differential effects of access to iodized salt
on subpopulations would help direct resources more appropriately.
This economic analysis is unique because it utilized Geographic
Information Systems to measure precise distance and geographic
information and connected this information with the district codes
of households in this sample. Used together as instruments, the GIS
data served to identify causal effects where a controlled experiment
was not possible. In addition, because the analysis used the 1998
NFHS, it covered a representative sample of the Indian population as
a whole. The timing of the survey captured the effect of five years of
universal salt iodization program in India and might have captured
the ban on non-iodized salt that had been instated in 1998.
The results of this study suggest that access to iodized salt posi-
tively impacted children’s height-for-age outcomes, especially for
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RESEARCH
Small Child Large Child Child Survival
5 6 7 8 9 10
OLS 2SLS OLS 2SLS OLS 2SLS
-0.00798** -0.159*** 0.00222 0.186*** 0.00167* 0.0281*
(0.00314) (0.0556) (0.00250) (0.0517) (0.000999) (0.0163)
23441 21388 23441 21388 23450 21396
children living in rural settings. The magnitude of the effect is sig-
nificant since increasing iodine concentration from 0 to 15 ppm
seemed to drive the average height-for-age standard deviation from
-1.91 (borderline stunted) to nearly +0.43 standard deviations above
the NCHS standard. Such an effect can have a significant effect on
the future productivity of the nation’s children. However, iodized
salt might not be the answer to the problem of childhood wasting.
and Medicine
Public Health
This is not a huge concern since the average weight-for-height was
not close to 2 standard deviations below the NCSH standard, which
defines someone as wasted. In addition, the results suggest that use
of iodized salt by mothers might reduce the likelihood of being born
small and increase the likelihood of being born large, especially for
girls and children born in rural areas. Finally, there seemed to be a
positive effect of iodine on child survival, especially for males.
It is important to note that only two of the five outcomes—height-
for-age and likelihood of being born large—passed the greatest num-
ber of robustness checks. This indicates that we can only begin to
extend the fetal origins of disease hypothesis to these two outcomes.
However, since even these two outcomes failed to pass the most strin-
gent robustness check, conclusions can only be made with some de-
gree of caution.
There are several measurement issues that this analysis faces. In
this sample of the NFHS-2, the reference population used in calcu-
lating anthropometric outcomes was the 1997 U.S. National Center
for Health Statistics (NCHS) standard. More recently however, a new
international reference population was released by the WHO in April
2006 and accepted by the Government of India, which may better as-
sess children regardless of ethnicity, socioeconomic status and type
of feeding. In addition, the set of child health outcome variables fo-
cusing on size of the newborn are especially prone to selection and
reporting biases, since mothers tend to self-report their children as
being bigger than they actually are, since size is considered a measure
of health. Finally, the way that I calculated child survival was simply
the number of children living over the total number of children born.
It is unclear whether such a measurement can really capture the in-
tricacies of child mortality early in life.
For the explanatory variables, an assumption was made that if
a household had iodized salt at the time of the survey, it probably
has had access to that level of iodized salt throughout the universal
salt iodization program, which became more active starting in 1992.
However, this assumption may not necessarily hold. The practices of
households in 1998 do not necessarily have to reflect the practices of
the household five years before.
The instruments used in this analysis showed high relevance in
the first-stage regression, yielding high F-stats and significant cor-
relations with the explanatory variable. However, the exogeneity re-
quirement was less convincing. Though precipitation and elevation
37
 RESEARCH
seem to be less of a problem, distance to the coast and distance to rail-
roads were arguably endogenous. Presumably the closer a household
is to a railroad or ocean, the more resources the household can access
and therefore the higher standard of living it may experience.
The robustness check limiting the set of instruments to only dis-
tance to railroad revealed insignificant results, showing that the main
findings might be sensitive to the instruments chosen. Moreover this
dataset was limited to district level data. Therefore, it is possible that
the instrument used in the most stringent specification did not have
enough variation to predict access to iodized salt accurately. Having
exact coordinates of households would enable a more robust analy-
sis.
Conclusion
This paper revealed that an increase in salt iodine levels led to pos-
itive and statistically significant child health outcomes—namely, a
1.168 standard deviation increase in height-for-age, a 15% decreased
likelihood of being small at birth, a 19% increased likelihood of be-
ing large at birth, and a 2% increase in child survival. The paper also
provided evidence that access to iodized salt had stronger effects on
female children and on children living in rural settings. Therefore,
and Medicine
Public Health
targeting certain populations could potentially have a more power-
ful impact on child health. This study is innovative in that it utilized
unique instruments constructed through GIS data to predict access
to iodized salt and isolate its causal effects on childhood health. Us-
ing this estimation strategy, the paper shows that the Indian govern-
ment would do well to continue promoting access to iodized salt in
order to secure the health and well-being of its children.
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Acknowledgements
I would like to thank the following people for their tremendous support
throughout this research process. Without their help and encouragement, this
original work would not have been possible. Erica Field, Assistant Professor of
Economics, Harvard University, for being a wonderful thesis advisor and contin-
ually guiding me in the right direction. Winnie Fung, PhD candidate in Econom-
ics, Harvard University, for being my thesis tutorial leader and helping me clarify
my questions and methodology. Konstantin Styrin, PhD candidate in Economics
and STATA Teaching Fellow, Harvard University, for answering my countless
STATA coding questions. Sebastian Linnemyer, PhD candidate in Economics
and Teaching Fellow, Harvard University, for his advice in the Ec980 Junior Tu-
torial, “Development, Education, and Health.” Scott Walker, Digital Cartogra-
phy Specialist, Harvard Map Collection, for spending so much time teaching me
about GIS, providing me with data and helping me construct map images. Bon-
nie Burns, GIS Specialist, Harvard Map Collection, for helping me identify the
1991 India Census Data. Fred Arnold and Bridgette James, DHS/NFHS Archives,
for providing me with district coding for the NFHS data. Joan Curhan, Debbie
Whitney, Suzanne Scudder, for supporting me in the Certificate in Health Policy
Program. The Cordeiro Family, for providing me with a generous grant to travel
to India to continue on-the-ground research in July 2008. Darpana Academy of
Performing Arts, for giving me the opportunity during my Fall 2007 semester
in India, to travel to Valsad (pictured above), a rural district in Gujarat, and be-
come involved in a UNICEF development project that inspired this thesis. Dr.
Chandrakant S. Pandav and Dr. Arijith Chakrabarty, International Council for
Control of Iodine Deficiency Disorders (ICCIDD), All India Institute of Medical
Sciences, New Delhi, for spending time sharing their insight with me on the most
recent accomplishments and challenges of the Universal Salt Iodization Program
in India. Dr.Rajan Sankar, Regional Manager for the South Asia branch of Global
Alliance for Improved Nutrition, New Delhi, for providing me with extensive
literature on the history of iodine deficiency. Mr. S. Sunderesan, Salt Commis-
sioner, and Mr. M.A. Ansari, Deputy Salt Commissioner at the Ministry of Salt
in Jaipur, Rajasthan, for communicating with me via email regarding questions
about the transportation and production of salt throughout the country. Tracy
Li, Peter Ganong, Chethan Bachireddy, Prithvi Shankar, and all my friends and
classmates for editing, giving me STATA and formatting tips, and helping make
this process enjoyable. My parents, for their unconditional love.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Olfactory bulb glomerular responses of

mice in different behavioral states
David Blauvelt
Harvard College ‘09, smhdavid@gmail.com

One of the first steps in processing a smell occurs at the glomerular layer, which is where axons of olfactory receptor
neurons make synapses on the dendrites of mitral cells. While olfactory receptor neurons’ pre-synaptic activity have
been extensively researched in anesthetized mice, little is known about odor-evoked synaptic activity in awake, freely
moving animals. This project attempts to address this problem by adapting a fiber optic bundle imaging technique
to the olfactory bulb. The results show that while behavior causes no consistent changes in olfactory receptor neuron
activity across all subjects and odors, it does seem to be linked to differences in the nature of odor responses in indi-
vidual mice. Importantly, this study is the first demonstration that a fiber optic imaging bundle can be used to image
the olfactory bulb of an awake, behaving animal.

Introduction
One of the fundamental concerns of neuroscience is to under-
stand how the brain perceives and processes the outside world. From
the first stage of detection by sensory nerves to the forging of a mem-
ory of an event, our brains are dynamically interacting with our sur-
roundings. Olfaction is one of the ways we sense the world, yet the
study of the olfactory system has many unanswered questions. How
do we process different odors? How do we forge and retrieve odor
Wilson and Mainen, 2006). PG cells receive stimulatory input from
ORNs, M/T cells, and even other PG cells. They then transmit inhibi-
tory signals reciprocally to the ORN axons and the M/T dendrites of
the exciting glomerulus. The other type of juxtaglomerular cell is the
short-axon cell, which indirectly inhibits M/T cells by exciting PG
cells. Contrary to what the name suggests, a short axon cell is a long-
range regulatory cell and may act on PG cells several glomeruli away
(Albeanu, 2008, Aungst et al., 2003, Wilson and Mainen, 2006).
Mitral/tufted cells also communicate with each other indirectly

memories? How do our different mental and physical states affect
how we respond to odor stimulation?
Sensing an odor begins with the olfactory receptor neurons
(ORN) located on the nasal epithelium (Wilson and Mainen, 2006).
A single mouse possesses approximately 44 million ORNs (Albeanu,
2008, Mombaerts, 2006, Wachowiak, 2004), with each ORN contain-
ing G-protein coupled receptors known as the olfactory receptors
(OR). In order to remain functionally distinct, a single ORN only
expresses one or perhaps a small number of different types of ORs
(Albeanu, 2008, Mombaerts et al., 1996). Every OR can detect mul-
tiple odors, and many types of ORs detect each odor. However, each
odor is sensed by a unique combination of ORs that allows it to be
distinguished from other odors (Malnic et al., 1999). The axons of
the stimulated ORN transmit the sensory signal to approximately
two locations in a single olfactory bulb known as glomeruli, which
are simply the synapses joining the ORN axons with the dendrites
of mitral/tufted (M/T) cells, which output the signal to the olfactory
cortex (Albeanu, 2008, Mombearts et al. 1996). Since the glomeruli
are spatially defined functional clusters, the glomerular layer can be
viewed as an odor map for which each odor is encoded by a unique
combination of glomerular activation (Mombaerts et al., 1996). Once
the signal reaches the glomerular layer, it is passed to the mitral cell
layer, which contains the M/T cells (Wilson and Mainen, 2006).
The structure of the olfactory system, however, is not a simple re-
lay. Rather, the system is subject to lateral modulation from regulato-
ry cells. Each glomerulus is surrounded by different types of regula-
tory juxtaglomerular (JG) cells, including periglomerular (PG) cells
and short axon (SA) cells (Albeanu, 2008, Wachowiak and Shipley,
2006). Periglomerular cells, the more populous of the interneurons
in the glomerular layer, are inhibitory regulators (Albeanu, 2008,
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Neuroscience
and directly. M/T cells form dendrodendritic synapses with granule
cells in the external plexiform layer, and can inhibit other M/T cells
through granule cells (Wilson and Mainen, 2006). M/T cells may
also directly influence their neighbors through a process known as
spillover. When the M/T cells release glutamate through their den-
drites, some of the glutamate excites dendrites of neighboring M/T
cells (Isaacson, 1999, Wilson and Mainen, 2006).
Finally, the olfactory system is influenced by centrifugal inputs
from the cortex and other areas of the brain (Isaacson, 1999). These
inputs may be affected by the behavior of the mouse, genetic predis-
positions, and other factors. Centrifugal influences can be separated
into three major categories. First, odor intake is affected by areas of
the brain outside of the olfactory bulb. Passive sniffing rate is deter-
mined by central pattern generators in the medulla (Ramirez and
Richter, 1996, Wilson and Mainen, 2006) while active respiration
is controlled by the forebrain (Wilson and Mainen, 2006). Second,
the bulb receives reciprocal feedback from output regions such as the
olfactory cortex. Finally, changing levels of neuromodulators such
as norepinephrine, acetylcholine, and serotonin can affect odor re-
sponses (Wilson and Mainen, 2006).
The complexity of the olfactory system, designed to accomplish
what may seem like a simple task, is astounding. This intricacy makes
the system susceptible to outside influences such as alterations in be-
havior. Most in vivo imaging research on the olfactory system exam-
ines the bulb while an animal is in an anesthetized state. However,
anesthesia greatly limits the scope of the field because it makes it im-
possible to study the effect of behavior on olfactory response.
One way in which an animal’s behavioral state may affect its re-
sponse to odors is by influencing the internal bulb activity. Recently,
a team headed by Kenasku Mori studied the dendro-dendritic activ-
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 RESEARCH
ity in the external plexiform layer (Tsuno et al., 2008). They found
that mitral-granule activity was strongest in anesthetized animals
and was progressively weaker in lightly sleeping, awake and immo-
bile, and awake and mobile animals.
In addition to direct effects upon the olfactory bulb, behavior may
affect centrifugal inputs. For instance, while the sniffing rate of an
anesthetized animal is passively controlled by the brainstem, a be-
having animal has the ability to actively control this rate. Further-
more, the levels of norepinephrine, acetylcholine, and serotonin can
be altered by different behavioral states (Wilson and Mainen, 2006).
As an example, Shea et al. (2008) found that norepinephrine release,
when coupled with odor presentation, acts in the olfactory bulb to
cause suppression of paired odor responses.
To more fully understand the effects of different behaviors on
odor responses, the olfactory bulb needs to be monitored while the
animal is awake and behaving. While electrical activity in behaving
animals has been extensively studied, they are limited to single cell
readouts as opposed to imaging studies, which allow for large-scale
population readouts. Sub-glomerular resolution imaging studies of
the olfactory bulb in awake animals have been performed on head-
fixed animals (Carey et al., 2004), but are limited. Furthermore, to
date, sub-glomerular resolution imaging of the olfactory bulb in
freely behaving animals has not yet been accomplished. The present
study attempts to address this issue by offering a way to image the
bulb in a freely behaving mouse.
Two important advances have made it possible to explore odor
responses in freely behaving mice. The first was the creation of trans-
genic mice expressing synaptopHluorin in the ORNs, which allowed
the visualization of pre-synaptic glomerular activity in the olfactory
bulb. pHluorins is a mutant form of GFP that was sensitive to pH and
would fluoresce in a neutral environment but not in an acidic envi-
ronment. SynaptopHluorin (spH) was then made by fusing pHluorin
Neuroscience
to synaptobrevin (Miesenböck et al., 2000). Synaptobrevin is a vesicle
protein required for the release of neurotransmitters into the syn-
apse. The idea is to take advantage of the acidic environment of the
neurotransmitter vesicles (pH ~5.7). When inside the vesicles, spH
would not fluoresce, but once a vesicle binds with the cellular mem-
brane to release neurotransmitters into the neutral pH of synaptic
space (~7.4), spH would fluoresce (Miesenböck et al., 1998). This laid
the groundwork for the use of synaptopHluorin as a genetically en-
coded molecular probe that would allow detection of neural activity
using simple fluorescence microscopy.
Though there are many other ways to image ORN synaptic activ-
ity such as nasal calcium dye injections, bulk calcium dye loading,
and intrinsic signaling (Toga and Mazziotta, 2002, Albeanu, 2008),
synaptopHluorin was used because of three significant advantages.
First, it is genetically encoded unlike methods involving calcium
dyes, which need to be exogenously loaded. Second, the signal is
mostly specific to pre-synaptic activity. Finally, because it is localized
to the axon termini, spH makes it possible to more easily distinguish
individual glomeruli.
The second necessary development for this study was the intro-
duction of an imaging technique using a flexible fiber optic imag-
ing bundle attached to the skull. This method was chosen over other
methods such as the head-fixed method (Carey et al., 2004) specifi-
cally because it allows the mouse to freely move around while keep-
ing the fluorescence image in the focal plane of the microscope.
The first fundamental question that this study attempts to answer
is whether the fiber technique is a viable way to image freely behav-
ing animals. This technology is still in its very early stages, and has
never been used to image the olfactory bulb. However, after adapting
and optimizing the procedure, this technique will presumably offer a
40
Volume 2 Issue 2 | Fall 2009
good way to image freely behaving animals.
The second question of this study is whether behavior has an effect
on the pre-synaptic activity of glomeruli. As discussed earlier, there
are several ways that behavior could influence ORN output. Thus,
it seems likely that behavior will cause some change in glomerular
activity, although attributing observed changes to specific individual
behaviors as well as parsing out the causes is beyond the scope of
this study. In order to test this hypothesis, the aforementioned fiber
optic technique was used to image transgenic mice expressing syn-
aptopHluorin in the glomeruli. In addition, a customized odor rack
was employed to expose the mice to different odors while monitoring
the fluorescence response in real-time. The same mice were imaged
in both an awake and an anesthetized state, and the responses were
compared.
Fundamentally, the goal of this study is simply to open the doors
to a burgeoning field of new imaging techniques while answering
some questions about how behavior can affect olfactory sensory ac-
tivity. First, this project attempts to determine whether a fiber op-
tic imaging bundle provides a way to image the olfactory bulb of an
awake, freely-moving animal. Second, this study uses the bundle to
track olfactory receptor neuron pre-synaptic activity in anesthetized
and freely-behaving mice to find similarities and differences based
on behavioral state.
Methods
Subjects and surgery
The subjects used were transgenic adult (postnatal days 60-100)
synaptopHluorin mice, including both heterozygous and homozy-
gous as well as male and female mice (Bozza et al., 2004). Mice were
anesthetized for surgery with a cocktail of ketamine and xylazine
(ketamine - 100 mg/kg, IP, Fort Dodge Animal Health #440761; 10
mg/kg xylazine, IP, Phoenix Pharmaceutical, Inc. #4111505). The
mice were mounted on a stereotaxic frame and the skin was cut to
expose the skull. A small hole, approximately 1.5-2 mm in diameter,
was opened over the right olfactory bulb by thinning the bone in a
circle and removing the piece. A flexible fiber bundle (Schott Inc.,
#1137189) with a 1.45 mm outer diameter was lowered onto the brain.
The other end of the bundle was placed in the focal plane of a wide-
field fluorescence microscope fitted with a 10x objective and a high
speed imaging camera (SensiCam, Cooke Corp.). The bundle was
lowered using an XYZ translator until glomeruli became clear. The
fiber bundle provided a continuous two-way light path from the mi-
croscope to the glomeruli and back to the microscope, allowing the
mouse to move around away from the microscope while still keep-
ing an image of the glomeruli within the focal plane of the micro-
scope. Once the bundle was in the proper place on the brain, it was
cemented onto the skull with RelyX Luting Plus (3M ESPE, #3525).
The mouse was allowed to recover for several hours before the analge-
sic, Buprenorphine HCl (0.5 mg/kg, BD, IP, Bedford Labs, #1208141),
was given. Anesthetized imaging occurred immediately after surgery
while the mouse was still anesthetized. Behavioral imaging was per-
formed after recovery.
Imaging fiber optic bundle
There are two issues regarding the properties of an imaging fiber
bundle that need to be addressed. The first is the pixelation of the
image. The bundle was composed of thousands of microscopic fibers,
each 8 microns in diameter. This led to pixelation that was visible un-
der 10x magnification. However, since the pixels were much smaller
than the glomeruli (approximately 90 microns), the pixelation did
not significantly interfere with the overall quality of the image. The
second issue is the lack of focusing optics in the bundle itself. Ide-
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Figure 1. Odor rack schematic. During the cleaning phase, air passes
though valve 6, cleaning the tubing. Air and contaminants are flushed
from the system though valve 3, the exhaust valve. During the air1 phase,
either valve 1 or 2 is opened. Odor fills the system but goes out through
valve 3. In addition, valve 5 is opened, allowing air to flow to the animal.
During the odor phase, valve 1 or 2 remains open, but valves 3 and 5 are
closed, replaced by the opening of valve 4, which allows odor to flow to the
animal. During air2, valve 4 is closed and valves 3 and 5 are reopened. In
addition valve 6 is opened, starting the cleaning process. This process is
repeated for each odor.
ally, one would use microscopic lenses to focus the light and prevent
cross-contamination of signals from nearby glomeruli. Fortunately
this was not a problem, since the distance between the bundle and
glomerular layer was sufficiently small. As demonstrated by the re-
sults, the images obtained were clear enough to distinguish individ-
ual glomeruli.
Odor delivery apparatus
In order to systematically present odors, a computer-controlled
olfactometer (Figure 1) was designed and constructed. The apparatus
contained tygon tubing (McMaster-Carr 5046K11) and two main sets
of solenoid valves (ASCO Scientific AL4124). The first set consisted
of ten valves, each associated with a single odor. When a valve was
opened, air would pass through the valve, going into a test tube con-
taining the odor and out via another path, carrying the odor with it.
The odor would then travel to a second set of valves, which included
an exhaust valve, an odor valve, and an air valve. The odor could ei-
ther go through the odor valve or the exhaust valve. If the odor valve
was open, the odor would be presented to the animal. Alternatively, if
the exhaust valve was open, the odor would leave the system through
a ventilation system. The air valve was connected to an air line, allow-
ing clean air to be presented to the animal. Aside from the two main
sets of valves, there was a cleaning valve.
Each trial involved four different phases that were repeated: clean-
ing, air presentation 1, odor presentation, and air presentation 2.
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RESEARCH
During the cleaning phase, the cleaning valve was opened to allow
fresh air to flow through the system. In addition, the air valve was
open, providing fresh air to the animal. During air presentation 1, the
air valve remained open and images were taken to obtain an average
baseline fluorescence image for comparison to odor images. During
the odor phase, the exhaust and air valves were closed, and the odor
valve was opened. The odor flowed through the second set of valves
to the animal. Finally, during air presentation 2, the odor valve was
closed, and the air valve was re-opened, allowing the fluorescence to
return to baseline while images were taken to track the return.
Software and experimental design
The odor delivery control and image acquisition was performed
by a software program written in LabView (National Instruments)
adapted by Tomokazu Sato from a similar program by Edward Soucy.
Each experiment could be altered by changing the settings of the pro-
gram. During the course of the experiment, the mouse was placed in
Neuroscience
a small chamber to minimize movement during awake imaging and
to maximize and homogenize odor exposure. The air in the cham-
ber was constantly evacuated throughout the experiment. However,
some delay in clearing an odor from the chamber was expected.
While the concentration was not measured, it was assumed that the
odor was expunged from the chamber quickly, likely on the order of
a few seconds or less.
Analysis
Analysis was done primarily using ImageJ (National Institutes
of Health) and Microsoft Excel. Time course image stacks were col-
lected and used to track the responses in real time. Ratio images were
used to quickly identify the presence or absence of responses as well
as the strength of response. These images are displayed as functions
Figure 2. Odor responses can be visualized with the fiber bun-
dle. A) Image of the olfactory bulb as seen through the fiber bundle. Ar-
rows point to sample glomeruli. B) Ratio image indicating ΔF/F values.
Image is inverted with dark spots indicating an increase in fluorescence.
Responding glomeruli can be seen as dark circles. Odor used was isopro-
pyl tiglate; subject was the mouse from A. C) Enlargement of the image
enclosed by the square in A. The contrast has been increased to more easily
visualize fluorescence differences. The circle encloses a glomerulus at rest-
ing fluorescence. D) Similar enlargement showing the same glomerulus
after odor stimulation. Contrast was increased by the same amount as in
C. Comparing the two images reveals a greater amount of fluorescence
after odor stimulation. This glomerulus is also the dark spot in the middle
of B. E) Graph tracking fluorescence over time. Gray arrow indicates the
start of odor stimulation (10 seconds) and the black arrow indicates the
stop (30 seconds). This time course was not obtained from the same glom-
erulus as in C and D.
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 RESEARCH
Figure 3. Olfactory bulb’s size and shape make it difficult to get
reliable results. A) Movement artifact caused by horizontal movement
of the brain. As the glomeruli shift, their original position becomes darker
because of the loss of base fluorescence. By contrast, their new position is
brighter. This is seen as a characteristic dark and bright pattern in the ratio
image. B) Movement artifact caused by vertical movement of the brain.
In this case, the glomeruli came more into focus, causing fluorescence in-
crease. Vertical movement is problematic, because it often manifests as an
odor response. Note the halo effect. C) Image of the olfactory bulb taken
immediately after surgery. Blood vessels and glomeruli are in focus. D)
Image of the same olfactory bulb taken one day after surgery. While many
glomeruli are still visible, others (arrows) may disappear out of focus.
of the inverse of ΔFluorescence/Base Fluorescence (ΔF/F). Hence,
dark spots indicate an increase in ΔF/F. Ideally, one would like to
subtract out all fluorescence that is not attributable to glomeruli.
However, since ratio images compared images taken within minutes
of each other, it was assumed that background fluorescence changed
minimally.
Results and Discussion
Efficacy of fiber bundle imaging
Using a fiber optic bundle to image the brain of an awake, behav-
ing animal is an underdeveloped technique. Hence, there are several
potential complications that may arise in its application to this study.
First, the fiber method has never been used to image glomeruli. It is
hard to predict whether individual glomeruli will be distinguishable
because of their proximity to each other and because of variable opti-
cal properties of the imaging fiber. Secondly, due to less constraining
structural support, the olfactory bulb may be more subject to move-
Neuroscience
ment when the mouse moves its head. If the bulb were constantly
moving relative to the fiber, it would be difficult to maintain clear
images throughout a chronic experiment. Furthermore, movement
of the brain during the course of imaging may introduce motion ar-
tifacts, interfering with results. A third potential problem with the
fiber technique is that the stressful nature of the surgery may cause
the mice to experience stress, pain, or fear upon recovery. This may
cause them to alter their behavior in response, which could affect not
only odor intake, but also neural response. While these are interest-
ing behaviors that can be studied, too high a level of stress, pain, or
fear may limit other behaviors such as natural exploration.
The first goal in the development of the technique was to repro-
duce detailed images comparable to simple wide-field images. The
concerns about pixelation and lack of optics mentioned earlier would
affect the quality of the images. However, the fiber technique was suc-
cessful in this respect, and consistently produced clear images, often
showing clear glomeruli (Figure 2A).
The next important step was to be able to consistently see glom-
erular odor responses. (For the rest of this paper, mentions of “glom-
erular responses” means a change in ORN pre-synaptic activity as a
result of odor stimulation.) This was also successful (Figure 2B-E),
but not as much as simply achieving clear images. While several ani-
mals showed clear responses, others did not. Often individual ani-
mals or even litters do not respond very well to odor stimulation for a
multitude of possible reasons such as sickness, obstruction of the na-
sal passages, or other variables. Furthermore, the surgery to implant
the fiber bundle is very invasive, and often damage to the dura matter
of the brain could cause a failure to respond. This could be due to ac-
tual damaging of the glomeruli or, more likely, due to decreased im-
age quality caused by dura matter damage. Finally, the bundle does
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Volume 2 Issue 2 | Fall 2009
not cover the entire bulb, so even strong odors may only stimulate
glomeruli outside of the field of view. In order to correct for this, a
test trial was run before cementing the bundle. If the testing revealed
limited responsiveness, the fiber was moved and retested until either
responses were seen or it was determined that the mouse would not
respond, in which case it was euthanized.
The final step was to determine whether the fiber technique was
suitable for imaging awake, freely behaving animals. Strong responses
similar to those seen in anesthetized animals were observed. Howev-
er, there were several shortcomings, as predicted. One major problem
is the small size and high curvature of the olfactory bulb, which cre-
ates problems with maintaining focus. The olfactory bulb has space
to move around, and during the awake trials, images often contain
motion artifacts due to brain movement relative to the fiber (Figure
3A,B). There were two types of motion artifacts observed. The most
common was caused by horizontal movement of the brain. When
the brain shifted horizontally, so too did the glomeruli in the field of
view. The movement was manifested in ratio images as a pattern of
very dark and bright spots (Figure 3A). Another type of movement
artifact was caused by vertical movement of the brain. Vertical move-
ment caused glomeruli to come in and out of focus. If a glomerulus
came into focus, the ratio image would show a dark spot at the new
position (Figure 3B).
Another focusing problem that was caused by the size and shape
of the bulb was simply maintaining the focus over a long time period.
Given the mobility of the bulb, it was hard to keep glomeruli in focus
even for a day. By the time a mouse fully recovers, the clarity of an
image may be lost (Figure 3C,D). While several glomeruli may still
be visible, others will be lost. This is one reason to keep the surgery
and recovery as short as possible. Usually, an entire experiment can
be finished within one day, preventing problems such as this.
Glomerular pre-synaptic activity in anesthetized and behaving
mice
As discussed earlier, one of the fundamental concerns of this
study is to determine whether behavior has an effect on ORN output.
To answer this question, images from three mice were analyzed. The
images from these three mice were chosen because they were the only
ones that matched a set of three criteria necessary to enhance the
probability of getting reliable results. First, the images remained clear
in both the behaving and anesthetized trials. Second, clear glomeru-
lar responses were observed. Finally, the animals were behaving rela-
tively normally after recovery and did not display signs of excessive
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Volume 2 Issue 2 | Fall 2009
stress or pain.
The first characteristic of glomerular response studied was the pat-
tern of the responses. Glomerular activation patterns for individual
odors were compared in the same animal to determine if the patterns
were similar in anesthetized and behaving animals. It was predicted
that the patterns would be the same. While behavior may affect the
feedback loops that can modulate the signal, it seemed unlikely that
behavior would alter odor-ORN binding affinities or the wiring of
the ORN. In all three mice it was found that the patterns were con-
served in behaving animals (Figure 4).
The next property studied was whether or not the signal strength
was significantly different when the animal was awake as compared to
when the animal was anesthetized. It was predicted that there would
be a difference, although whether it would be stronger or weaker was
unknown. ΔF/F values were obtained for several responding glom-
eruli in each of the three animals and averaged over 10 trials. Then,
the mean values from the anesthetized and behaving conditions were
compared. However, since ORN output can vary significantly for
different odors and even different glomeruli, comparison was only
done between the same glomeruli in each animal for identical odors.
Also, in order to correct for motion artifacts, data from trials with
large motion artifacts were removed. Somewhat contrary to what was
predicted, the results (Figure 5) indicated that only one glomerulus
Figure 5. ORN output is similar in the anesthetized and behav-
ing conditions. Shown is a bar graph pairing the mean ΔF/F values for
the anesthetized and behaving conditions for various animals (A), odors
(O), and glomeruli (G). These 11 glomeruli were chosen because they
showed some response in either the awake or the anesthetized condition.
Only A1-O2-G3 showed a statistically significant difference between the
anesthetized and behaving conditions (p<0.05 2-tailed paired t-test). Odor
1 (O1): Isopropyl Tiglate; Odor 2 (O2): Ethyl Valerate. Error bars denote
standard error.
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RESEARCH
Figure 4. Glomerular response patterns are similar in both
anesthetized and behaving animals. Shown are the Z-stack projec-
tion averages derived from ΔF/F images for each of the three mice over 10
trials. Each row represents images from a different mouse. The left column
displays images taken from an anesthetized mouse and the right column
displays images from the same mouse while behaving. Some of the image
slices were removed from the projections because of large motion artifacts.
While some motion artifacts remain, the response map appears to be unaf-
fected by behavior.
responded to one odor, ethyl valerate, significantly more strongly in
the anesthetized condition (p<0.05 2-tailed paired t-test). The other
11 glomerulus-odor combinations examined had statistically insig-
nificant differences.
While the pattern and intensity of the responses appeared to be
unaffected by behavior, it was predicted that the nature of the re-
sponse over time might be affected. In order to evaluate this, time
course data was examined for patterns across glomeruli and animals.
Unfortunately motion artifacts greatly limited the quantity of usable
data, making it difficult to draw global conclusions
One major difference noted in the time courses was that the slope
of the fluorescence change during odor presentation varied. In ani-
mal 2, stimulation with ethyl valerate in the anesthetized condition
caused a fluorescence change with a steeper slope (Figure 6). By con-
trast, in animal 3, stimulation with isopropyl tiglate in the anesthe-
tized condition caused a change with a shallower slope (Figure 7).
For both animals, this change was seen across all glomeruli but only
during the first repeat. Subsequent trials were too variable to draw
any conclusions. In addition, data would ideally come from the same
odor. Hence, this difference cannot be attributed to animal differenc-
es or odor differences. Nevertheless, the differences between the two
conditions in both animals are strikingly large. Interestingly, it seems
Neuroscience
that the slopes all fall around 0.05 for both animals in the anesthe-
tized conditions. By contrast, it seems that the variance comes mostly
from the behaving condition, which has a slope of only 0.025 in ani-
mal 2 but approximately 0.1 in animal 3. There are several possible
explanations for this. One possibility is that animal 3 may have had a
higher sniffing rate than animal 2. It is possible that animal 3 found
isopropyl tiglate more pleasing than animal 2 found ethyl valerate.
Animal 3 could have also been more exploratory or aware than ani-
mal 2, which may have not only increased its sniffing rate but also its
mental awareness. A further study could test this theory by monitor-
ing sniffing rate and comparing it to the slope of the response.
The final characteristic examined was rate of recovery from odor
stimulation. Only animal 2 was analyzed because the data from ani-
mals 1 and 3 had too much noise or large motion artifacts during re-
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 RESEARCH Volume 2 Issue 2 | Fall 2009

B.
Glomerulus 1 Glomerulus 2
Slope R2 Slope R2
Anesthetized 0.046 0.91 0.053 0.97
Behaving 0.025 0.71 0.025 0.92

Figure 6. Animal 2 has a steeper odor response slope in the

anesthetized condition. A) Time courses for the two responding
glomeruli in animal 2. Gray arrow indicates stimulus start; black arrow
indicates stimulus end. B) Table showing the slopes and R-squared values.
The interval for which each slope was calculated varied based on the start
and end of the response. The slopes for the anesthetized condition were
roughly twice those of the awake condition. Slope is expressed as change
in fluorescence over time.

points used to create the trendline. It can be argued, however, that

covery. Also, other than a few exceptions that will be discussed later,
only the first trial was analyzed for the same reasons. To quantify the
rate of recovery, the downward slope was calculated. It is important
to note that the data included in this analysis is somewhat arbitrary
because there is no time point defined as the peak and each glomeru-
lus peaks at a different time. For both odors and across glomeruli,
the odor recovery was slower during the behavior trial (Figure 8). In
fact, for isopropyl tiglate, the odor responses did not peak over the
course of the entire trial. The small differences in slope and the low
R-squared values are less than ideal for drawing solid conclusions.
Neuroscience
total sum of squares ought to be based on all data points, since that is
the true variability of fluorescence. In this case, the R-squared values
would be higher. All of this would suggest that even low R-squared
values do not necessarily signify an erroneous trend, especially in the
cases where the slope is very close to zero. The difference in recov-
ery time could be the result of several factors. One possibility is that
active exploration after the stimulus presentation may play a role in
odor intake or neural response. The expunging of the odor from the
stimulus chamber does not happen instantly. Rather, after the odor
presentation, a weak vacuum evacuates residual odorants out of the
chamber. It is unclear at what concentration or time point the mouse
is no longer able to sense the odor, so it is possible that even as the
concentration dwindles, the mouse increases its sniffing rate, caus-
ing the effect observed. However, as mentioned earlier, while odorant
concentration was not measured, it is assumed that it was expunged

However, when viewed in combination with the time course graphs, relatively quickly. Given this, another explanation is that the mouse
there does seem to be a trend for faster recovery under anesthesia. may actively suppress inhibition in an attempt to try to maintain sen-
Furthermore, the lowest R-squared values were seen in the behav- sation of the smell even after the odor is cleared.
ing condition, and as discussed earlier, behavior seems to cause large
fluctuations in the signal as the animal recovers from odor stimula-
tion. Furthermore, since the slope is often close to horizontal, these
vertical fluctuations can significantly affect the correlation coefficient
because the mean variance in the y-direction is determined almost
completely by these fluctuations. By contrast, as the slope of the trend-
line gets steeper, the total variance in the y-direction increases. This
means that the proportion of the total variance that is determined by
the variance of these fluctuations decreases, thus increasing the R-
squared value. Finally, the R-squared value was calculated based on a
total sum of squares that only took into account variance of the data

Figure 7. Animal 3 has a steeper odor response slope in the be-

having condition. A) Representative time courses from glomerulus 1 of
animal 3. Gray arrow indicates stimulus start; black arrow indicates stimu-
lus end. B) Table showing the slopes and R-squared values. The interval for
which each slope was calculated varied based on the start and end of the
response. In this case, the slopes for the behaving condition were about
twice those of the anesthetized condition, which is the opposite of the re-
sult found in animal 2.

B.
Glomerulus 1 Glomerulus 2 Glomerulus 3 Glomerulus 4
Slope R 2
Slope R 2
Slope R 2
Slope R2
Anesthetized 0.038 0.97 0.061 0.99 0.043 0.96 0.031 0.96
Behaving 0.074 0.96 0.091 0.98 0.119 0.96 0.076 0.96

44 The Harvard Undergraduate Research Journal

Volume 2 Issue 2 | Fall 2009 RESEARCH

Conclusions and Future Directions sensitive dye, GCaMP, in mitral cells.

From the beginning, this study has had two goals. The first goal
was to explore the use of a fiber optic imaging bundle as a viable way
to image the olfactory bulb in awake, freely-moving mice. The second
was to investigate any differences in glomerular response based on
whether the mouse was anesthetized or awake and freely-behaving.
This project is the first demonstration that a fiber optic bundle can
be used to image the olfactory bulb of awake, freely-behaving mice.
The results illustrate that not only can clear images showing distinct
glomeruli be attained, but also changes in fluorescence can be ob-
served, captured, and quantified. Furthermore, the unique advantage
that the fiber bundle offers over other awake imaging methods such
as the head-fixed method is that mice are able to freely explore their
environments. Given results that indicate that exploratory behavior
may have a significant effect on odor response, the fiber technique
offers a way to further investigate this possibility.
However, since this method is still in its early stages, some prob-
lems remain. One issue not indicated by the presented results is that
the surgery required to install the bundle on the mouse is incredibly
difficult. Obtaining and maintaining clear images is a sensitive pro-
cess requiring a clean and rapid surgery. Also, as shown in the results,
motion artifacts could interfere with results. In order to make the
fiber technique practical, it is necessary to optimize the technique to
address issues such as motion artifacts. However, the success of this
technique in its first application to the olfactory bulb is a testament to
its potential as an invaluable tool for imaging behaving animals.
The second major finding of this study is that generalized free be-
havior does not seem to cause any consistent differences in glomeru-
lar responses across all animals, odors, and glomeruli. Both the pat-
tern of the response as well as the overall strength of response were
The final major finding was that behavior causes localized changes
individual to different mice, glomeruli, or odors. In two animals, a
wider variability was observed after the average response began to
stabilize. Differences in the rate of odor response were also seen in
two mice. One mouse responded more quickly when it was behaving
and the other responded more slowly. Finally, a difference in odor
recovery rate was observed. For both odors presented, one of the mice
recovered from odor stimulation more slowly when it was behav-
ing. Interestingly, for one of the odors, differences were found even
amongst trials for the same glomeruli. In two of the trials, relative
to when it was anesthetized, the mouse recovered more slowly when
behaving, but in the third, it recovered more quickly. Furthermore,
the recovery rate in the anesthetized condition did not vary much.
Rather, the discrepancy was caused by a large variation in recov-
ery rate between free-behavior trials. This trend of large variations
in odor response in the freely-behaving condition was also seen in
the fluctuations of fluorescence, as mentioned earlier. In addition it
was seen in the rate of odor response. While the slopes for the anes-
thetized condition were similar for both animals, the slopes for the
freely-behaving condition were dramatically different. Thus it seems
that while differences are individualized and seemingly unpredict-
able on a global scale, the general trend seems to be that behavior
causes variation in the nature of the odor response.
The field of imaging awake animals holds an exciting future. Little
is known about how behavior and brain activity are linked and count-
less questions remain unanswered. However, as mentioned earlier,
before these questions can be addressed, the fiber technique needs to
be optimized. One way the method can be improved is by enhancing
the image quality. There are several potential ways to accomplish this.
Adding focusing optics such as a gradient-index (GRIN) lens would

unchanged by the behavioral state. As discussed earlier, one of the
main reasons for looking at ORN synaptic output was because ORNs
are more upstream than the rest of the olfactory system. Knowing
that odor responses do not appear to be globally modulated at the
level of the ORN, it is now possible to study the mitral cells to see if
a global change occurs in that layer. If a difference is observed, one
can more reliably attribute the change to mitral cell modulation as
opposed to a modification in ORN activity. One way to explore this
possibility would be to use transgenic mice expressing the calcium-
Neuroscience
not only increase the image quality by reducing cross-contamination
of light, but it would also allow the fiber to be placed above the bulb as
opposed to directly on it. In addition, adding a mechanism by which
the fiber can be focused after a surgery is performed would decrease
the number of experiments that fail because of bulb movement.
The preliminary results of this study inspire several future stud-
ies that can further explore some of the findings presented. Since the
results indicate that exploratory behavior, possibly sniffing rate, may
have an effect on odor response, a study that concurrently monitors

Figure 8. Animal 2 recovered from odor stimulus faster in the

anesthetized state. A) Representative time courses taken from the
first trial of glomerulus 1 and odor 1, isopropyl tiglate. Gray arrow indi-
cates stimulus start; black arrow indicates stimulus end. B) Chart showing
the slope and R-squared values for each glomerulus stimulated by odor
1. Note the positive slopes in the behaving condition. C) Chart showing
the slope and R-squared values for each glomerulus stimulated by odor 2,
ethyl valerate.

B.
Glomerulus 1 Glomerulus 2 Glomerulus 3 Glomerulus 4
Slope R 2
Slope R 2
Slope R 2
Slope R2
Anesthetized -0.006 0.66 -0.003 0.33 -0.008 0.62 -0.003 0.25
Behaving 0.003 0.28 0.003 0.46 0.003 0.27 0.002 0.15

C.
Glomerulus 1 Glomerulus 2
Slope R 2
Slope R2
Anesthetized -0.006 0.60 -0.017 0.95
Behaving -0.002 0.07 -0.011 0.83

www.thurj.org 45
 RESEARCH
sniffing rate and glomerular response could shed some light on this
variation. One potential outcome is that sniffing rate may match
closely with the level of fluorescence observed, which would explain
why different mice had different odor responses while freely behav-
ing. To further explore this, one could use calcium dyes to more
clearly pair sniffing with ORN output. While synaptopHluorin is a
good indicator of overall ORN activity, it cannot be used to image
individual ORN action potentials. By contrast, calcium dyes react
quickly to ORN activity, and one can visualize events on a much
shorter time scale, including individual action potentials.
As discussed earlier, the biggest difference between the head-fixed
technique and the fiber bundle technique is that with a fiber bundle,
mice can freely move and behave. Thus, it would be interesting to ex-
amine whether the two techniques produce differences in glomerular
response. Hypothetically, any significant differences can be attribut-
ed to exploratory behavior, and this may help to resolve whether the
differences seen in this study are attributable simply to being awake
or rather to being able to move around and freely explore the envi-
ronment.
Finally, one could explore the effect of training on odor responses.
A basic study could compare mice that are previously exposed to
the experimentation setup to naïve mice. It is conceivable that mice
that trained in this fashion may reduce their exploratory behavior
because the environment they are experiencing is not a novel one.
Another study could compare mice trained to a certain odor to mice
that are just trained to general odor stimulation or even naïve mice.
This might also have an effect on how a mouse approaches and ex-
plores the different odors presented.
This project is the first step towards understanding the effect of
behavior on the olfactory bulb’s response to odor stimulation. While
the field is new and there is a large amount of unexplored territory,
hopefully this study will lay the groundwork for similar future stud-
Neuroscience
ies. Given the promising results of this project, the fiber technique
has the potential to offer a way to understand the olfactory bulb in the
larger context of the whole brain and even the entire body.
References
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Albeanu, D.F. (2008) Precision and diversity in an odor map on the olfactory
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Aungst, J.L., Heyward, P.M., Puche, A.C., Karnup, S.V., Hayar, A., Szabo, G.,
and Shipley, M.T. (2003). Center–surround inhibition among olfactory
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Belluscio, L., and Cummings, D.M. (2008) Charting Plasticity in the Regen-
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Bouret, S., and Sarah, S.J. (2004). Reward expectation, orientation of atten-
tion and locus coeruleus-medial frontal cortex interplay during learning.
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Edmondson, J., and Axel, R. (1996). Visualizing an Olfactory Sensory
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The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Analysis of the role of Bhlhb5 and Prdm8

in neural development
Stephanie I. Mok
Harvard College ‘09, moksi@mail.nih.gov

In neural development, Bhlhb5 and Prdm8 may have a cooperative role in the same regulatory processes underly-
ing neuronal differentiation and maturation. Three potential models of interaction are proposed. The three models
are: 1) Bhlhb5 and Prdm8 regulate the same target genes, but do not bind together in complex, 2) Bhlhb5 and Prdm8
interact in complex to regulate the same gene targets, and 3) Prdm8 and Bhlhb5 each regulate the transcription of
unique proteins that are critical mediators of a common pathway. To address the hypothesis that Bhlhb5 and Prdm8
are crucial in the same neurons in development, the protein expression patterns of Bhlhb5 and Prdm8 were examined
in the mouse brain and spinal cord via immunohistochemistry. Results show that Bhlhb5 and Prdm8 do not bind
directly together in complex. Moreover, Prdm8-expressing neurons are significantly lost in the spinal cord of Bhlhb5
mutants, Bhlhb5 and Prdm8 are highly co-expressed in the brain and spinal cord, and Prdm8 mutants exhibit loss
of the corticospinal tract. These results suggest that Bhlhb5 and Prdm8 are crucial regulators of a common pathway
that underlies neuronal development in the mammalian central nervous system.

Introduction This was indicated by a significant decrease in balancing skills when

The bHLH family of transcriptional regulators
One group of transcriptional regulators found to be critical in
the early development of the central nervous system is a family of
proteins known for their highly conserved Basic-Helix-Loop-Helix
or BHLH structural domains. Many members of the BHLH fam-
Bhlhb5 mutants were placed on a rotating rod and fell off at much
higher rates than wildtype animals (Figure 1B).
While the nervous systems of Bhlhb5 mutants appeared normal at
a gross level, closer inspection revealed several defects at the cellular
level. For instance, a statistically significant loss of Bhlhb5-expressing
neurons in the most superficial laminae of the dorsal horn of the spi-

ily have been found to perform crucial regulatory roles in nervous
system development in a broad array of cellular types (Massari and
Murre, 2000). A particular BHLH gene of interest is Bhlhb5, which
has been found to be transiently expressed in subpopulations of post-
mitotic neurons throughout the central nervous system (Ross et al.,
unpublished data). These findings suggest that the Bhlhb5 transcrip-
tion factor may have a regulatory role in cellular differentiation and
maturation during neuronal development of key pathways necessary
for proper movement and sensory mechanisms.
Bhlhb5 mutants have phenotypes indicating deficits in the ner-
vous system
Investigators in the Greenberg Lab examined the potential role of
Bhlhb5 in neural development through the generation of a Bhlhb5
knockout mouse (Bhlhb5 -/-). They found that Bhlhb5 mutants ex-
hibited a series of physical and behavioral deficits (Figure 1: Supple-
mentary Figures). Behavioral phenotypes observed include self injury
due to excessive spot-grooming and the subsequent development of
open sores, or skin lesions (Figure 1A). Moreover, in behavioral tests
that evaluated sensitivity of Bhlhb5 mutants to chemical stimulation
through exposure to capsaicin, a natural irritant found in chili pep-
pers, Bhlhb5 mutants were found to demonstrate decreased response
(measured by paw licking) to the harmful stimulant. Similarly, Bhl-
hb5 mutants also exhibited decreased sensitivity to thermal stimu-
lation as indicated by the decreased rate of paw withdrawal when
placed on a hot plate (Ross et al., unpublished data).
An unusual behavioral phenotype observed in the Bhlhb5 mu-
tants was a “handstand” behavior in which the mice walked forward
on their forelimbs, while retracting their hindlimbs to display a tran-
sient handstand posture (Figure 1C). Bhlhb5 mutants also displayed
lack of motor coordination when compared to wildtype controls.
www.thurj.org
Neuroscience
nal cord was observed in Bhlhb5 mutants when compared to that of
wildtype controls (Figure 2). In addition, the corticospinal tract was
missing in Bhlhb5 mutants (Ross et al., unpublished data) (Figure
3). The corticospinal tract (CST) consists of the bundle of neuronal
fibers extending from the motor cortex of the brain into the spinal
cord, and is therefore a key factor in regulating motor coordination
in animals. The distinct absence of the CST in mutants thus indicates
a potential role of Bhlhb5 in the formation of motor circuits in de-
velopment. To identify the genes that are mis-expressed in Bhlhb5
mutants, Affymetrix array analysis examining RNA levels in Bhlhb5
knockout versus wildtype was performed. Prdm8 was the most dra-
matically misregulated gene (Sup. Figure 1) (Ross et al., unpublished
data). In fact, Prdm8 was found to be up-regulated significantly in
Bhlhb5 mutants (p<0.05) (Ross et al., unpublished data), thus sug-
gesting that Bhlhb5 may directly repress the transcription of Prdm8
(Figure 4: Supplementary Figures).
Similarity of phenotypes in Bhlhb5 and Prdm8 mutants
The Affymetrix RNA array results from the Bhlhb5 mutants sug-
gested that Prdm8 may be an important transcriptional regulator in
the same neuronal pathways as that of Bhlhb5. A putative transcrip-
tion factor that is a member of the PR Domain family known for its
histone methyl-transferase activity, Prdm8 has not been researched
broadly and little is known about its role in the central nervous sys-
tem.
However, Prdm8 analysis in the retina was being explored by in-
vestigators at the McInnes Lab; intriguingly, Prdm8 knockout mice
exhibited similar behavioral phenotypes and deficits when compared
to that of Bhlhb5 mutants (Sup. Figure 2). Like Bhlhb5 knockout
mice, the Prdm8 mutants also displayed the unique “handstand”
posture during movement. Furthermore, Prdm8 mutants also devel-
47
 RESEARCH
Figure 2. BHLHB5-expressing neurons are missng from a sub-
population of cells in the superficial layers of the spinal cord
dorsal horn. A) Control BHLHB5 cre/+ mice exhibit BHLHB5-express-
ing neurons marked by a reporter gene (eYFP) as shown. B) Mutant BHL-
HBS cre/- mice exhibit fewer BHLHB5-expressing neurons as marked by
a reporter gene (eYFP) as shown. The most superficial layers of the spinal
cord are marked by layers A and B (outlined in white). (Ross et al., unpub-
lished data).
Figure 3. Bhlhb5 mutants are missing the corticospinal tract. A)
Neuroscience
PKC-gamma immunostaining in BHLHB5 wt mice marks the CST (indi-
cated by white arrow). B) PKC-gamma immunostaining in BHLHB5 mut
mice exhibit no expression of PKC-gamma, thus demonstrating loss of the
CST(idicated by white arrow). (Ross et al., unpublished data)
oped extensive skin lesions like the Bhlhb5 mutants. The combina-
tion of these observations in conjunction with the RNA Affymetrix
array results indicating that Prdm8 is the most mis-regulated gene
in Bhlhb5 mutants suggested that Prdm8 may be involved in a com-
mon pathway with Bhlhb5 in the development of the central nervous
system in mice.
What is the mechanism through which Bhlhb5 and Prdm8 regu-
late transcriptional pathways such that removal of either factor in-
duces the same phenotypic deficits in knockout mice? I propose three
models of interaction to answer this question (Figure 4).
Three chief objectives were addressed: 1) Which model of interac-
tion between Bhlhb5 and Prdm8 is best supported by the observed
data? In Figure 8, several models of interaction that may characterize
the true collaborative function of Bhlhb5 and Prdm8 in neuronal de-
velopment are presented. Is there evidence to support the hypothesis
that Bhlhb5 and Prdm8 regulate the same target genes, yet do not
bind together directly in complex (Figure 4A)? Do they interact di-
rectly with each other to regulate the same gene targets (Figure 4B)?
Or, do Prdm8 and Bhlhb5 each regulate distinct groups of genes that
are critical mediators of a common pathway (Figure 4C)?
2) Is there evidence to suggest a mechanism of interaction between
Bhlhb5 and Prdm8 in the regulation of processes important neuronal
development? Do protein expression patterns of Bhlhb5 and Prdm8
in various regions of the brain and spinal cord indicate that these two
factors may be crucial in the same neurons during differentiation and
development?
48
Volume 2 Issue 2 | Fall 2009
Figure 4. Propsed models of Prdm8 and Bhlhb5 regulation of
target genes. A) Bhlhb5 and Prdm8 bind to distinct regions of DNA,
yet repress the same target genes. B) Bhlhb5 and Prdm8 interact directly
with each other in complex to co-repress the same target genes. C) Bhlhb5
and PRdm8 each repress transcription of distinct target genes (X and Y,
respectively), which are key proteins involved in a common pathway. In
this model, both Bhlhb5 and Prdm8 act as indirect regulators of the same
downstream processes, yet Bhlhb5 and Prdm8 do not interact with each
other or target directly the same genes.
3) To design a Prdm8 fusion protein construct for the develop-
ment of an antibody that accurately recognizes Prdm8 protein in
immunhistochemistry and immunoprecipitation experiments and
allows for visualization of endogenous Prdm8 protein in fixed tissue
and neural lysates.
Materials and Methods1
Immunoprecipitation of Bhlhb5 or Prdm8 in transfected 293T
cells
Supernatant of 293T cell lysate was incubated with primary Bhl-
hb5 (rat) antibody (or anti-Myc epitope (mouse)) antibody. Blocked
Protein-A agarose beads were added to the cell lysate supernatant,
pelleted from the cell lysate, and washed. Bhlhb5 protein (or Prdm8
protein) was then precipitated and bound to the beads through pull-
down.
Design and generation of Prdm8-directed antibody
Multiple Prdm8 GST-fusion protein constructs were devised that
consisted of all 6 combinations of 3 highly conserved domains in the
full length Prdm8 sequence (labeled as Domain 1, Domain 2, and
Domain 3). Primer-targeted PCR amplification was conducted, trans-
forming constructs into competent cells, which were maxiprepped
and sequenced. Constructs were then digested with restriction en-
zymes, ligated into pGEX vectors, and transformed the vectors into
Top10 Competent Cells. The researcher then cultured in high volumes
the correctly transformed GST-PRDM8 constructs, maxiprepped,
and then transformed them into BL21 (E. coli) cells.
1
(see Supplementary Materials for full Materials and Methods)
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Induction and purification of Prdm8 GST-fusion protein con-
structs
Colonies of transformed BL21 cells were diluted, cultured in high
volumes, and induced with IPTG. Cells were spun down, lysed, and
incubated with Protein-G sepharose beads. Protein-coated beads
were spun down, washed, and the protein eluted from the beads. A
Bradford protein assay was conducted to quantify the eluted protein
from the beads, and the purified protein was sent for injection into
rabbits. To observe induction of each of the 6 PRDM8 GST-Fusion
protein constructs in BL21 cells, cells were lysed in Western Lysis
Buffer and protein sizes were examined via SDS-PAGE and coomass-
ie staining (using a GelCode Blue Stain Reagent).
Immunoprecipitation of endogenous Bhlhb5 and Prdm8 protein
in neurons
The supernatants of the neural lysate of Prdm8 mutant and wild-
type mice were incubated with primary Bhlhb5 (rat) (or Prdm8 (rab-
bit)) antibody. Blocked Protein-A agarose beads were added to the
cell lysate supernatant, and the beads were pelleted from the neural
lysate and washed. Bhlhb5 protein (or Prdm8 protein) was precipi-
tated bound to the beads through pull-down.
Western blot analysis of immunoprecipitation results
Lysates from the immunoprecipitation experiments were analyzed
via SDS-PAGE and western blot using Prdm8 and Bhlhb5-specific
antibodies. Prdm8 rabbit antibody was used to probe the upper half
of the blot (Prdm8 protein band is approximately 72 kDa in size in
neurons). For immunoprecipitation experiments conducted on 293T
cell lysates, an anti-Myc (mouse) antibody to probe for the C-termi-
nal epitope tag attached to Prmd8 transfected in cultured 293T cells
was used. Bhlhb5 rabbit antibody was used to probe the lower half of
the blot (Bhlhb5 protein is approximately 35 kDa in size).
Immunohistochemistry analysis
To analyze the cell-specific expression patterns of Prdm8 and Bhl-
hb5 in cortical and spinal cord tissue sections, immunohistochem-
istry analysis was performed on fixed tissue. Whole specimens were
frozen in blocks of optimum cutting temperature (OCT) formula,
and sagittal and coronal sections 20 microns thick were collected
utilizing a cryostat, and the sections were transferred onto slides. Im-
munostaining analysis was conducted to visualize proteins of inter-
est.
Results
No co-immunoprecipitation of Bhlhb5 or Prdm8 in 293T cells
To test the hypothesis of whether or not any evidence of protein-
protein interaction between Bhlhb5 and Prdm8 could be observed,
immunoprecipitation experiments were conducted. In this experi-
ment, the researcher performed antibody-specific pull-down of a
particular protein (Bhlhb5) via adhesion to beads and precipitation
from the whole cell lysate. The precipitate was probed with antibod-
ies specific for its partner protein (Prdm8) to observe for any co-im-
munoprecipitation. Through western blot analysis (Figure 5), no evi-
dence was found of a direct interaction between Bhlhb5 and Prdm8.
Antibody-specific immunoprecipitation of the Bhlhb5 protein was
observed, and any associated Prdm8 pull down via probe identifica-
tion of the Myc-epitope tagged to the C-terminal of the PRDM8 pro-
tein was analyzed. As seen in Figure 9, there is no observable protein
band at approximately 98 kDa in the lane containing the precipitate
pulled down via Bhlhb5 interaction. Conversely, immunoprecipi-
tation of Prdm8 via its Myc-epitope tag exhibited no pull-down of
Bhlhb5 in the precipitate (which would have been indicated by a band
~37 kDa in size).
Design and development of Prdm8 GST-fusion protein constructs
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RESEARCH
Figure 5. Western blot of immunoprecipitation conducted in
293T cells. 293T cells were transfected with both BhlHb5 and myc-
tagged Prdm8. First lane displays IP for Bhlhb5, and no co-IP of Prdm8
is observed. Second lane displays IP for PRdm8 via its myc epitope tag,
and no co-IP of Bhlhb5 is observed. Bottom half of blot(divded by dotted
line) was blotted with antibody for Bhlhb5, upper half of blot probed with
antibody for Myc-epitope tag of Prdm8.
In order to conduct immunoprecipitation experiments on endog-
enous Bhlhb5 and Prdm8 protein from neurons, use of a Prdm8 an-
tibody was required. Due to the lack of available Prdm8 antibody,
however, a series of Prdm8 GST-Fusion protein constructs were de-
signed and developed. Single constructs to be purified were selected
Neuroscience
and injected into rabbits for Prdm8 antibody production.
Sup. Figure 3 exhibits the design of the 6 different protein domains
cloned into vectors with GST to create 6 individual GST-fusion pro-
tein constructs. Sup. Figure 4 displays the induction results for each
of the Prdm8 GST-Fusion protein constructs in which the darkest
band in each lane corresponds to the appropriate sized protein band
for each of the Prdm8 fusion proteins. The middle region of the pro-
tein was found to be the only construct isolated in the soluble fraction
during the protein purification process. This region (termed Domain
2) was selected as the Prdm8 antigen to be injected into rabbits for
Prdm8 antibody production.
Designed antibody recognizes Prdm8 protein in neurons
To test for specificity and strength of the developed Prdm8 anti-
body to recognize endogenous Prdm8 in neural lysate, an experimen-
tal western blot on a wildtype whole mouse cortex and one mutant for
Prdm8 was conducted (Sup. Figure 5). Prdm8 antibody derived from
the serum of a rabbit injected with purified antigen (derived from the
Prdm8 Domain 2 GST-Fusion protein construct) was used to probe
for the presence of Prdm8 protein in the neural lysate. The Prdm8
antibody accurately identified the Prdm8 protein in the neurons as
predicted. This finding was verified in the western blot through the
observation of a ~72 kDa protein band, which corresponds to the ap-
proximate size of endogenous Prdm8 in neurons.
No co-immunoprecipitation of Bhlhb5 or Prdm8 in neurons
Unlike the previous experiment performed in which 293T cells
transfected with Bhlhb5 and Myc-Prdm8, this immunoprecipitaton
experiment was performed on proteins expressed from endogenous
genes in neural lysate drawn from the whole cortex of Prdm8 wild-
type and mutant mice. The advantage of this protocol is that any as-
sociated co-factors necessary to facilitate proper Bhlhb5 and Prdm8
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 RESEARCH
Figure 6. Western blot of BHLHB5 immunoprecipitation con-
ducted in neural lysate. Neural lysate from whole cortex of both
PRDm8 wt and Prdm8 mut P0 mice were utilized to IP for Bhlhb5 (both
BHLhb5 wt). First and second lane display neural lysate (1% NP-40 lysis
buffer) of Prdm8 mut and Prdm8 wt mice, respectively. Second lane clearly
indicates the presence of PRDM8 protein in the neural lysate prior to the
IP. Third and fourth lanes indicate IP for BHlhb5 in both Prdm8 mut and
Prdm8 wt mice, respectively. No evidence of co-IP of Prdm8 is observed
in the Prdm8 wt neurons.
protein interaction would also be available in the neural lysate to
maintain natural protein-protein interactions (such would not be
possible in 293T cells).
In this experiment, antibody-specific pull-down of a particular
protein (i.e. Bhlhb5) was conducted via adhesion to beads and pre-
Neuroscience
cipitation from the whole cell lysate. The precipitate with the anti-
body specific for the partner protein (i.e. Prdm8) was then probed
to look for any co-immunoprecipitation. As indicated in Figure 6,
there is no protein band at approximately 72 kDa (MW of Prdm8
protein) in the lane that indicates the precipitate pulled down via
Bhlhb5 interaction. Since Prdm8 is evident in the wildtype whole
cell lysate when compared to the Prdm8 mutant control, endogenous
Prdm8 is clearly present prior to immunoprecipitation. No Prdm8
was co-immunoprecipitated with Bhlhb5 in neurons. Similarly, im-
munoprecipiation was performed for Prdm8 and no associated pull-
down of Bhlhb5 in the precipitate was found (Figure 7).
Prdm8-expressing cells lost in the dorsal horn of spinal cord of
Bhlhb5 mutants
In previous research, Bhlhb5 mutants exhibited distinct pheno-
types that included skin lesion development, handstand behavior,
decrease in motor coordination, and decrease in sensitivity to ther-
mal or chemical stimulation (Ross et al., unpublished data). When
examined at the cellular level, Bhlhb5 mutants also exhibited a sig-
nificant loss of Bhlhb5-expressing neurons in the superficial layers
of the dorsal horn of the spinal cord (Ross et al., unpublished data).
Recent findings that Prdm8 mutants indicate striking similarities
comparable to those of Bhlhb5 mutants (i.e. skin lesion development
and handstand behavior) (McInnes, unpublished data) suggested the
theory that Bhlhb5 and Prdm8 may function using similar regula-
tory pathways of the central nervous system. As a result, it is possible
that these two transcription factors are expressed in the same neu-
rons during development. This reasoning led to the research hypoth-
esis that: 1) Bhlhb5 mutants also exhibit significant loss of Prdm8-
expressing neurons in the superficial layers of the dorsal horn of the
spinal cord, 2) Prdm8 mutants exhibit the loss of Prdm8 and Bhlhb5
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Volume 2 Issue 2 | Fall 2009
Figure 7. Western blot of PRDM8 immunoprecipitation conduct-
ed in neural lysate. Neural lysate from whole cortex of both Prdm8 wt
and Prdm8 mut P0 mice were utilized to IP for PRDM8. First and second
lanes display neural lysate (SDS lysis buffer) of Prdm8 wt and Prdm8 mut
mice (both Bhlhb5 wt), respetively. Presence of BHLHB5 protein in these
lysates is observed by the dark band at the 35kDa mark in each lane. Third
and fourth lanes indicate IP for PRDM8 in both Prdm8 wt and Prdm8
mut mice, respectively. No evidence of co-IP of BHLHB5 is observed in
the Prdm8 wt neurons.
expressing neurons in the dorsal horn of the spinal cord.
To test the first hypothesis, immunohistochemistry was per-
formed to compare Prdm8 expression in neurons in the dorsal horn
of the spinal cord between wildtype controls and Bhlhb5 knockout
mice at P0. A significant loss of Prdm8-expressing neurons in the dor-
sal horn of the spinal cords of Bhlhb5 mutants was detected(Figure
8A). The mean number of Prdm8-expressing neurons located in the
dorsal horn of the spinal cord in Bhlhb5 mutants and controls were
quantified to determine if a statistically significant difference ex-
isted. A two-tailed T-test with a confidence value of 95% (p-value =
0.0012) (Table 1) was utilized to perform the statistical analysis. A
significant decrease in Prdm8-expressing neurons in Bhlhb5 mutant
mice as compared to those of Bhlhb5 wildtype controls was observed.
Moreover, the Prdm8-expressing neurons were lost to the outer-most
laminae (superficial region) of the spinal cord dorsal horn.
Co-expression of Bhlhb5 and Prdm8 in spinal cord dorsal horn
To test the second hypothesis, immunostaining was conducted for
Bhlhb5 and Prdm8 in the spinal cord of Prdm8 wildtype controls
and mutants at P0. Wildtype mice exhibited high levels of Bhlhb5
and Prdm8 co-expression in the dorsal horn region of the spinal cord
(indicated by white dotted lines in Sup Figure 6A).
Co-expression of both transcription factors (indicated in the
“merged” panels of Sup. Figures 6A and 6B, where neurons express-
ing both Bhlhb5 and Prdm8 appear purple or blue-green in color,
respectively) was detected. Interestingly, in the analysis of immunos-
taining for Bhlhb5 and a GFP marker for Prdm8-expressing neurons
in Prdm8 mutants, a qualitative loss was observed of Bhlhb5 and
Prdm8 co-expressing neurons in the dorsal horn on the spinal cord
(Sup. Figure 6B).
Co-expression of Bhlhb5 and Prdm8 in the frontal cortex
Due to the similarity in phenotypes observed in both Bhlhb5 and
Prdm8 mutants (both behaviorally and at the cellular level in the spi-
nal cord), there is another key hypothesis. The third hypothesis is
that high levels of co-expression of both Bhlhb5 and Prdm8 would
also be observed in neurons located in the cortex where important
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Volume 2 Issue 2 | Fall 2009
Figure 8. BHLHB5 mutants exhibit significant loss of PRDM8-ex-
pressing meurons in dorsal horn of spinal cord. A) Immunostain-
ing analysis exhibiting loss of PRDM8 (marked in red) in subpopulations
of neurons located in the dorsal horn of the spinal cord (outlined in white).
B) Graphical representation of the significant loss of PRDM8-expressing
neurons in the dorsal horn of the spinal cord when comparing BHLHB5
mutants vs. wildtype mice.
neuronal components of sensory and motor circuits are derived and
send projections throughout the body. Immunostaining was con-
ducted for Prdm8 and Bhlhb5 in sagittal sections of wildtype mice
frontal cortex, which indicated high levels of co- expression for both
proteins in populations of neurons at P0 (Figure 9). Figure 9A ex-
hibits immunostaining for Bhlhb5 and Prdm8 in a wildtype control
brain and Figure 9B exhibits immunostaining for Bhlhb5 and Prdm8
in a Prdm8 mutant cortex. Enlarged boxes of each panel in Figure 9
demonstrate the co-expression of both proteins in the same neurons
in wildtype and Prdm8 mutants. In the analysis of the immunostain-
ing, no qualitative difference between Bhlhb5 and Prdm8 expression
patterns in the cortex was observed. Nevertheless, high levels of co-
expression was localized near the outermost layers of the frontal cor-
tex of both wildtype and Prdm8 mutants.
Prdm8 mutants and double heterozygotes are missing the CST
In previous research, the observation that both Bhlhb5 and Prdm8
mutants demonstrate similar behavioral phenotypes (i.e. develop-
ment of skin lesions, handstand behavior) (Ross et al., unpublished
data) led to the overarching theory that Bhlhb5 and Prdm8 may be
two transcriptional regulators involved in common regulatory pro-
cesses in neuronal development. This conceptual idea thus led to the
reasoning that the phenotypes observed in Prdm8 mutants mirror-
ing that of Bhlhb5 mutants would also extend to the cellular level
throughout the central nervous system. Therefore, based on the find-
ing that Bhlhb5 mutants exhibit distinct loss of the corticospinal tract
(CST) (Figure 3), Prdm8 mutants might also exhibit loss of the CST.
To test this hypothesis, immunohistochemistry analysis of spinal
cord sections of wildtype and Prdm8 mutant mice was performed
at P0. In the immunostaining analysis, the presence of the CST was
assessed through analyzing sectioned spinal cord tissue with a probe
that recognizes a protein marker, Protein Kinase C-gamma (PKC-
gamma), for the CST. PKC-gamma is highly expressed in the corti-
Mean # PRDM8-
BHLHB5 WT BHLHB5 MUT expressing cells in
Sample Size Sample Size PRDM8 WT
(St. Dev)
56 Neurons
N=20 N=20 (19.31608)
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RESEARCH
Figure 9. PRDM8 and BHLHB5 immunostaining in cortex. A)
Immunostaining analysis of PRDM8 expression (red) and BHLHB5 ex-
pression (blue) in the frontal cortex of wildtype mice (P0). High levels
of PRDM8 and BHLHB5 co-expression expression exhibited in the outer
layers of the cortex (shown as purple neurons in enlarged box). B) Im-
munostaining analysis of PRDM8-expressing neurons via GFP knockin
at Prdm8 locus (green) and BHLHB5 expression (blue) in the frontal cor-
tex of PRDM8 mutant (-/-) mice (P0). Large population of neurons co-
expressing PRDM8 and BHLHB5 also exhibited in the outer layers of the
cortex (shown as blue-green neurons in enlarged box).
cospinal tract (Moreno-Flores et al., 2006), which is composed of a
bundle of fibers derived from neurons originating in the motor cor-
tex of the brain (Purves, 2001). In the analysis of the immunostaining
experiment, the CST was found to be lacking in Prdm8 mutants (Sup.
Figure 7C).
The observation regarding the absence of the CST in Prdm8 mu-
Neuroscience
tants is identical to evidence demonstrating that Bhlhb5 mutants are
also missing the corticospinal tract, established through research
indicating that corticospinal axons may mis-project or stop growth
prematurely and do not reach targets in the dorsal spinal cord (Ross
et al., unpublished data). Therefore, the combination of past research
exhibiting the absence of the corticospinal tract in Bhlhb5 mutants
with this project’s findings that Prdm8 mutants may also lack the
corticospinal tract further support the overarching hypothesis that
Prdm8 and Bhlhb5 are both crucial factors in the development of cir-
cuit formation in the central nervous system. Intriguingly, it was also
noted that double heterozygous mice for Prdm8 and Bhlhb5 (Prdm8-
/+ Bhlhb5 -/+) also indicate loss of the CST through the absence of
PKC-gamma protein expression in spinal cord sections at P0 (Sup.
Figure 7B). However, this observation regarding the loss of the CST
in heterozygous animals is not observed in animals heterozygous for
only one gene.
Table 1. PRDM8 mutants exhib-
Mean # PRDM8-
it signifiacnt loss of PRDM8-ex-
expressing cells in 2-Tailed T Test P-
pressing neurons in dorsal horn
PRDB8 Mut Value*
of spinal cord. 2-Tailed statistical
(ST. Dev)
test (*2 sample unequal variance het-
eroskedastic) comparing te number
of PRDM8-expressing neurons be-
tween Bhlhb5 mut and wt mice in-
28 Neurons dicate a statistically significant differ-
(7.483314) 0.0011548 ence (p-value<0.05, confidence value
of 95%).
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Discussion
No evidence of Bhlhb5 or Prdm8 binding in 293T cells
Results from this experiment (Figure 9) did not present any evi-
dence of co-IP. Conversely, when immunoprecipitation for Prdm8
was conducted, no presence of Bhlhb5 was observed in the precipi-
tate. These results suggest that Bhlhb5 and Prdm8 do not interact,
and therefore provide evidence in support of models in the Figures
4A and 4C. However, this experiment was conducted in transfected
293T cells, where many endogenous factors necessary for regulato-
ry processes in neurons are absent. It is possible that other proteins
present in neurons but absent in 293T cells are necessary co-factors to
facilitate the complex formation of Bhlhb5 and Prdm8.
The immunoprecipitation experiment was replicated in neurons
where pull-down of endogenous Prdm8 and Bhlhb5 in their natural
substrate environments could be observed. To conduct this experi-
ment in neurons, a direct Prdm8 antibody was required. The previous
immunoprecipitation experiment conducted in 293T cells utilized a
Myc-epitope tagged construct of Prdm8. Thus, immunoprecipitation
and probing for Prdm8 in 293T cells could be performed via the use
of an anti-Myc antibody (rather than a direct antibody for Prdm8). A
chief obstacle in this study that arose was the lack of available Prdm8
antibody to use in immunoprecipitation and immunohistochemistry
experiments on neurons and fixepd tissue.
Design and development of the Prdm8 antibody
To resolve the issue of the absence of a Prdm8 antibody, a series of
Prdm8 GST-Fusion protein constructs was designed and developed
(Supp. Figures 3 and 4), from which a purified Prdm8 antigen could
be selected and injected into rabbits for production of an antibody.
No evidence of endogenous Bhlhb5 and Prdm8 binding in neu-
rons
Immunoprecipitation for Bhlhb5 in Prdm8 wildtype and mutant
Neuroscience
neurons from the whole cortex of mice demonstrated clear pull-
down of the Bhlhb5 protein, yet no evidence of any Prdm8 was found
in the precipitate (Figure 6). Similarly, no co-IP of Bhlhb5 was ob-
served when pull-down for Prdm8 was conducted (Figure 7). Again,
these results suggested that Bhlhb5 and Prdm8 do not interact, and
therefore provided evidence in support of models in the Figures 4A
and 4C.
Nevertheless, it is also possible that conditions of the immunopre-
cipitation experiment itself may have contributed to the dissociation
of proteins in complex from one another. Considering the multiple
steps involved in immunoprecipitation (i.e. washing of the beads,
incubation of the antibody, and incubation with beads), it is very
plausible that the nature of the protein-protein interaction between
Bhlhb5 and Prdm8 may be very sensitive to the concentration of the
lysis buffer ingredients, and that such may have affected the natural
affinity between Bhlhb5 and Prdm8. Future experiments to clarify
the interactive relationship between Bhlhb5 and Prdm8 should there-
fore call for a series of tests examining the effects of different lysis
buffer ingredients, concentrations, and wash conditions on the im-
munoprecipitation and co-immunoprecipitation of proteins.
Significant loss of Prdm8-expressing neurons in Bhlhb5 mutant
spinal cord
To further explore the relationship between Bhlhb5 and Prdm8 in
neural development, the expression patterns of each protein in both
Bhlh5 and Prdm8 mutants were characterized. Immunohistochemis-
try analysis was conducted on Bhlhb5 mutant mice, and a significant
loss of Prdm8 was observed in the dorsal horn of the spinal cord at P0.
The researcher confirmed this observation through immunostaining
analysis (Figure 8A) and statistical quantification (Table 1, Figure
8B). These results, in conjunction with past research that Bhlhb5 is
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Volume 2 Issue 2 | Fall 2009
significantly lost from populations of neurons in the superficial layers
of the dorsal horn of the spinal cord (Ross et al., unpublished data),
combine to support the primary hypothesis: that Prdm8 and Bhlhb5
may be important regulatory factors in the same pathways in central
nervous system development. With evidence from past research that
Bhlhb5-expressing neurons are lost in the dorsal horn of the spinal
cord, this finding that Prdm8-expressing neurons are also lost in the
same region of the spinal cord suggested that Prdm8 and Bhlhb5 may
be highly expressed in the same neurons.
High levels of co-expression of Prdm8 and Bhlhb5 in dorsal horn
of spinal cord
Due to the finding that a significant loss of Prdm8-expressing
neurons was observed in the same regions of the dorsal spinal cord
where a significant loss of Bhlhb5-expressing neurons are absent in
Bhlhb5 mutants, it was asked whether or not Bhlhb5 and Prdm8 co-
expression would also be observed in the dorsal horn of the spinal
cord. Sup. Figure 6A depicts immunostaining results from a wildtype
mouse spinal cord. A high degree of co-localization of Prdm8 and
Bhlhb5 protein expression in the dorsal horn of the spinal cord was
observed. Furthermore, when the wildtype immunostaining results
were compared to those from Prdm8 mutant spinal cord, a qualita-
tive loss was determined of neurons co-expressing both Prdm8 and
Bhlhb5 in the dorsal horn of the spinal cord (Sup. Figure 6B). The
finding that Prdm8 and Bhlhb5 are both highly expressed in the same
neurons thus provides further evidence in support of the hypothesis
that Bhlhb5 and Prdm8 are involved in the same pathways in nervous
system development.
Due to the lack of available Prdm8 mutant mice, there was not
a sufficient sample size to conduct a quantitative statistical analysis
to determine the significance of this loss of neurons co-expressing
Prdm8 and Bhlhb5 in the dorsal horn of the spinal cord. Future ex-
periments, however, should call for a quantification of the number of
co-expressing neurons in wildtype versus Prdm8 and Bhlhb5 mutant
mice, and statistical calculation to test for significance in difference
between the two groups.
High levels of co-expression of Prdm8 and Bhlhb5 in frontal
cortex
Due to the high levels of observed Bhlhb5 and Prdm8 co-expres-
sion in the spinal cord, in addition to the underlying theory that
these two genes are crucial in the development of the central nervous
system, Bhlhb5 and Prdm8 expression patterns in the mouse brain
were analyzed. Immunostaining analysis was conducted for Bhlhb5
and Prdm8 on sagittal sections of the mouse cortex. Expression was
observed for each of these proteins in cortical neurons, and for any
distinguishing features between Bhlhb5 and Prdm8 expression pat-
terns in wildtype versus Prdm8 mutant P0 mice. From the results
(Figure 9), high levels of Prdm8 and Bhlhb5 co-expression in the cor-
tex was found. The qualitative analysis further determined that the
areas of greatest co-expression were the outermost layers of the fron-
tal cortex. Qualitative examination of Bhlhb5 and Prdm8 expression
in the frontal cortex between wildtype and Prdm8 mutant mice did
not reveal any outstanding disparities in expression patterns. Nev-
ertheless, further analysis following the availability of more Prdm8
mutant mice would be necessary to examine critically the differences
in Prdm8 and Bhlhb5 expression patterns in the cortex of mutant
mice.
Prdm8 mutants and Prdm8-Bhlhb5 heterozygotes exhibit loss of
CST
From the finding that Bhlhb5 mutants exhibit distinct loss of the
corticospinal tract (CST) (Figure 3), it was hypothesized that Prdm8
mutants would also exhibit loss of the CST. Immunostaining analy-
sis was conducted utilizing a CST marker (PKC-g) (Sup. Figure 7C),
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
which indicates the distinct loss of the CST in Prdm8 mutants. This
result supportes the hypothesis that Prdm8 and Bhlhb5 mutants are
both missing the CST and further provides evidence in support of the
theory that Prdm8 and Bhlhb5 are crucial in the development of neu-
rons involved in the same pathways of the central nervous system.
More intriguingly, however, the loss of the CST in double heterozy-
gous mice (Bhlhb5 -/+ Prdm8 -/+) (Sup. Figure 7B) was noted. This
finding is particularly striking, since no loss of the CST was observed
in mice heterozygous for only one gene (i.e. Bhlhb5 -/+ Prdm8 +/+
or Bhlhb5 +/+ Prdm8-/+). Therefore, the finding that a heterozygous
copy of both Prdm8 and Bhlhb5 could result in the absence of the
CST, a phenotype found only in Bhlhb5 or Prdm8 full knockouts,
suggestes that Bhlhb5 and Prdm8 may have an additive impact where
the absence of a single copy of both Prdm8 and Bhlhb5 in mice may
be equivalent in phenotype to missing both copies of either gene. The
discovery of the absence of the CST in double heterozygous mice is
exceptionally intriguing and prompts the hypothesis that such mice
will also exhibit other identical phenotypes at the cellular (i.e. sig-
nificant loss of Bhlhb5 or Prdm8-expressing neurons) and behavioral
(i.e. skin lesion, handstand) levels. This hypothesis thus calls for fu-
ture experiments to explore the phenotypes of double heterozygous
mice through a series of behavioral assays (i.e. rotarod balance exper-
iments, observation of self-injurious behavior, thermal and chemical
stimulation assays) and examination of cellular expression patterns
(i.e. analysis of Bhlhb5 and Prdm8-expressing neurons in the spinal
cord and brain) and comparing such results to those of Prdm8 or
Bhlhb5 single gene knockout mice.
Future Directions
Although results gathered in this study does not suggest direct
interaction between Bhlhb5 and Prdm8, it is still not clear what the
precise nature of their regulatory relationship may be. Three poten-
tial models of interaction were proposed, of which one (Figure 4B)
was not supported by the immunoprecipitation results. However, two
other models (Figures 4A and 4C) are possible. Therefore, a future di-
rection of this study would be to distinguish between the two mecha-
nisms or identify other alternative models that may best describe the
relationship between Bhlhb5 and Prdm8.
Conclusions
The major finding in this study was that no indication of direct
interaction between Bhlhb5 and Prdm8 proteins was observed. The
models in Figures 8A and 8C are potential mechanisms of interaction
between Bhlhb5 and Prdm8 in transcriptional regulation. In these
models, Bhlhb5 and Prdm8 do not bind to each other in complex,
targeting either the same genes (Figure 4A) or distinct genes in the
same pathway (Figure 4C). Nevertheless, due to the nature of the
experimental conditions and the possible sensitivity of the proteins’
affinities for each other, it is necessary that further experiments be
conducted before the model in which Prdm8 and Bhlhb5 interact in
complex (Figure 4B) may be rejected.
Despite the lack of evidence identifying the precise model of inter-
action between Bhlhb5 and Prdm8, immunostaining results provide
strong evidence in support of the overarching theory that Bhlhb5 and
Prdm8 function as important regulatory factors in the same pathway
of neuronal development in the central nervous system. This con-
clusion was drawn from four critical findings: 1) Prdm8-expressing
neurons are significantly lost in the dorsal horn of the spinal cord in
Bhlhb5 mutants, 2) high levels of co-expression of Prdm8 and Bhlhb5
are observed in the dorsal horn of the spinal cord, 3) dramatic loss of
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RESEARCH
neurons co-expressing Prdm8 and Bhlhb5 is observed in the dorsal
horn of the spinal cord, and 4) loss of the CST is observed in Prdm8
mutants and double heterozygous mice (Bhlhb5 het, Prdm8 het). The
combination of these four findings demonstrate that Prdm8 mutants,
like Bhlhb5 mutants, express similar Prdm8 and Bhlhb5 expression
patterns in the brain and spinal cord. This study additionally sug-
gests that the missing subpopulation of neurons in the dorsal horn of
the spinal cord co-express both Bhlhb5 and Prdm8, thus providing
evidence in support of the hypothesis that Bhlhb5 and Prdm8 are
crucial factors in a common pathway underlying neural differentia-
tion and development.
Technical difficulties encountered included the lack of available
mice of desired Prdm8 and Bhlhb5 genotypes. With a greater number
of Prdm8 mutants, double heterozygotes, and double mutants, more
immunostaining experiments could be conducted to discern for any
nuances in phenotype that distinguish Bhlhb5 mutants from Prdm8
mutants. Furthermore, the availability of more mice of desired mu-
tant genotypes would allow for statistical analysis to be performed
so that the significance of Bhlhb5 and Prdm8 co-expression may be
calculated.
In sum, the overarching motivation that propelled this research
was the pursuit of a better understanding of the processes underly-
ing central nervous system development in mammals. The striking
phenotypes exhibited by both Bhlhb5 and Prdm8 mutant mice was
the first signal that a cooperative relationship may exist between the
two proteins, and that their function was critical in neural develop-
ment. These results provide support for the view that a novel relation-
ship between Bhlhb5 and Prdm8 exist in the same neural pathways;
they also help illustrate a process within the formation of the central
nervous system that have significance for the future understanding
of not only how the mammalian nervous system develops, but how it
matures and ages with time.
Neuroscience
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Hamik, A., and Jain, M.K. (2008). Variety is the splice of life. Journal of Mo-
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Hand, R., Bortone, D., Mattar, P., Nguyen, L., Heng, J.I.-T., Guerrier, S.,
Boutt, E., Peters, E., Barnes, A.P., Parras, C., et al. (2005). Phosphoryla-
tion of Neurogenin2 Specifies the Migration Properties and the Dendritic
Morphology of Pyramidal Neurons in the Neocortex. Neuron 48, 45-62.
Hughes, S.M. (2004). Muscle Differentiation: A Gene for Slow Muscle? Cur-
rent Biology 14, R156-R157.
Jiang, X., Tian, F., Du, Y., Copeland, N.G., Jenkins, N.A., Tessarollo, L., Wu,
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moter 4 Activity and Neuronal Excitability. J. Neurosci. 28, 1118-1130.
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The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Still images of a motion picture: Using

static crystal structures to understand the
behavior of a DNA glycosylase
Kimberly (Wei-Wei) Oo
Harvard College ‘09, ms.kimberlyoo@gmail.com

The cell has developed a number of defenses against DNA damage; glycosylases, for example, remove damaged bases.
Human 8-oxoguanine glycosylase 1 (hOGG1) is responsible for the excision of the damaged base oxoguanine (oxoG).
In this study, we present the first reported structure of hOGG1 containing guanine in a fully wild type active site. In
addition, we also present the structure of a novel catalytic intermediate during the excision of oxoG.

Introduction (Crenshaw 2009; David 1998). hOGG1 is a bifunctional enzyme, act-

Maintaining the integrity of the genome is crucial for the sur-
vival of an organism. DNA is vulnerable to damage from a variety
of sources, both extrinsic and intrinsic to the cell (Lindahl 1993;
Friedberg 2003). Unless the cell can repair this damage, it will cause
mutations in the genome. Thus, DNA repair mechanisms in the cell
are extraordinarily important (Lindahl 1993). One common kind of
DNA damage is the oxidation of guanine to 8-oxoguanine (oxoG) by
reactive oxygen species (Neeley 2006; Henle 1997), which are com-
monly produced by ionizing radiation, exposure to transition metals,
or even as byproducts of aerobic respiration (Henle 1997; Bjelland
2003). Although the Watson-Crick face of oxoG is identical to that of
guanine and can still base pair with cytosine, it can also rotate around
its glycosidic bond and base pair with adenine through a Hoogsteen
interaction (Oda 1991) (Figure 1). During replication, DNA poly-
merase preferentially adds adenine across from oxoG, which after
two rounds of replication, causes a G:C to T:A transversion, thereby
introducing a mutation into the genome (Hsu 2004; Shibutani 2001).
Human 8-oxoguanine glycosylase 1 (hOGG1) locates and excises
oxoG from the genome as part of the base excision repair pathway
ing as a glycosylase, which breaks the N-glycosidic bond to the base,
and a β-lyase, which nicks the DNA on the 3' side of the sugar (Boi-
teux 2001). The enzyme diffuses along the DNA until it locates oxoG.
It induces a bend in the DNA and extrudes oxoG out of the DNA
helix and into its active site (Bruner 2000). The structural intermedi-
ates involved in flipping out the base from the DNA helix make up
the base extrusion pathway. Then, hOGG1 excises oxoG, creating an
abasic site, and nicks the DNA (Dodson 1994). Other members of the
base excision repair pathway then remove the sugar and insert a new
guanine nucleotide, repairing the DNA (Vidal 2001).
One goal of the Verdine lab is to answer the “search problem,”
namely, how hOGG1 distinguishes oxoG from other bases. Although
hOGG1 can distinguish between damaged and normal bases while
they are within its active site through specific contacts, it is unclear
whether it is necessary to individually flip out each base into the ac-
tive site to find rare oxoG substrates hidden in the genome (Bruner
2000). Such a process seems unlikely since hOGG1 slides along DNA
at a velocity approaching the upper limit for one-dimensional diffu-
sion (Blainey 2006). However, when oxoG is intrahelical, there are no
obvious structural distortions to the DNA duplex that could be used

Chemical
Biology
to distinguish between oxoG and guanine (Bowman 2008; Lipscomb
1995; Oda 1991).
To determine if hOGG1 can distinguish between guanine and
oxoG while the bases are intrahelical, it would be ideal to capture
“encounter complexes” of hOGG1 on an intrahelical oxoG and gua-
nine. With these structures, one could compare the interactions be-
tween the protein and DNA to determine if the interactions differed
between oxoG and guanine. Thus, our initial goal was to solve the
structure of hOGG1 with an intrahelical guanine.
Another goal of our lab is to elucidate the catalytic mechanism
hOGG1 uses to cleave out oxoG. In general, a bifunctional glycosy-
lase uses a nucleophilic residue to displace the undesired base from
the sugar, opening the ring and forming a Schiff base. β-elimination
of the 3' phosphate through deprotonation of the C2' hydrogen cleaves
the DNA strand and forms an α, β unsaturated Schiff base (David
1998), which is then hydrolyzed to release the glycosylase from the
DNA, completing the mechanism. The structures of several interme-
diates have been solved to bring insight into the specific mechanism
Figure 1. The cause of the mutagenic potential of oxoG. Repro- hOGG1 uses (Fromme 2003; Radom 2006; Chung 2004).
duced from Crenshaw 2009. Here, we present two crystal structures of hOGG1-DNA com-
www.thurj.org 55
 RESEARCH
plexes. The first is a structure of hOGG1 containing guanine within
its active site, the “G complex.” This structure was solved while we
attempted to solve a structure of hOGG1 with an intrahelical gua-
nine. The G complex was completely unexpected because previous
studies indicated that guanine would be excluded from the active site
(Banerjee 2006). In spite of being in the active site of a catalytically
active hOGG1, the guanine was not excised out of the DNA. Thus,
this study presents the first structure of hOGG1 with an undamaged
base in the wild-type active site. The second structure presented is a
structure of hOGG1 bound to a trans-α,β-unsaturated aldehyde, a
previously unreported intermediate along the catalytic pathway.
Methods
Because hOGG1 has no specific preference for an undamaged base,
to capture a structure with guanine it is necessary to restrict hOGG1
to that guanine so that the protein-DNA complex would be homo-
geneous enough for crystallographic studies. We formed a disulfide
bond between a mutated cystine on hOGG1 and a modified thiol-
containing base in the DNA, creating a disulfide crosslink that cova-
lently tethers the two together, restricting hOGG1 to the desired base
(Banerjee 2005) (Figure 2). This disulfide crosslink was necessary to
determine the structure of many of the intermediates captured thus
far (Banerjee 2005; Banerjee 2005; Radom 2007). Our lab has also
employed this strategy to determine the structure of other systems
suffering from similar problems (Huang 2000; Huang 1998).
A number of intermediates along the base extrusion and catalytic
pathways have already been captured. In the base extrusion pathway,
crystal structures have been solved containing: guanine extruded
out of the helical stack, but folded back into the major groove of the
DNA (intermediate 1) (Banerjee 2006); oxoG (int. 2) (Radom 2007)
and guanine (int. 3) (Banerjee 2005) in an “exo site,” with the base
extrahelical, but on a part of hOGG1 adjacent to the active site; oxoG
entering the active site (int. 4) (Radom 2007); and oxoG fully in the
active site (int. 5-8) (Bruner 2000; Banerjee 2005; Radom 2007; Nor-
man 2003). An overview of the previously captured intermediates in
the base extrusion pathway is summarized in Figure 3.
In the earliest intermediate so far crystallized in the pathway,
(int. 1), guanine was extruded out of the helical stack into the major
groove (Banerjee 2006). The guanine was placed adjacent to oxoG,
but a disulfide crosslink formed between N149C and the cytosine
opposite from the guanine was short enough to restrict hOGG1 to
Chemical
Biology
4 2
C4Cysteamine
Cysteine 292
hOgg 1 S292C
Crosslinking A
Figure 2. Functionalization of adenine and crosslinking with
hOGG1 at the S292C site
56
Volume 2 Issue 2 | Fall 2009
interrogate the guanine. The crosslink forced the guanine out of the
DNA helix because the crosslink physically occupied the same place
the intrahelical guanine would have occupied. The extruded gua-
nine formed a noncannonical base pair with the neighboring oxoG,
stabilizing guanine in the major groove. Likewise, in the structure
containing guanine in the exo site, (int. 3), the same disulfide cross-
linking site was used, which biased the guanine to be extrahelical
(Banerjee 2005).
These two results seemed to imply that when forced out of the he-
lix, guanine should have no strong preference for any particular site
extrahelical to the DNA, as both the exo site and the noncannonical
base pair do not seem offer a particularly large amount of stabiliza-
tion (Banerjee 2005; Banerjee 2006). Thus, it is likely that it is more
favorable for guanine to be within the DNA helix, where there are
specific interactions stabilizing it. As a result, moving the crosslink
to a different site that would not sterically displace guanine from the
DNA helix might allow the capture of a structure containing an in-
trahelical guanine (Radom 2006).
In this study, the crosslinking site was between S292C and an
adenine four bases down from the guanine. This site had previously
been employed to solve the late-stage intermediate structure (int. 4)
with oxoG partially in the active site of hOGG1 (Radom 2007). In
addition, the structure of a catalytically inactive mutant of hOGG1
crosslinked to oxoG containing DNA at the S292C site (int. 7) is
almost completely identical to the structure of the mutant without
the crosslinking site (int. 5) and the structure of the mutant with
the N149C crosslinking site (Bruner 2000; Radom 2007) . Thus, the
S292C crosslinking site seemed like it might not force the guanine to
be extrahelical. As a result, we hoped that crystallization of hOGG1
crosslinked through S292C to guanine-containing DNA would allow
the structural determination of a complex containing an intrahelical
guanine. In reality, the structure captured was that of hOGG1 with
guanine in its active site. An overview of the disulfide crosslinking
strategy is presented in Figure 4.
Several intermediates along the catalytic pathway have also been
captured. An overview of the theorized catalytic mechanism sum-
marizing the previously captured intermediates is presented in Fig-
ure 5. Two intermediates in the catalytic mechanism for hOGG1 have
been captured using the technique of borohydride trapping (Fromme
2003; Radom 2006). Intermediates containing iminium ions can be
reduced by sodium borohydride to the amine, halting catalysis at a
particular step and allowing for the characterization of a normally
Figure 3. An overview of the previously captured intermediates
in the base extrusion pathway. hOGG1 is shown in purple, DNA in
blue, and the interrogated base in red. The intermediates captured thus are
categorized by: the location of the interrogated base; the crosslinking site,
if there is one; and other mutations/information. PDB accession codes: 1.
2I5W29, 2. 2NOF31, 3. 1YQK30, 4. 2NOZ31, 5. 1EBM15, 6. 1YQR30, 7.
2NOL31, 8. 1N3C32.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

borohydride (V) (Radom 2006).

Figure 4. An overview of the crosslinking strategy. A) Crystal
structures of: (int. 1). K249Q hOGG1 (green) bound DNA (blue) with
oxoG (red); (int. 9). S292C hOGG1 (purple) crosslinked to DNA with
guanine (green); (int. 3). N149C hOGG1 (yellow) crosslinked to DNA
with guanine. B) and C) A comparison of the S292C and N149C crosslink
sites.
fleeting intermediate (Zharkov 2002; Gilboa 2002). In hOGG1, the
ε-amino group of Lys 249 is a nucleophile and displaces the oxoG
base to create the initial Schiff base (II). This intermediate has been
captured by treatment with sodium borohydride and the structure
determined by X-ray crystallography (III) (Fromme 2003). In ad-
dition, the intermediate after β-elimination – the α, β unsaturated
Schiff base (IV) – was captured by treatment with sodium cyano-
Although borohydride trapping has been useful in determining
the structures of intermediates, one limitation of borohydride and
other chemical trapping methods is that they can only capture inter-
mediates with certain functional groups. A more general technique
is needed to discover all the intermediates in the catalytic cycle. To
address this, Drs. Chris Radom and Seongmin Lee in the Verdine
lab developed a “time resolved” crystallography technique (Radom
2006). Instead of chemically trapping an intermediate, the interme-
diate would be freeze-trapped by cryoprotection of crystals in liquid
nitrogen. In brief, DNA containing a bulky photocleavable adduct
attached to oxoG was crosslinked to hOGG1 by a disulfide tether be-
tween N149C and the cytosine opposite from oxoG and crystallized.
The structure of a complex containing this bulky adduct has been
solved, indicating that the bulky adduct prevents oxoG from entering
the active site and instead places it at the exo site. Flashing the crystal
with ultraviolet light cleaves the adduct off and allows oxoG to enter
the active site of hOGG1. Therefore, all the hOGG1 synchronously
begins excising the oxoG. After a certain length of time, the crystal is
frozen with liquid nitrogen, and the low temperature (77K) prevents
further catalysis from occurring. As crystals are usually cryoprotect-
ed to protect against radiation damage and increase the diffraction
quality and resolution, this method of freeze-trapping the crystals is
very convenient (Rodgers 1994; Henderson 1990). Ideally, this allows
one to capture the various intermediates along the catalytic pathway
by varying the time between photocleaving the adduct and freezing
the crystal (Radom 2006).
This technique has been used to capture a late stage intermediate
in the base extrusion pathway (Lee 2008). A crystal was cryoprotect-
ed immediately after photocleavage of the adduct, and the structure
of that complex was solved. The oxoG was almost fully inserted into

SN1-like

SN2-like
NaBH4

I. Before Catalysis II. Initial Schiff Base III. Reduced Schiff

Base
E2-like E1cb-like

Chemical
Biology
Hydrolysis

VI. α, β Unsaturated Aldehyde IV. α, β Unsaturated Schiff Base

(This work)

Ring Closing NaBH 3CN

VII. Ring Closed Sugar V. Reduced α, β Unsaturated Schiff Base

Figure 5. An overview of the catalytic pathway of hOGG1. Red indicates that the structures have been crystallized. The geometry of double bond
in IV and V can be either cis or trans. PDB accession codes: I. Same as (int. 5)-(int. 8), III. IHUO, V. Not submitted, and VII. 1M3H.
www.thurj.org 57
 RESEARCH Volume 2 Issue 2 | Fall 2009

the active site of hOGG1, but the active site had not yet made any of and addition of additives, without much improvement.
the contacts with oxoG that have been observed in other structures To find a different crystal form that might be more amenable to
containing oxoG in the active site. Thus, this structure validates this crystallographic studies, ten 96-well Nextal screening suites (Qiagen)
technique as a way to capture otherwise fleeting intermediates. were used to screen the hOGG1 complex over a variety of different
This study used the time resolved crystallography technique to conditions. However, the crystals that resulted from this large-scale
solve the crystal structure of a catalytic intermediate obtained by screen were almost all either needles or dendrites and were also dif-
freezing the crystal 30 minutes after photocleavage. It is hereafter re- ficult to reproduce in the 24-well format. As a result, this effort did
ferred to as the “30 minute structure.” not yield any useful results.
Eventually, a crystal yielding diffraction data of sufficient qual-
Results and Discussion ity was produced. The complex was crystallized with the addition of
additives in a hanging drop vapor diffusion format with the 24 well
Crystallization of the G complex OptiSalts Screen (Hamptom). The resolution of the structure from
The crystallization of the G complex was surprisingly difficult. this crystal was 3.05 Å. Unfortunately, reproduction of this condition
The previous structures of base extrusion intermediates had all been did not produce higher resolution data, so the 3.05 Å structure was
crystallized in similar conditions, approximately 150 mM calcium used.
chloride, 17% polyethylene glycol 8000, and 100 mM sodium cacody- Structure of the G complex
late pH 6.0 within the 24 well hanging drop vapor diffusion method Charisse Crenshaw collected X-ray diffraction data for the G com-
(Bruner 2000; Banerjee 2005; Banerjee 2006). However, when these plex and scaled it to 3.05 Å (Figure 6). She solved the structure using
conditions were used, the crystals were either too thin to be useful in the phases calculated from a previously solved hOGG1 structure, the
X-ray crystallography or severely branched. In addition, they were K249Q, Q315A mutant containing oxoG in the active site (PDB ac-
fragile and broke apart while being transferred to cryoprotectant. cession code: 2NOH) (Radom 2007). Surprisingly, this structure was
The screens were broadened to look at the effects of different salts almost identical to structures containing oxoG in the active site, ex-
(magnesium acetate and magnesium chloride), temperatures (4 and cept that guanine was in the place of oxoG (heavy atom RMSD=0.401
20 °C), drop volumes, and protein complex to well solution ratios, Å with (int. 5), the structure of catalytically inactive hOGG1 (K249Q)
without much change in crystal behavior. In addition, other crystal- bound to oxoG containing DNA) (Bruner 2000).
lization techniques were tried, such as sitting drop vapor diffusion, Conformational changes
the use of oils to slow vapor diffusion, macroseeding, streakseeding, When hOGG1 binds oxoG in its active site, it undergoes confor-
mational changes that allow its residues to make contact with oxoG
hOGG1 dDXL G-complex (Space group: P6522, Cell parameters: a = b = 90.91
(Crenshaw 2009; Radom 2007). In a structure without oxoG in the
Å, c = 211.63 Å, α = β = 90.0°, and γ = 120°) active site, such as in the structures along the base extrusion pathway,
Data collection a the α-O helix is held away from the active site in an “open” conforma-
Source APS 24ID tion (Banerjee 2005; Radom 2007). In this conformation, His 270 on
the α-O helix forms a salt bridge with Asp 322 and an aryl-π inter-
Wavelength (Å) 0.97921
Resolution (Å) 50-3.05 (3.16-3.05)
R sym (%) a
13.4 (69.7)
Total no. of obs. 130,934
No. of unique obs. 10,527 (1,009)
Completeness (%) 99.7 (100.0)
<I> / σ <I> 45 (2.1)
Refinement
Chemical
Biology

Resolution (Å) 50-3.05

No. reflections 10,500
No. of non-hydrogen atoms 3,067
Rwork (%)b 22.09
Rfree (%) c
28.72
Mean B value (Å 2)d 22.8038
Rmsd bond (Å) d
0.0063
Rmsd angle (°)d 1.244
Ramachandran plot analysis e
82.4, 17.3, 0.4, 0.0
(% most favored, additional allowed,
generously allowed, disallowed)
a
R sym = Σ|I–<I>|/ ΣI where I is the integrated intensity of a given reflection.
b
Rwork = Σ|F(obs)–F(calc)|/ΣF(obs).
c
Rfree = Σ|F(obs)–F(calc)|/ΣF(obs), calculated using 7.5% of the data
d
From CNS.
e
From PROCHECK.
Figure 7. A comparison of the open and closed active sites. The
Figure 6. Data collection and refinement statistics of the G com- G complex is purple. A) open active site (int. 3) is orange. B) closed active
plex. Reproduced from Crenshaw 2009. site (int. 5) is green.
58 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
action with Phe 319 (Figure 7a). However, in a structure containing
oxoG in the active site, such as the structures captured with catalyti-
cally inactive mutants or the structures of the catalytic intermediates,
the α-O helix moves towards the active site into a “closed” conforma-
tion, and His 270 instead forms a salt bridge with a phosphate of the
DNA backbone, freeing Phe 319 to form a π-stacking interaction with
oxoG. Furthermore, Gln 315 hydrogen bonds with the Watson-Crick
face of oxoG (Figure 7b) (Bruner 2000; Radom 2007).
In the structure of the active site G complex, the aforementioned
residues occupy the same positions as the residues in the “closed”
conformation, with the exception of His 270, which is rotated 90°.
However, this rotation does not affect its ability to form a salt bridge
with the DNA phosphate, and it functions in the same manner as the
histidine in the “closed” conformation.
Discrimination between oxoG and guanine
The previous interactions are characteristic of the “closed” con-
formation and stabilize both oxoG and guanine. However, based on
quantum mechanical calculations using (int. 3) and (int. 5), there are
two sources of discrimination between oxoG and guanine in the ac-
tive site of hOGG1 that might destabilize guanine binding in the ac-
tive site (Banerjee 2005). First, there is a hydrogen bond between the
Gly 42 carbonyl and the oxoG N7 that is lost with guanine. This is
calculated to stabilize oxoG by about 3.5 kcal/mol. Second, the salt
bridge between Lys 249 and Cys 253 causes a dipole that interacts
favorably with the dipole of oxoG. However, since guanine has the
opposite dipole, this would be a repulsive interaction, favoring oxoG
by about 3.3 kcal/mol.
In the active site G complex, the positions of Gly 42, Lys 249 and
Cys 253 stay roughly the same, along with the rest of the residues in
the active site, when compared to (int. 5) (Figure 8). The loop contain-
ing Gly 42 was previously found to be rigid, which may explain why it
does not move to relieve the posited repulsion. However, the distance
between the Cα carbonyl of Gly 42 and N7 increases slightly (2.7 Å
in (int. 5) to 3.2 Å in the active site G complex) by the base shifting
down, which may relieve some repulsion. In addition, it is possible
that the N7 of guanine is protonated, which would recreate a favor-
able interaction. This is unlikely, however, because N7 is not basic
www.thurj.org
RESEARCH
and is unlikely to be perturbed enough to be protonated. The other
structures containing oxoG in the active site have distances similar
to that of (int. 5): 2.9 Å (int. 6) (Banerjee 2005), 2.8 Å (int. 7) (Radom
2007) and 2.7 Å (int. 8) (Norman 2003), indicating that the increased
distance to 3.2 Å may be significant. The last structure (int. 7) indi-
cates that the increase is not directly due to the crosslinker, though it
does not rule out a more subtle effect.
The salt bridge also appears to be intact in the G complex. The
distance between Lys 249 and Cys 253 increases slightly from 2.6 Å in
(int. 5) (Bruner 2000) to 2.8 Å in the G complex, which is still consis-
tent with a salt bridge (Petsko 2003). Thus, it is unlikely that these re-
pulsive interactions were completely removed, though the repulsion
may be lessened slightly. Therefore, it is unclear as to why guanine
would be inserted in the active site, as is seen in this structure.
Crosslinking bias
The authors presenting (int. 2) and (int. 4), which contain oxoG in
the exo site or partially in the active site, respectively, posed a similar
question (Radom 2007). When the crosslinking site was moved from
N149C to S292C, the oxoG moved further along the extrusion path-
way. It was proposed that the crystal packing of the particular crystal
form of (int. 4) bends the DNA to favor an extrahelical base (Radom
2007; Radom 2006). However, this leaves unresolved why in (int. 4),
oxoG moved further along the base extrusion pathway instead of re-
maining in the exo site, as presumably the crystal forms and packing
interactions were the same (since the crystallization conditions were
similar and the space groups identical).
Here, we propose that DNA bending is indeed the key difference
but that the DNA is bent because of the crosslinker, not because of
crystal packing. The distance between S292 and the adenine, which
is the distance a crosslinker would need span if it were placed at the
S292C site varies depending on the stage of the base extrusion path-
way (Figure 9). The structures of the early intermediates (1 and 3)
require a larger span (10.1 Å and 9.6 Å), while the structures of later
intermediates (5, 6, and 8) require a smaller span (8.1 Å and 8.3 Å).
However, in the nine lowest energy conformations of the four-carbon
crosslinker used at the S292C site, the largest span is 8.46 Å, which is
too short to span the distances of the early intermediates (Figure 8).
Chemical
Biology
Figure 8. The effect of the S292C crosslink. A) and B) (int. 1) pink,
(int. 3) yellow, (int. 8) green, (int. 5) blue, and (int. 6) purple. The length of
the crosslink correlates with the position of the base along the base extru-
sion pathway. C) and D) The lowest energy conformations of the crosslink
used at the S292C site. Figure reproduced from Crenshaw 2009.
59
 RESEARCH Volume 2 Issue 2 | Fall 2009

Thus, it is likely that the early intermediates, such as the exo site or
intrahelical intermediates, are disfavored by the S292C four-carbon
crosslinker, forcing the base into the active site.
Furthermore, in the previously solved structures containing the
S292C crosslinker, (int. 4) and (int. 7), the distances spanned are 8.32
Å and 6.98 Å respectively. In the latter case, it is well under the maxi-
mum distance, explaining why there was no observable perturbation
of that structure when compared to (int. 5), the structure without a
crosslink.
Based on this evidence, it is probably not possible to capture an in-
trahelical base with a four-carbon crosslink, and a longer one is nec-
essary. This also raises the possibility that different crosslink lengths
can be used to capture different stages along the base extrusion path-
way, though care must be taken to ensure that the intermediates cap-
tured are biologically relevant.
Synthesis of a C8 crosslink
An 8 carbon (C8) crosslink was synthesized to test this possibility.
However, solubility problems prevented it from being able to func-
tionalize the DNA. C8 was activated with Aldrathiol to reduce the
size and make it less nonpolar. However, this was difficult to purify
and was not ultimately used, as a shorter crosslink, such as a 5 or
6 carbon crosslink, would probably be a simpler and equally useful
tool. (See Supplementary Information for more detail.)
Structure of the 30 minute structure
The X-ray diffraction data for this structure was collected, scaled
to 2.44 Å, and solved (Figure 9). Overall, this structure resembles pre-
viously solved catalytic intermediates. Several characteristics of this
structure should be noted. First, oxoG is not present in the active site.
hOGG1 dDXL G-complex (Space group: P6522, Cell parameters: a = b = 91.99
5Å, c = 211.293 Å, α = β = 90.0°, and γ = 120°)
Data collection a
Source APS 24ID
Wavelength (Å) 0.97921
Resolution (Å) 50-2.44 (2.53-2.44)
R sym (%) a
9.0 (100)
Total no. of obs. 220,303
No. of unique obs. 20,494 (1981)
Completeness (%) 99.3 (99.4)
<I> / σ <I> 40.6 (2.1)
Chemical
Biology
Second, C3' and the 3' phosphate are not covalently bound. Third,
C1' and Lys 249 are not covalently bound. Fourth, the C2'-C3'double
bond is trans. Fifth, the structure is in the “open” conformation as-
sociated with base extrusion intermediates, instead of the “closed”
conformation associated with catalysis. The first four characteristics
point to this structure being a late stage intermediate in catalysis, af-
ter the hydrolysis of the Schiff base, but before the isomerization to
the end-product structure (Figure 10). However, the fifth characteris-
tic seems to contradict this conclusion.
A late stage intermediate
Most of the characteristics of this structure favor it as a late stage
intermediate. During refinement, when oxoG was modeled into the
active site, a strong negative density resulted in the σA-weighted fo-fc
electron density maps, indicating that there is not enough electron
density to indicate that oxoG is in the active site (data not shown).
Instead, three water molecules were placed in the active site. The exis-
tence of water molecules within the active site in the absence of oxoG
is consistent with other structures, including catalytic intermediates
(III) (Radom 2006), (V) (Radom 2006), and (VII) (Chung 2004) and
base extrusion intermediates (int. 2) (Radom 2007) and (int. 3) (Ba-
nerjee 2005).
In addition, there is no electron density between the C3' and the 3'
phosphate. Furthermore, the distance between C3' and the oxygen of
the 3' phosphate is 5.7 Å, much larger than a normal C-O bond. Thus,
in this structure, C3' and the oxygen of the 3' phosphate are no longer
covalently attached and β-elimination of the phosphate has already
taken place (Figure 10).
Similarly, there is no electron density between C1' and Lys 249,
and the distance between the terminal amine and C1' is 4.4 Å, in con-
trast with the normal distance of a C=N bond. Thus, lysine 249 is no
longer involved in a Schiff base with C1' and has been hydrolyzed off
the sugar (Figure 10).
Taken together, these three details of the structure indicate that
this intermediate is after (IV) in the catalytic pathway. Thus, this
structure is the latest intermediate currently captured along the cat-
alytic pathway. Furthermore, this structure provides evidence that
hOGG1 is able to catalyze the excision of oxoG even when it is con-
strained by crystal packing forces.
The geometry of the C2'-C3' bond
In (III), the structure of the reduced initial Schiff base, the oxoG is
already excised but remains bound within the active site of hOGG1,
and its N9 is positioned within 4 Å of C2' – a plausible distance for

Refinement
Resolution (Å) 50-2.44
No. reflections 20,492
No. of non-hydrogen atoms 2,975
Rfree (%)c 26.27
Mean B value (Å ) 2 d
51.51
Rmsd bond (Å)d Main chain–1.406, Side chain–1.988
Rmsd angle (°) d
Main chain–2.393, Side chain–3.167
Ramachandran plot analysise 86.4, 12.8, 0.7, 0.0
(% most favored, additional allowed,
generously allowed, disallowed)
a
R sym = Σ|I–<I>|/ ΣI where I is the integrated intensity of a given reflection.
b
Rwork = Σ|F(obs)–F(calc)|/ΣF(obs).
c
Rfree = Σ|F(obs)–F(calc)|/ΣF(obs), calculated using 7.5% of the data
d
From CNS.
e
From PROCHECK.

Figure 9. Data collection and refinement statistics of the 30 Figure 10. The active site of the 30 minute intermediate. In pur-
minute structure. Figure reproduced from Crenshaw 2009. ple is the α,β-unsaturated aldehyde that formed from the sugar of oxoG.
60 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
it to deprotonate C2’ (Fromme 2003). Furthermore, addition of
oxoG analogues 8-bromoguanine (8-bromoG) and 8-aminoguanine
(8-aminoG) can accelerate strand scission of an abasic site. X-ray
crystal structures of the initial borohydride trapped Schiff base with
the free oxoG purified out and 8-bromoG or 8-aminoG soaked into
the hOGG1-DNA complex show that 8-bromoG and 8-aminoG oc-
cupy the same position that oxoG occupies in (III), indicating that
oxoG may behave similarly to 8-bromoG and 8-aminoG (Fromme
2003). Taken together, this indicates that oxoG likely deprotonates
C2' and causes the elimination of the 3' phosphate.
Molecular modeling calculations have indicated that the depro-
tonation of the C2' proR hydrogen by oxoG is favored (80% proR
to 20% proS) (Fromme 2003). Nevertheless, it is unclear if elimina-
tion of the hydrogen would lead to a cis- or trans-α,β-unsaturated
Schiff base. Because the phosphate is antiperiplanar to the proR hy-
drogen, it is possible that the elimination is concerted and more E2-
like, and thus the elimination of the proR hydrogen would lead to a
trans-α,-β-unsaturated Schiff base. However, it is also possible that
the elimination instead forms the enamine first and then eliminates
the phosphate (thus is more E1cb-like), which may lead to either the
trans or cis product. In addition, molecular modeling found that the
trans product is more stable than the cis product, which is consistent
with elimination reactions in general, but although this supports the
trans product, it is possible that this reaction is under kinetic con-
trol (Fromme 2003). Furthermore, although the end-product struc-
ture (VII) contains the sugar is in the ring-closed form, and thus the
double bond between C2' and C3' must be cis eventually, this does not
preclude the formation of a trans double bond in the initial elimina-
tion, since this bond can later isomerize to the cis form (Chung 2004).
Unfortunately, the crystal structure containing the α,β-unsaturated
Schiff base caught by borohydride trapping (V) was similarly incon-
clusive. Models containing both the cis, trans, or saturated (from
potential overreduction by sodium cyanoborohydride) bonds fit into
the electron density of that structure, with little difference in the R free,
so it was not possible to distinguish between the possibilities (Radom
2006). Complicating the matter, it is also possible that hOGG1 may
produce a mixture of both the trans or cis products, which may be the
reason for the ambiguity of (V).
The structure presented in this study resolves this issue. In this
structure, the bond between C2' and C3' was determined to be trans.
The sugar of oxoG could be in one of three conformations: cis, trans,
or ring closed. To determine which of the sugar forms was likely, we
modeled in the three different forms, and after performing one round
of energy minimization and individual B factor refinement in CNS
Figure 11. Electron density maps of the 30 minute structure. A) The trans product is purple. B) The cis product is green. C) The ring closed
product is yellow. Maps are σA-weighted 2Fo-Fc map contoured at 0.6 σ.
www.thurj.org
RESEARCH
(Brunger 1998; Brunger 2007), found that the trans-α,β-unsaturated
aldehyde best fit the electron density (Figure 11). In addition, the R free
values after the round of refinement were 0.2602 for the trans, 0.2613
for the cis, and 0.2630 for the ring closed, indicating that of these
three, the trans best described the structure. In addition, even though
the cis fit the second best, it is likely that the cis intermediate may be
too short lived to capture, because it can very favorably turn into the
ring closed form since it is already in the conformation to do so. Thus,
the comparison is between the trans and ring closed forms, and the
trans intermediate fits the best.
Thus, the bond between C2' and C3' appears to be trans, implying
that the β-elimination of the 3' phosphate occurs to give the trans
product. Furthermore, this structure also indicates that isomeriza-
tion of the trans to the cis product occurs on the aldehyde and not the
Schiff base, since in this structure the Schiff base has already been
hydrolyzed.
It is likely that this isomerization occurs through a conjugate ad-
dition of a nucleophile, and then the subsequent rotation and conju-
gate elimination of that nucleophile to form the cis-α,β-unsaturated
aldehyde (Figure 12B). Although there has been no direct evidence of
this isomerization, the existence of a religated product (VIII) formed
from the addition of 8-aminoG to a crystal containing the end prod-
uct structure (VII) implies that the phosphate is able to undergo a
conjugate addition, albeit assisted by 8-aminoG (Figure 12A) (Chung
2004). Thus, it is possible that phosphate or a different nucleophile
such as water, may be able to undergo conjugate addition if driven
by an ultimate thermodynamic preference for the ring-closed struc-
ture.
The “open” conformation
Surprisingly, this structure is in the “open” conformation. The α-O
helix is held away from the active site and His 270 is interacting with
Asp 322 and has an edge-face interaction with the π-system of Phe
319, which moves it away from the position it occupies in the “closed”
conformation. In addition, Gln 315 is also away from its position in
the “closed” conformation. In contrast, the previous two catalytic in-
termediates were in the “closed” conformation (Figure 13).
At this time, it is unclear whether the “open” conformation of the
30 minute structure is the natural conformation or if it has been ar-
tificially introduced by the system. Biochemical experiments done by
Charisse Crenshaw have indicated that the disulfide crosslink, which
is one of the differences between this structure and the previous cata-
lytic intermediates, does not seem to have a significant affect on the
Chemical
Biology
conformation of the DNA, in contrast to the role it played in the G
complex. However, this does not preclude bias introduced from other
61
 RESEARCH
8-aminoG accelerated
ring opening Conjugate Addition
VII. Ring Closed Sugar
Conjugate Addition Rotation Conjugate Elimination
VI. α, β Unsaturated Aldehyde
Figure 12. A possible isomerization mechanism. A) This is a proposed mechanism for the formation of VIII (PDB accession code 1M3Q39) from
VII. B) This is the proposed mechanism for the formation of VII from VI.
aspects of the freeze trapping system or from another unidentified
bias from the crosslinker.
The previous two structures obtained using this freeze trapping
technique were in the “open” conformation; however, it is unclear if
this is because the structures were within the base extrusion pathway
or if this system biases hOGG1 to the “open” conformation. In addi-
tion, it is possible that the “closed” conformation is only necessary
for the initial part of catalysis and the conformation may move to
the “open” form later during catalysis. Because this structure does
not contain oxoG in the active site, the π-stacking interaction be-
tween Phe 319 and oxoG, which stabilizes the “closed” structure, is
lost. Thus, it is possible that by this stage in the reaction, hOGG1 has
moved back into the “open” form.
Moreover, although the end-product structure, which would be
after this intermediate, does contain hOGG1 in the “closed” form
(Chung 2004), this may be due to the mutation used to crystallize
that structure, D268E, which had previously been known to affect
the nearby residues 269-271 (Norman 2003). Because the interaction
between His 271 and the DNA is a key part of the “open” conforma-
tion, the mutation may be biasing the structure of the molecule to
the “closed” form. Again, however, it is not clear whether the “open”
conformation is an accurate description of this intermediate or if it is
an artifact brought on by the system used.
Chemical
Biology
Figure 13. The 30 minute structure is in the open conformation.
The 30 minute structure is in blue, with the sugar from oxoG in purple. III,
a closed structure, is in green, with its oxoG and sugar in orange. Here π
interactions are shown with dashes.
62
Volume 2 Issue 2 | Fall 2009
Tautomerization
VIII. Religated Product
Ring Closing
VII. Ring Closed Sugar
Conclusion
In the active site G complex, guanine is not cleaved out, even
though the protein is catalytically active, and the active site of the
guanine is in the “closed” conformation and presumably ready for
catalysis. This is surprising because excision of oxoG by hOGG1
occurs within the 30 minute structure. The G complex and the 30
minute structure are in the same crystal forms and are thus presum-
ably affected by the same crystal packing forces. Furthermore, the
S292C crosslink – at least by itself – does not prevent catalysis, as
hOGG1 with the S292C crosslink has been shown to excise oxoG in
biochemical studies (Crenshaw 2009). However, guanine does not
seem to have been excised, as the N glycosidic bond and the 3' and 5'
phosphodiester bonds are all intact. In addition, biochemical experi-
ments have shown that hOGG1 with the S292C crosslink does not
excise guanine.
Thus, the G complex raises the possibility that there is a “catalytic
checkpoint,” or a way that hOGG1 can prevent excision of guanine
even in the likely rare case that guanine is placed in the active site.
Such a catalytic checkpoint is not a ubiquitous feature of glycosylases:
Uracil DNA glycosylase, (which sterically excludes thymine from en-
tering the active site) when modified to allow thymine to enter the
active site, will excise thymine (Bennet 2006). However, human thy-
mine DNA glycosylase does use a catalytic checkpoint. It allows both
thymine from T:G mismatches and cytosine from C:G matches into
the active site but only excises thymine because thymine is a better
leaving group than cytosine (Otwinowski 1997). OxoG is intrinsical-
ly a better leaving group than guanine, and it is possible that hOGG1
discriminates between oxoG and guanine at the level of catalysis, and
not only during the search process.
The 30 minute structure is a previously uncharacterized late stage
intermediate in the catalytic mechanism of oxoG excision. In addi-
tion, this intermediate confirms that elimination of the phosphate
creates the trans α, β unsaturated Schiff base. It also lends support
to the validity of the freeze trapping technique. However, in order
to confirm whether the freeze trapping technique is valid, the struc-
tures of the other crystals for which data has already been collected
should be solved. It is important to confirm that the stage in catalysis
the intermediates are captured at correlates with the interval between
photocleavage of the adduct and cryoprotection; if not, the progres-
sion of catalysis is too stochastic, and thus the crystals are likely to
be too heterogeneous to be characterized accurately. Additionally,
it would be useful to ensure that early intermediates captured using
this methodology are in the “closed” form, like the other early inter-
mediates; otherwise, it would indicate that the freeze trapping system
artificially warps the structures.
The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

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www.thurj.org 63
ONLINE ABSTRACTS Volume 2 Issue 2 | Fall 2009

More research papers are available

online at www.thurj.org
Y-Box binding protein 1 is a novel substrate of
Granzyme A
David Kopelman ‘09, dbkopelman@gmail.com

Granzymes are the cell death effector serine proteases in the granules of natural killer (NK) cells and
cytotoxic T lymphocytes (CTL) that target cells during an immune response. Among the five human gran-
zymes, Granzyme A (GzmA) is the most abundant. It initiates caspase-independent cell death that is mor-
phologically indistinguishable from apoptosis. Once delivered by killer cells into infected cells or tumor cells
that have been targeted for elimination, GzmA cleaves a number of proteins inside the cell, including multiple
components of the SET complex, to induce apoptosis. The Lieberman Laboratory performed yeast two-hybrid
screens that identified Y-box binding protein 1 (YB-1) as a protein that specifically interacts with two SET
complex proteins, SET and pp32. YB-1 is a multifunctional nucleic acid-binding protein whose overexpression
has been implicated in multiple cancers. We investigated the possibility that YB-1 might also be a substrate
of GzmA. Treatment of lysate from cells over-expressing YB-1 with GzmA and treatment of whole cells with
GzmA and perforin (PFN) demonstrated dose-dependent proteolysis of YB-1. Purified recombinant YB-1
protein was cleaved by GzmA in the arginine-rich region between R234 and R253.

Consolidation and quality:

An examination of the effect of hospital
consolidation on the quality of care over time
Tariq Nazir Ali ‘09, tariqali@post.harvard.edu

The study of hospital consolidation and its effect on quality of patient care has been of great interest in
both the economic and legal communities as consolidation activity surged in the mid-1990s. Previous studies
have reached conclusions that the impact on quality of hospital mergers and acquisitions is either inconclusive
or is detrimental. Hospital care before and after hospital consolidation is examined from 1993 to 1998 from
patient data across 14 states. Using inpatient mortality and length of stay for CHF patients as quality of indica-
tors, the study incorporates time lag variables to test for any time variance in the effect of hospital consolida-
tion on quality of patient care. Initially, in the first year post-merger, ospital consolidation results in an initial
increase in inpatient mortality and has a negligible effect on length of stay. In subsequent years post-merger,
there is a significant decrease in inpatient mortality and length of stay—both indicating an improvement in
quality of care. These results seem to counter the conclusions of existing literature and thus, invite further
study.

64 The Harvard Undergraduate Research Journal