You are on page 1of 65

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.

Undergraduate
With thisresearch
issue of istheimportant because it helps
Harvard Undergraduate studentsJournal
Research navigate
wethe journey
continue to from
see being
science students scientific
the impressive to becoming full-fledged scientists.
accomplishments of Harvard The great inventor
College students. and scientist Edwin Land
noted that by participating in undergraduate research students bring to fruition their own special,
unique way of thinking and
Undergraduate seeing
research in environments
is important becausethat support
it helps and navigate
students encourage thetheir intellectual
individuality
journey from and creativity.
being scienceProviding
students Harvard College
to becoming studentsscientists.
full-fledged with moreThe opportunities
great to engage
with facultyand
inventor in cutting
scientistedge
Edwinresearch
Land is one of
noted the
that byhighest priorities
participating of the College. research
in undergraduate
students bring to fruition their own special, unique way of thinking and seeing in
The articles in
environments thatthis issueand
support ofencourage
the Harvardtheir Undergraduate Researchand
intellectual individuality Journal show that our
creativity.
undergraduates
Providing Harvardare already
Collegemaking
studentsimpressive
with morecontributions
opportunities to new scientificknowledge
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 CONTENTS

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

www.thurj.org
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 FEATURE

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


into research at the field station, stopping in midair at the instant
Today, Biewener’s research Biewener stresses the importance of they are changing direction. Flying
interests are primarily focused on studying animal movements in more at slower speeds actually requires the
smaller animals, particularly on avi- natural environments. The repetitive bird to generate more force, which is
an flight. When he came to Harvard walking motion on a treadmill, after why hovering is so difficult for most
from the University of Chicago in all, is quite different from navigating birds. Biewener hopes that laser im-
1999, a new wind tunnel, essentially a rocky, winding path, and the same is aging can shed some more light on
an aerial treadmill, was constructed true of the constant speeds in a wind the mechanisms behind these tricky
to study birds in flying. The birds, tunnel versus natural flight. One of turns.
usually cockatiels, are released and the ongoing projects in Biewener’s However, avian flight is only
photographed in the wind tunnel, lab studies the take-off and landing one area of research in Biewener’s
which can generate winds up to 65 of birds, in which case wind tunnels lab. Other active research proj-
miles per hour. are not very useful. Instead, doves are ects include guinea fowl walking,
Although the wind tunnels and set up indoors to fly back and forth, frog swimming, and locomotion in
treadmills have factored extensively and the displacement of air is tracked goats.
by laser imaging. In the same
way that sunlight reveals Chasing Goats
dust in the air, laser lights up
“The immediate plan is to chase
a thin sheet of air, and when
a goat around!” laughs doctoral stu-
a mist is sprayed, particles in
dent Carlos Moreno when asked
the laser light can be tracked
about his work on African pygmy
by a camera to calculate the
goats, another project supervised by
flow field around the bird.
Biewener. “It’s pretty nonscientific.
Transducers, tiny devices
You just holler and clap and make a
that measure the flexion and
lot of noise. And they run away down
extension of muscles, are
the corridor,” Moreno says about his
also implanted in the birds to
goat-chasing techniques. The goal
study how they use muscles
is to make the goat perform evasive
in different maneuvers.
maneuvers in order to study the bio-
The same laser imag-
mechanics of turning and dodging
ing set-up is used to study 90
movements. Chasing the goat is only
degree and 180 degree turns
the easy part, though probably the
in flight, the mechanism of
most physically strenuous.
which is still poorly under-
Prior studies on goats at the
stood. 180 degree turns are
field station have looked at bone
especially interesting because
bending in turns. Strain gauges are
they involve the bird almost
glued to the bone to detect bending,
www.thurj.org 7
FEATURE Volume 2 Issue 2 | Fall 2009

instead of names. The animals are


What about their horns? “Handles well cared for, especially by Pedro
Ramirez, the animal care technician
growing out of their heads,” says who has worked at the field station
for [twenty] years – “He’s the reason
Moreno. that science can happen around here”
says Moreno – but it’s inevitable that
and the goat is shooed around an en- ing the act of a gazelle running from most animals will eventually reach
closed space. Results showed that of a cheetah. A tiny slippage can be the their terminal surgery.
two bones in the leg, the radius has difference between life and death for Technologies developed for
less variability in bending than the the gazelle. In contrast to a treadmill, these studies have also spilled into
metacarpal. The radius is a curved these more variable environments in experiments on human movement
bone, so bending mostly happens which goats run allow for more in- and evolution. Professor Dan Lie-
in one direction, in contrast to the sight into how animals turn and ac- berman, from the department of
straight metacarpal that can bend celerate, as a gazelle running from a Human Evolutionary Biology, has
every which way. cheetah would. adapted the goats’ force plates to an-
The remnants of a “cinderblock Goats are chosen for the stud- alyze the dynamics of barefoot hu-
mountain” once implanted with ies because they are representa- man running.
force plates, stand next to the goat’s tive quadrupeds, and also because
runway as a reminder of past climb- they’re quite harmless. They don’t Bees Can’t Fly? Unraveling
ing studies. Current studies con- bite or kick, and are easily made to Insect Flight
tinue to focus on the biomechanics run. What about their horns? “Han-
of 90-degree turns. A goat is chased dles growing out of their heads,” says Just outside the Station’s main
down a plywood runway embedded Moreno. Chasing goats also seems lab space is a large tent-like struc-
with force plates that detect the force to be a favored activity of Duchess, ture with a running pond and a few
exerted by the goat on the ground. a pet dog who sometimes makes in plants. In early May, this greenhouse
Additionally, a camera captures the appearance at the field station. She is rather empty, but as summer rolls
movement of the goat’s joints, spe- stands in contrast to the dozens of around, the plan is to bring in local
cially labeled with joint markers. other nameless animals that popu- dragonflies to study how they chase
The act of chasing a goat down late the field station, since usually, prey. “Dragonflies do amazing aerial
a plywood runway may sound silly the scientists here are careful to keep chases,” says Professor Stacey Comb-
in itself, but it simulates evolution- a certain distance from their lab es, Assistant Professor of Organis-
arily relevant behaviors by mimick- animals, labeling them by numbers 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
8 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 FEATURE

done in collaboration with Professor


Whether or not an insect can fly higher Rob Wood’s Microrobotics Lab at the
School for Engineering and Applied
in the tree canopy, where there is Sciences, which builds miniature ro-
botic insects.
turbulence, may affect its success at The use of robotics is a theme
mating or evading capture. that runs through other research
projects too. Until recently, Biewen-
with a fan blowing out into the open sects beat their wings so fast as to er’s lab group worked in collabora-
air, which causes air to curl in and out create a negative vortex around their tion with Boston Dynamics, the
creating turbulence. There she found wings, thus generating lift. Combes biorobotics company famous for Big
that instead of retracting their large is interested in taking the study of Dog, an amazingly agile robot dog.
legs to reduce drag at high speeds, insect flight outside of controlled lab Data from the goat studies as well
the bees actually stick out their legs spaces like a vat of mineral oil and as other dog studies at the Concord
to reduce rolling and pitching, much marrying biomechanics with ecol- Field Station went into figuring out
like a figure skater sticking out his ogy, thus the dragonfly habitat out- the mechanics of Big Dog’s move-
arms to slow a spin. side. Turbulence, of course, is found ments.
Combes’ research builds on in any natural environment, and an Researchers at the Concord
foundational work done in the past insect’s ability to fly in turbulence is Field Station continue to undertake
few decades. Until twenty years ago, likely to have evolutionary impor- a wide range of studies on the biol-
the aerodynamics of insect flight tance. Whether or not an insect can ogy and biomechanics of movement.
was a mystery. Insect flight works fly higher in the tree canopy, where The insights they obtain have appli-
differently from the more familiar there is turbulence, may affect its suc- cations in diverse fields, like robotics
aerodynamics of a bird or airplane, cess at mating or evading capture. and human locomotion. Throughout
in which air flows faster over the top Additionally, Combes stud- its forty year history, the Station has
than the bottom of the wing to cre- ies the shape and material proper- harbored countless animals and sci-
ate lift, and the myth once circulated ties of insect wings, which bend and entists, and today it continues to be a
that “insects can’t fly.” The mystery flop remarkably in flight, though the center for novel research. Harvard’s
was solved by studying robotic wings aerodynamic effects of this flexibil- ‘Animal House’ parties on.
in mineral oil, which found that in- ity are unclear. Some of this work is

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

10 The Harvard Undergraduate Research Journal


Volume 2 Issue 2 | Fall 2009 FEATURE

also complete problem sets, take ’09, jokes that you can spot a PRISE the PRISE email list continues to be
orgo midterms, go to lecture, and Fellow in a crowd because “They’re filled with questions and the Fellows
participate in a slew of other extra- the ones that simultaneously crowd often end up as lab partners or din-
curricular activities, PRISE “creates around both the cheesecake and the ner dates, continuing to engage with
an environment for a concentrated science!” each other and with science.
group of scientists to share their ex- Adds Carol Suh, PRISE Fellow “PRISE provides early interdis-
periences and support each other on in Summer ’08 and Administra- ciplinary opportunities to network
a daily basis,” said Llacer. tive Fellow in Summer ’09, “Some with peers to experience science
Besides living in Leverett, the people have told me that one of the more broadly and to make connec-
Fellows are provided with a stipend special things about a PRISE fellow tions outside more narrow or tra-
for lunch and also eat dinner togeth- is that you can have a discussion ditional definitions of scientific re-
er, where the chatter often revolves on the most random science topics search,” adds Llacer.
around the important questions for hours at a time, even if it’s about Outside of their labs, the Fel-
lows are able to propose and receive
“One of the special things about a funding for a number of science and
social activities—a unique aspect of
PRISE fellow is that you can have a dis- the program that allows the Fellows
to take the initiative.

cussion on the most random science As Hsu recalls, “There were


countless fun things PRISE did –
whale-watching, getting hit in the
topics for hours at a time, even if it’s eye at point blank range by a water
balloon that didn’t explode, try-
about compost and fertilizer.” ing to figure out what exactly a 3-D
shadow of a 4-D object is, and ex-
of the day—including how to get compost and fertilizer.” ploring the creepy tunnels at Boston
an overlapping three-piece PCR to This kind of conversation is Harbor Islands.”
work and the good movies coming just what Director Gregory Llacer This past summer, for example,
out next week. hopes for every summer; indeed, Hsu was part of the team that orga-
This may seem unusual, but Jer- his favorite part of PRISE is not just nized a trip to Tanglewood, the sum-
emy Hsu, a PRISE Fellow in Summer the summer, but the entire year, as mer home of the Boston Symphony

Credit: Kate Xie THURJ ‘09

www.thurj.org 11
FEATURE Volume 2 Issue 2 | Fall 2009

Orchestra, to see them perform a little nerve-wracking, is the first volume of applicants, interviews are
with Yo-Yo Ma, and part of the team time they get to learn in detail what not feasible, so the committee relies
that planned a canoeing trip down all their newfound friends had been- on the written materials the appli-
the Charles river (cleverly named working on over the past ten weeks: cants present.
‘PRISE takes over the Charles’). “It was a remarkable experience to 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, to diagnose, treat, and pre- participants in the near
the PGP aims at “recruiting vent illness.” future.
volunteers who are willing Ten notable names in
to share their genome se- science, like Steven Pinker, For more infor-
quence and many types of Esther Dyson and George mation, go to:
personal information with Church himself, have al- h t t p : / / w w w.
the research community ready volunteered to share personalge-
and the general public, so their entire genomic se- nomes.org/
that together we will be quences with the world at
better able to advance our large. They are the mem- The
understanding of genetic bers of PGP-10, the first 3D Model
and environmental con- phase of the project, of DNA
tributions to human traits which may ultimate-
Image:
and to improve our ability ly enroll 100,000 more Wikipedia

ing obsessed with how we would actually sequence all this Q: What criteria were involved in selecting the PGP10
DNA. Sequencing struck me as very unsolved, but very (the first 10 experimental subjects of the Personal Ge-
solvable. At that time no one used computers in biology, nome Project)?
except for maybe a little bit in crystallography or neurosci-
ence. There was actually a bit of a sidetrack at this point A: They were board members of genomic companies or
that some people find amusing. I finished college two years a chief scientific officer or CEO of a sequencing instru-
early and then flunked out of graduate school. I was work- mentation facility. The IRB (Institutional Review Board)
ing so hard on the crystal structure of tRNAs. I figured I wanted it to be very likely that they knew what they were
had already proven that I could take hard courses and do getting themselves into by having published about genom-
well in them; did I have to do it again? So, I flunked out of ics or made public statements. We wanted them to be di-
Duke and for some reason Harvard let me into graduate verse within that definition, so that if I was missing some-
school. thing, they could provide another set of advisers. There
are certain things in science that you just can’t do yourself.
Q: What kind of interdisciplinary projects are you One of the problems with the old way of thinking was
working on now? 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
A: Probably the most interdisciplinary project we have is the person with the answers is a school teacher in England
the Personal Genome Project. We have actually had to in- - not American, and with no scientific background. The
novate in these fields. We got to the point that we needed most likely person to think outside of the box is the least
a lot of holistic human data. Many previous studies had likely person to have the credentials.
been very “tunnel-vision”: looking at this liver enzyme or
this liver disease. By specifically recruitinh people that are Q: Perhaps I could ask you about some of your other
okay with public disclosure, We made a new paradigm in projects? Could you tell us something about your ‘ag-
medical research. 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 companies would make a kit for you. And then even that
years. We have one of the best comparative zoology da- wasn’t enough. And eventually they would build a device
tabases. What we ideally want are two animals that are re- that would implement the kit. This has made it easier for
ally close to each other in sequence but have very different scientists to use a wide variety of tools, but you can’t al-
longevities. Two species that have very similar sequences ways hide in the ivory tower. You need to make sure that
are the mouse and the naked mole rat. The mouse is an you have enough control over whatever method you are
ideal human surrogate. It turns out that the typical mouse developing, and work with a company to ensure they don’t
lives just over two years and the naked mole rat lives about botch it. You don’t want to micromanage your company
twenty years. So what we are doing is taking big chunks of just like you don’t want to micromanage your lab, but you
DNA from the mole rat and putting them into the mouse, do want to manage it.
replacing the mouse gene with the mole rat gene to see if
we can increase its longevity. Q: You have a very eclectic range of topics. How do
you keep track of them?
Q: How direct is your involvement with businesses
and companies, and what is your motivation for de- A: Well, to me they all fit into one thing. There are a rela-
veloping biotech products? tively small number of applications that make sense to me.
For example, I say if you have to pick a disease, what is the
A: YYou can choose to just publish [your research] and thing that everyone dies of? When you are between the age
hope that someone pays attention, which is increasingly of 20 and 45, not much happens to your body. It is only
not the case. In the early days of molecular biology, com- once you start aging that almost every organ system starts
panies would make the enzymes for you [the academic fading. Aging is really the fundamental problem and all the
researcher]. And then that wasn’t enough. So then 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 Volume 2 Issue 2 | Fall 2009

Uncovering
Implicit Biases Do you know your
By Jen Jian Gong ‘12
own mind?
M
any of us believe we are more impartial than age and religion to expose a myriad of implicit biases
our historic predecessors, whether it be in These tests (located online at https://implicit.harvard.
matters of race, gender or ethnic equality. edu/implicit/demo/) are an eye-opening experience
Landmark achievements like the Universal Declara- for many subjects. Since its launch in 1998, over 4.5
tion of Human Rights, the end of apartheid or the rise million online visitors have completed the web-based
of a black president to the highest office in America version of IAT, generating an immense data set on the
might seem like enough reason to believe that human- prevalence of prejudice. The data yield a clear conclu-
ity’s biases are a thing of the past, at least in the de- sion: implicit biases not only exist, they are pervasive
veloped world. Recent scientific discoveries, however, across large sets of the general population.
suggest that they are not. Currently, Banaji’s research focuses on how implicit
Harvard psychology professor Mahzarin Banaji is a attitudes and preferences first came to be. “We know
leader in the field of implicit social cognition, which that many of them are learned by us after we are born…
investigates what she has termed “implicit biases”- un- but our minds seem infinitely ready to learn them, for
conscious prejudices that persist even as our explicit some reason.” This curious “readiness” has led Banaji
attitudes evolve. She is the current Richard Clarke and her colleagues to conduct studies utilizing young
Cabot Professor of Social Ethics and is one of the prin- children, as well as adults, in order to look for pat-
cipal investigators of “Project Implicit.” She is joined terns in the appearance and development of implicit
in this project by Brian Nosek, from the University of attitudes. They studied children’s explicit attitudes to-
Virginia. wards certain groups, but also measured their uncon-
According to Nosek, the goal of Project Implicit is scious biases using an IAT. They found that younger
“to understand thoughts and feelings that exist out- children spoke more openly about their preferences,
side of awareness and control.” The project functions while older children and adults tended to mask their
as a virtual laboratory, where online visitors can assess biases with more socially acceptable language. How-
their own implicit attitudes using the so-called Implic- ever, “on the IAT, if white American adults are show-
it Association Test (IAT). The IAT requires subjects to ing a certain level of preference for whites over blacks,
rapidly categorize pairs of stimuli, using differences in then young children in that group are showing the
response time as an indicator of implicit bias. If, for exact same preference.” These IAT data seem to in-
example, a subject holds an unconscious bias against validate the assumption “that young kids should not
Illustration by Amanda Lu

a certain group, then one would expect the subject to really show some of those biases, because they are still
have more difficulty associating positive stimuli with evolving.” In fact, implicit bias is present in what looks
images of that group’s members than with images of to be exactly the same form in children (as young as
members belonging to a preferred group. This diffi- age 6) as it is in adults. Disturbingly enough, implicit
culty manifests itself in slower response times, making biases may begin to develop at a far younger age than
the IAT a reliable indicator of bias. we previously thought.
This technique has been used to explore many forms In addition to trying to understand biases from a
of prejudice, using group classifications like race, sex, developmental perspective, Banaji and her colleagues

16 The Harvard Undergraduate Research Journal


Volume 2 Issue 2 | Fall 2009 FEATURE

have begun using neuroimaging techniques to under- Scholars are also beginning to recognize the relevance
stand how our brain activity varies when we distinguish of this research to their own fields. Professor Jerry Kang,
between different groups of people. Collaborating with from the University of California, Los Angeles Law
her is Jason Mitchell, an Assistant Professor in the De- School, has collaborated with Banaji to explore the ways
partment of Psychology. As Banaji explains, “Most of us that such scientific findings should shape the study and
feel that we treat people from different groups largely practice of law. For example, one collaborative paper
the same, that our minds focus on them in the same addresses affirmative action policy and findings from
way. Our job is to look at how early in the sequence our implicit social cognition that might affect its implemen-
brains start doing something different when we look at tation. “Right now, civil rights and racial justice talk
somebody.” seems to be stuck in the mud,” Kang says. Rather than
In one of their recent studies, published in the journal utilizing “new philosophical arguments about justice,”
Neuron, subjects heard descriptions of people of varying perhaps the findings from research into the brain and
political views and were asked questions about them. mind will eventually provide “insurmountable evidence
The study showed that “many liberals that we are not in fact ‘colorblind’.”
literally use a different set of neurons For Banaji, the an- While it is true that a growing body
when they’re thinking about Joe, the
liberal, versus Jeff, the conservative.”
swer is a resound- of psychology literature is revealing our
unconscious tendencies to think about
Most of the questions the researchers ing yes: we are groups of people differently, the im-
asked about the characters did not portant question seems to be whether
touch on political beliefs, for exam- adaptable beings, or not these biases can be changed. Is
ple: ‘Do you think he does his laun-
dry every week?’ The neuroimaging
and these implicit there a cure for the mental virus?
For Banaji, the answer is a resounding
data from these studies showed that biases are not set yes: we are adaptable beings, and these
there was more activity in the ven- implicit biases are not set in stone. She
tral medial prefrontal cortex (mPFC) in stone. believes there is a “very hopeful mes-
when subjects were asked to make judgments about sage that’s coming from a lot of work: that our systems
those with similar political leanings. When they were are highly adaptable, that we are very malleable.”
asked questions about those with dissimilar views, there She suggests that a diverse campus like Harvard’s is
was more activity in the dorsal mPFC. also a good laboratory in which to test the malleability
This research is clearly relevant to our daily lives, of our minds. Experiences with different groups of peo-
with far-reaching educational and judicial implications. ple can challenge our deep-seated biases and, perhaps,
Banaji receives many phone calls about her work – and begin to change them. “If we’re indeed adaptable, then
not just from other psychologists. Individuals in busi- these simple notions of who’s conservative and who’s
ness and law want to know more about these uncon- liberal will pose for us opportunities to see if our minds
scious prejudices because they have the potential to play can take that kind of leap.”
an integral role in their own jobs. “People don’t know Thanks to the IAT and similar research endeavors, we
that there is this stuff in their mind that ends up affect- are more aware of implicit biases than past generations.
ing how they treat people,” she explains. “Because their So, Banaji asks, “now that we know they exist…will we
intention is to do the right thing, they become really do with this information what we do with new medi-
disturbed when they learn that maybe they’re not acting cal information?” We now “know that there are things
in the interest of their patient.” happening between us that we can’t see…and that it’s
Banaji sees these implicit biases as “mental viruses”, costing our society.” Because we can no longer claim ig-
analogous to any other illness or physical virus we have norance, she argues, “the standard for us is different.”
discovered. Since everyone is susceptible to implicit The first step towards combating these viruses, Bana-
bias, Banaji says, “when your role is to be public de- ji firmly states, is “awareness, awareness, awareness.”
fender or a social worker, you especially don’t think of Since they operate outside conscious awareness, it is
yourself as harming anyone. But imagine that you too not always easy to acknowledge our biases. But, they are
carry those viruses. Are you really able to do your job as consequential nonetheless, affecting who we marry, be-
well as you might be able to?” friend, hire or convict.
www.thurj.org 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


Effective regulation of the acute and chronic phase by the host’s
HIV-1 infection immune system gives the patient a much better prognosis for the dis-
Human Immunodeficiency Virus 1 (HIV-1) is a retrovirus that ease (Vanhems and Beaulieu, 1997). Patients with little loss of CD4+
preferentially infects human nucleated cells with tropism towards T-cells have slow disease progression, hence validating the impor-
CD4+ T or monocyte-derived cell lines (Callaway et al., 1999; Sup. tance of an efficient immune response (Sheppard et al., 1993). For
Fig. A). After infection, the virus integrates its retro-transcribed this study, I was interested in aspects of the cell-mediated immune
DNA into the host cell’s genome and can either reproduce numerous response because cell types involved in this response that undertake
active progeny or remain quiescent (Stevenson et al., 1990; Hauber et anti-HIV specific functions could increase the host’s capability to
al., 1987). Cells with dormant virus, known as viral reservoirs, can control HIV-infection (Schmitz et al., 2001).
linger in the host indefinitely (Chun and Fauci, 1999). To initiate cell-mediated responses, circulating dendritic cells
Of all the Peripheral Blood Mononuclear Cell (PBMC) subsets, (DCs) in the bloodstream can phagocytose the foreign pathogens,
researchers implicated CD4+ T-cells and monocytes as key reservoirs process them, and present the viral peptides to immature CD4+ and
of virus during infection (Smith et al., 2003; Brenchley et al., 2004). CD8+ T-cells in order to begin their maturation process (Randolph
Severe depletion of CD4+ T-cells occurs during the acute stage of HIV et al., 2008). Mature CD8+ T-cells become cytotoxic T-lymphocytes
infection with around 40-50% lymphocyte loss (Hel et al., 2006; Sup. (CTLs) that target and kill infected cells or produce other antiviral
Fig. B). However, monocytes do not drastically decline in numbers responses, and mature CD4+ T helper cells either assist in the recruit-
as CD4+ T-lymphocytes do (Douek, 2007). During chronic infec- ment of CTLs, aid in the production of neutralizing antibodies, or
tion, viral levels in the blood rise, the immune system weakens, and produce cytokines that enhance the immune response (Betts et al.,
consequently AIDS symptoms, like Kaposi’s sarcoma, occur (Douek, 2001; Hel et al., 2006). Thus, infection of CD4+ T-cells greatly weak-
2007). Progression to AIDS is faster among HIV-infected patients ens the immune system. Also, it gives a rationale for the success of
with drawn out symptoms during acute infection due to a weaker long-term non-progressors who have highly efficacious T-cell-medi-
immune response (Pedersen et al., 1989). ated responses against HIV (Paroli et al., 2001).
18 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Antigen processing (Groothuis et al., 2005). CTLs primed by DCs recognize the epitope
One of the most important events in a cell-mediated immune re- presented as well as the MHC-I molecule (Frahm et al., 2007; Fig. 1).
sponse is antigen processing, which encompasses the degradation, If the CTL can recognize a non-self peptide, like an HIV-1 epitope, it

Immunology
transport, and presentation of both self and foreign proteins to CTLs. kills the infected cell or produces other antiviral responses.
There are two major antigen processing pathways: endogenous and The exogenous pathway
exogenous. Professional antigen presenting cells (APCs), like DCs, use this
The endogenous pathway pathway during the initial activation of the immune response to ex-
Defective, newly synthesized, or intracellular proteins can be tracellular pathogens. Here, MHC class II molecules on APCs present
tagged with multiple copies of ubiquitin, a protein that targets them HIV-1 epitopes to CD4+ T-cells after cathepsins peptidase degrada-
for proteolysis in the proteasome (Schubert et al., 2000). Three of the tion (Pajot et al., 2007).
proteasome’s beta subunits bear the protease’s active sites: caspase- Antigen processing in PBMC subsets
like, chymotrypsin-like, and trypsin-like, which cleave proteins after It is generally assumed that antigen processing across cell types is
acidic, hydrophobic, and basic amino acids, respectively (Kopp et al., similar, though one can deduce that differences in presented peptides
1997). can lead to variations in the immune response. For example, cellular
After proteasomal processing, the endogenous pathway utilizes production of more antigenic peptides should lead to stronger im-
peptidases in the cytosol, such as tripeptidyl peptidase II (TPPII), mune responses. Despite the multi-layered comprehension of antigen
thimet oligoendopeptidase (TOP), and aminopeptidases, to further processing, the process is not well understood in PBMC subsets in-
degrade the peptide products (Groothuis et al., 2005). Recently, an in fected by HIV. Since HIV-1 targets certain PBMC cell subsets, such
vitro intracellular experiment demonstrated that TPPII was neces- as CD4+ T-lymphocytes and monocytes, it is necessary to understand
sary for the generation of an HIV-1 epitope presented through the both the type of immune response that these subsets elicit and the
endogenous pathway in DCs (Seifert et al., 2003). Like TPPII, TOP processes by which they are generated.
can generate C-termini that are different from peptides produced by Although direct comparisons of antigen processing in CD4+ T-
the proteasome, but TOP prefers to cleave shorter peptides (Oliveira cells and monocytes have not been previously reported, some work
et al., 2001). Saric and colleagues determined that TOP was a criti- has been published about differences in antigen processing between
cal intracellular endopeptidase that degraded proteasomal products other cell types. First, mouse fibroblast and dendritic cell lines in-
of 9-17 amino acids (Saric et al., 2004). The large family of cytosolic fected with lymphocytic choriomeningtitis virus (LCMV) presented
aminopeptidases further processes these peptide products (Hattori different viral peptides to CTLs, resulting in non-identical cell type-
and Tsujimoto, 2004). However, an experiment on the capability of specific immune responses (Butz and Bevan, 1998). The presented
mice lacking leucine aminopeptidase to present viral peptides found peptides varied in their antigenicity; thus immune responses to DCs
that that this peptidase was not necessary to generate peptides for the and fibroblasts varied, showing the possibility of functional dif-
MHC-I pathway (Towne et al., 2005). This evidence does not contra- ferences in antigen processing among cell types. Another study in
dict previous findings that implicate aminopeptidases as contribu- mice found that dendritic and non-dendritic cells presented different
tors to antigen processing. Rather, it suggests that many peptidases epitopes to CTLs during primary and secondary infection of influ-
contribute to the processing of viral peptides. enza (Crowe et al., 2003). This differential presentation affected the
After cytosolic processing, peptides around 9 amino acids long immune response through the generation of distinct memory CD8+
with a preferred C-terminal residue can be transported to the en- T-cells for each cell type. Both of the aforementioned suggest cell-
doplasmic reticulum (ER) by transporter associated with antigen type-specific variations in antigen processing that affect the immune
processing (TAP) (Larsen et al., 2005). In the ER, degradation by response, making this of high interest to HIV research since HIV-1
ERAP-1 may continue before the peptide is loaded onto an MHC has tropism for select cell types.
class I molecule. Stronger binding affinity of the peptide in the MHC When this is applied to immune responses to HIV-1, where in-
binding groove displaces the complex from Tapasin, a protein that fection is subset-specific and depletion of lymphocytes is skewed to-
anchors MHC-I in the ER. Released, MHC-I/peptide travels through wards CD4+ T-cells, one realizes the need to identify and describe
the trans-golgi pathway to be presented at the surface of the cell 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.
Figure 1. Endogenous pathway of antigen processing. 1) HIV in-
A thorough understanding of HIV pathogenesis will require
tegrates its genome into host 2) Transcription of HIV genes and translation
knowledge about the immune response that HIV-infectible cells, such
of protein occur 3) Ubiquitination and degradation of HIV protein in the
proteasome 4) Peptide products are further degraded in aminopeptidase, as CD4+ T-cells and monocytes, can elicit. First, the two tropisms of
TOP, and TPPII 5) Optimal epitope is transported into the ER through HIV-1 strains are for CD4+ T-cell lines and for monocyte lineage cells
TAP 6) and is subjected into further degradation in ER by ERAP, but can (Weiss, 2008). For instance, CD16+ monocytes, which are precursors
be loaded onto MHC-I heavy chain/Tapasin complex 7) Tapasin disso- to DCs and macrophages, have increased permissivity to HIV-1 in-
ciates from completely assembled MHC-I/peptide complex 8) Complex fection with HIV-seropositive individuals having elevated numbers
moves through Golgi to 9) cell surface. 10) T-cell receptor (TCR) of CTL of this cell population (Ellery et al., 2007). Besides infectibility, both
and co-receptor, CD8, recognize MHC-1/peptide complex. CD4+ T-cells and monocyte cell lines can serve as latent reservoirs of
www.thurj.org 19
RESEARCH Volume 2 Issue 2 | Fall 2009

HIV. The creation of reservoirs begins during primary infection in anti-mouse and anti-rabbit antibody coupled to horseradish per-
resting CD4+ T-cells, and there is evidence that monocyte-derived oxidase (GE Healthcare) was diluted to 1/6000 in 5% milk and 0.1%
macrophages in the gastrointestinal tract can serve as reservoirs for NP40 and incubated for 40-45 minutes in room temperature with the
Immunology

the virus during chronic infection (Chun et al., 1998; Smith et al., membrane. Membrane was washed again as stated above. For imag-
2003). ing, ECL Plus Western Blotting Detection Reagents and Amersham
As important as CD4+ T-cells and monocytes are during HIV in- Hyperfilm ECL were used following manufacturer’s instructions (GE
fection, there is little known about the differences in their antigen Healthcare).
processing that can potentially affect the immune response. It is Peptidase inhibitors and substrates
known that gene expression between the PBMCs and T-lymphocyte Caspase-like, chymotrypsin-like, and trypsin-like active sites of
subsets varies in HIV-seropositive individuals (McLaren et al., 2004). the proteasome were inhibited by the proteasome inhibitor MG132
This introduces the possibility of variations in the expression of an- using a 50mM stock solution in DMSO (Sigma-Aldrich, St. Louis,
tigen processing genes and in the activity of their protein products, MO). Aminopeptidase activity was inhibited by Bestatin hydro-
leading to functional differences. Such variations between subsets chloride using a 12mM stock solution in DMSO (Sigma-Aldrich, St.
targeted by the virus may affect the kinetics, the amount, or the iden- Louis, MO). Cpp-AAF-pAb, the TOP inhibitor, was diluted in DMSO
tity of peptides presented to CTLs and may alter the capacity of CTLs for 1mM stock solution (Bachem, Torrance, CA). The TPPII inhibitor
to kill infected CD4+ T-cells or monocytes. Butabindide oxalate was diluted with DMSO for a 10mM stock solu-
tion (TOCRIS Bioscience, Northpoint, UK). TPPII, chymotrypsin-
Materials and Methods like, and trypsin-like activities were measured with H-Ala-Ala-Phe-
Amc, Suc-LLVY-Amc, and Boc-LRR-Amc [where Amc represents
Blood processing 7-amido-4-methyl-coumarin] respectively and were resuspended
Blood was drawn from healthy and HIV-infected donors (Massa- in DMSO to produce stock solutions of 10mM, 50mM, and 100mM
chusetts General Hospital, Boston, MA). PBMCs were isolated from (Biochem Bioscience, Torrance, CA). The aminopeptidase substrate
blood using Ficoll-Hypaque (Sigma, St. Louis, MO) density gradient Leu-AMC and the proteasome caspase substrate ZLLE-Amc were
separation after a 30-minute spin at 1500rpm at room temperature. obtained from Calbiochem (San Diego, CA) to be resuspended in
Cells were washed three times in Hank’s Balanced Salt Solution DMSO for respective stock solutions of 50mM and 50mM. The TOP
with 10mM HEPES and 1% PSG. After each wash, cells were spun at substrate Mcc-PLGPK-Dnp was resuspended in DMSO for a stock so-
1500rpm for 10 minutes at room temperature. lution of 10mM. All inhibitors and substrates were stored at –20°C.
Isolation of PBMC subsets Antigen Processing Peptidase Activity Assays
CD4+ T-lymphocytes and monocytes subsets were isolated from For proteasome peptidase activities, 3µg of whole cell extract
PBMC and magnetically immunosorted using the EasySep® Human was incubated in a buffer containing 20mM HEPES, 50mM KAc,
CD4+ T-cell Enrichment Kit and EasySep® Monocyte Enrichment Kit 5mM MgCl2, 1mM DTT, and 1mM ATP at room temperature for
according to the manufacturer’s instructions (StemCell, Vancouver, 30 minutes. To ensure the specificity of the assay, 1μM MG132 was
Canada). Enriched cells were washed three times in Dulbecco’s Phos- incubated with the extract. After the incubation, substrates for the
phate Buffered Saline (Sigma) at 1500rpm for 10 minutes at room different proteasome active sites were added and fluorescence was
temperature. Dry cell pellets were kept at –80°C until cytosol extrac- immediately measured every 5 minutes over 1 hour at 37°C with a
tion. Victor-3 plate reader (Perkin Elmer, Boston, MA). 75μM ZLLE-Amc
Cytosolic extracts was used to measure caspase-like activity, 100μM Suc-LLVY-Amc
Cell pellets were resuspended in a Digitonin buffer (50mM HEP- was used for chymotrypsin-like activity, and 25μM Boc-LRR-Amc
ES, 50mM KAc, 5mM MgCl2, 1mM DTT, 10% glycerol, 1mM ATP, was used for trypsin-like active site activity. Aminopeptidase, TPPII,
0.5mM EDTA, 0.0125% Digitonin) for 5 minutes to allow sufficient and TOP fluorescence assays were conducted in similar conditions,
detergent permeabilization and then spun at 20,000rcf for 15 min- excluding DTT and ATP in the buffer. Specificity was determined
utes. Protein concentration in both methods was measured with by incubation with 120μM Bestatin, 10μM Cpp-AAF-pAb, or 1μM
the DC Protein Assay Kit from Bio Rad Laboratories (Hercules, CA). Butabindide for aminopeptidase, TOP, and TPPII activities respec-
Whole cell extracts were kept at –80˚C until use. tively. Following the 30 minute incubation, respective substrates
Actin normalization were added: 50μM Leu-Amc, 20μM Mcc-PLGPK-Dnp, and 100μM
To standardize the amount of extract used in experiments, actin H-Ala-Ala-Phe-Amc for aminopeptidase, TOP, and TPPII. Fluores-
in each sample was compared to a sample known to contain 3µg cy- cence was read as stated above. Excitation and emission wavelengths
tosol. The normalized amount of each sample was the volume of cell for trypsin-like, aminopeptidase, and TOP fluorescent products were
extract with an actin content that matched 3µg of actin in the known 345nm and 405nm, respectively. Those wavelengths for caspase-like,
sample after Western Blot analysis. chymotrypsin-like, and TPPII fluorescent products were 380nm and
Western Blot Analysis 460nm, respectively.
All samples were subjected to Western Blot analysis. First, whole Synthetic peptides and RP-HPLC peptide analysis
cell extracts were mixed with laemmli buffer and heated at 85°C for Peptides were made by the AAPPPTEC Apex 396 multiple peptide
10 minutes. Lysates were run through a 12% SDS-PAGE gel at 100V synthesizer at the MGH peptide core (Massachusetts General Hospi-
through stacking gel and 125V through running gel. The gel was tal, Boston MA). Peptides were then purified by reverse-phase high
transferred to PVDF transfer membrane (GE healthcare) at 25mA pressure liquid chromatography (RP-HPLC) and their sequences
overnight. The membrane was blocked at room temperature for at were verified by mass spectrometry, which showed greater than 95%
least 30 minutes in 5% milk and 1% NP40. Primary anti-beta actin purity (Partners Proteomics, Cambridge, MA). Eluted peptides pro-
antibody (Abcam, Cambridge, MA) was diluted to 1/20,000 in 5% duced defined peaks on the RP-HPLC where the area under the peak
milk and 0.1% NP40, added to the membrane, and incubated for 1 was proportional to the amount of peptide in the analyzed sample.
hour at room temperature. Following the primary, the membrane Defined peptide peaks were calibrated using a 4.6x50mm 3mm C18
was washed 5 times with 0.1% NP40 every 10 minutes. Secondary column (Waters, Milford, MA).
20 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Intracellular peptide degradation assays Results


PBMC, CD4+ T, and monocyte whole cell extracts from the same
healthy donor were resuspended in buffer containing 20mM HEPES, Fluorogenic assays of antigen processing associated peptidases

Immunology
137mM KAc, 1mM MgCl2, and 1mM ATP in nano-pure H2O. Subse- As protein degradation of HIV-1 proteins is crucial to the even-
quently, peptide was added to the solution, at which point the incuba- tual presentation of viral peptides to CTLs, I sought to characterize
tion at 37°C began. At 0, 10, and 30 minutes, aliquots of the reaction the activity of peptidases associated with antigen processing—pro-
containing 30μg whole cell extracts and 6nmol peptide were stopped teasome-caspase-like, -chymotrypsin-like, and -trypsin-like; amino-
with 0.3% Trifluoroacetic acid (TFA) (Sigma Aldrich, St. Louis, MO). peptidase; TOP; and TPPII—in CD4+ T-cells and monocytes. To do
6nmol of pure peptide in same buffer and TFA conditions was used this, I utilized an in vitro fluorescence-based hydrolytic assay already
to identify the elution time and amount of the undigested peptide developed by others in the group. Here, I incubated cytosolic extracts
through RP-HPLC. All peptides were eluted using a gradient solution from peripheral blood mononuclear cells (PBMC), CD4+ T-cells, and
of two buffers. The first was 0.05% TFA in nanopure H2O, while the monocytes with substrates that fluoresce upon cleavage by specific
second buffer varied depending on the peptide. For peptides 5RK3, peptidases (Figure 2). The measured fluorescence of the sample was
A3-RK9, B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-K11, the second directly proportional to the activity of the peptidase over time. Af-
buffer either contained 50% acetonitrile (AcN) and 0.01% TFA or ter a 30-minute pre-incubation with peptidase-specific inhibitors,
100% methanol and 0.05% TFA. The second elution buffer for B7-FL9, I found that the average specificity of substrates to their respective
B7-TL10, and B7-FR10 contained either 100% AcN and 0.03% TFA peptidases in PBMC, CD4+ T, and monocyte subsets was between
or 100% methanol and 0.05% TFA. Integrated peaks from digested 80% and 95% (Sup. Table 1). I must note that cleavage of Leu-Amc in
peptides were compared to the undigested peptide. Identities of some monocytes was about 50% specific to aminopeptidases, which indi-
digested peaks were verified by mass spectrometry. cated that another peptidase along with aminopeptidase cleaved Leu-
Antigenicity of produced peptides Amc. However, fluorescence from cleavage in monocytes was much
Peptide products from the 17-mer (gag) intracellular degradation greater than in other cell extracts that we considered the discrepan-
assay were purified with 10% trichloroacetic acid precipitation and cies to be negligible (data not shown).
diluted in RPMI-1640 without serum. Subsequently, pH was adjusted CD4+ T-cells and monocytes have different peptidase activity
to 7.4. HLA-A3+ B cells derived from HeLa cells served as target cells levels
and were labeled with 51Cr and pulsed with 0.5ug/ml of the peptide After ensuring the specificity of the fluorescence assay, I sought
products for 30 minutes at 37°C without serum. CTL clones specific to characterize the activity of the proteasome, aminopeptidase, TOP,
for RK9 were incubated for 4 hours with the B cells at a 4:1 effector- and TPPII in two PBMC subsets targeted by HIV: CD4+ T-cells and
target ratio. Cell lysis was defined as [(51Cr release from B cells incu- monocytes. Prior research indicated that activities of the antigen
bated with digested products - spontaneous release) / (total release – processing machinery varied according to cell type (Butz and Bevan,
spontaneous release)]. Cell lysis values were also compared to that of 1998). Therefore, we hypothesized that the proteasome, aminopep-
HLA-A3+ B cells that were incubated with undigested 17-mer peptide tidase, TOP, and TPPII would show differences in activity between
at a 0.5µg/ml concentration. CD4+ T-cells and monocytes. Using blood from 8 healthy donors, I
Statistical analysis purified the PBMC and immunosorted CD4+ T-cells and monocytes.
Maximum slope in the fluorescence assays was calculated with With equal amounts of cell extracts normalized through western
Workout 2.0 using a liner kinetic fit of the fluorescence at different blots against actin and GAPDH, I compared peptidase activities of
time intervals (Perkin Elmer, Boston, MA). The differences in maxi- whole PBMC, CD4+ T-cells, and monocytes by using the fluorescence
mum slopes and fluorescence levels between CD4+ T-lymphocytes emitted by the sample at 1 hour after the 37°C incubation. All assays
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 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
Figure 2. Intracellular fluorescence-based hydrolytic activity activities were measured in whole cell extracts from PBMC (•), immuno-
assay. Cell extracts were incubated with specific fluorogenic substrates at sorted CD4+ T-cells (■) and monocytes (▶) after a 1 hour incubation with
37°C for 1 hour. Cleavage of those substrates produced fluorescence. Pep- peptidase-specific substrates at 37°C. Fluorescence was compared using
tidase activity is proportional to fluorescence. Sample proteasome-trypsin a non-parametric Mann-Whitney statistical test. Significant differences
assay shows fluorescence level over time. Slope and 1-hour fluorescence between CD4+ T-cells and monocytes were found. *p<0.05, **p<0.01,
are indicated on graph. ***p<0.001. n=8.
www.thurj.org 21
Immunology
RESEARCH 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
Figure 4. Faster peptidase kinetics in monocytes. The degradation peaks where the integral under the peak is proportional to the amount of
of fluorogenic substrates specific for either the proteasome: caspase-like or that peptide. B) HLA-A3+ B cells were pulsed with peptide degradation
trypsin-like active sites, aminopeptidase, TOP, or TPPII were measured in products and used as targets in the 51Cr release assay. CTLs specific for the
whole cell extracts of PBMC (•), CD4+ T-cells (■), and monocytes (▶) over HLA-A3+ -restricted epitope RK9 (orange) were used as effectors.
1 hour at 37°C. The maximum slopes of degradation in CD4+ T-cells and
monocytes were compared using non-parametric Mann-Whitney statisti- sentative sample shows the observed differences in slope over time
cal analysis. Monocytes showed significantly higher kinetics than CD4+ (Figure 2). I used the average of the maximum slope from 3 experi-
T-cells. *p<0.05, **p<0.01, ***p<0.001. n=8. ments as my comparison values. Peptidases in monocytes have sig-
nificantly faster degradation capacities than those in CD4+ T-cells
were repeated three times, and the arithmetic mean of all trials was (Figure 4). Again, the proteasome chymotrypsin-like subunit failed
used as the final value for comparison. A representative sample shows to show a statistically significant difference between the two subsets,
the typical fluorescence pattern with monocytes having a higher but other members of the group observed differences when using
fluorescence at the 1-hour time point than CD4+ T-cells (Figure 2). a larger sample size (data not shown). These differences in antigen
Overall, I found that proteasome-caspase-like, proteasome-trypsin- processing activities and kinetics imply that monocytes are capable
like, aminopeptidase, TOP, and TPPII had significantly higher ac- of processing intracellular peptides to a greater extent than CD4+ T-
tivity in monocytes than in CD4+ T-cells (Figure 3). In proteasome- cells, which may have downstream effects on the recognition of the
chymotrypsin-like activities, I did not find a significant difference, infected cell.
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 7. Peptide products from intracellular 5RK3 digestion.


Whole cell extracts of PBMC, CD4+ T-cell, and monocytes were used to
Figure 6. Intracellular degradation of 5RK3 to produce RK9. digest an HIV-1 Gag p17 fragment (RWEKIRLRPGGKKKYKL aa 15-31),
Whole cell extracts of PBMC (+), CD4+ T-cell (■), and monocytes (◊) were called 5RK3 (uppermost rectangle). It contains an HLA-A3 restricted
incubated with 5RK3, an HIV-1 Gag p-17 fragment, at 37°C. At 2, 10, 30, epitope, RK9 (black text); N-terminal flanking regions are to the left; C-
60, 120 minutes, samples were collected and eluted through RP-HPLC for terminal flanking regions are to the right. Degradation products at 2 and
analysis and quantification. A) Monocytes showed faster degradation of 60 minutes that were found through mass spectrometry are listed. Prod-
5RK3 than CD4+ T-cells. B) Monocytes produced the HLA-A3 restricted ucts with dotted outlines are unique to the indicated cell type. Shaded
optimal epitope, RK9, faster and at sustained levels for over 2 hours. Iden- products are previously described antigenic epitopes. Monocytes showed
tities of 5R3 and RK9 were confirmed by mass spectrometry. shorter and more antigenic degradation products than CD4+ T-cells.
22 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Intracellular degradation of longer peptides to produce optimal assay allowed me to attribute any observed differences in the prod-
HIV-1 epitopes ucts between CD4+ T-cells and monocytes to the antigen processing
After ascertaining significant differences in activity and kinetics machinery. We sent the products for mass spectrometry analysis to

Immunology
of antigen processing peptidases between CD4+ T-cells and mono- identify the peptide sequences. I found that peptides produced from
cytes, we wanted to know if those differences affect HIV-1 epitope 5RK3 in monocytes were shorter than those from CD4+ T-cells, pre-
processing. To do this, I utilized an in vitro intracellular degradation sumably allowing for a better fit on MHC-I (Figure 7). In addition,
system where I incubated whole cell extracts of PBMC, immunosort- monocyte degradation products contained already identified optimal
ed CD4+ T-cells, or monocytes with a 17-amino-acid sequence from HIV-1 epitopes: HLA-B27 restricted IK9 (IRLPPGGKK) and HLA-
HIV-1 Gag p17 (RWEKIRLRPGGKKKYKL, aa 15-31), called 5RK3 A3 restricted KK9 (KIRLPPGGK), RK9 (RLRPGGKKK), and RY10
in this experiment, for 0 to 120 minutes (Figure 5a). When intracellu- (RLRPGGKKKY) (Korber et al., 2008; Fig. 7). This alluded to differ-
lar peptidases degrade 5RK3, one can visualize the peptide products ences in the CTL recognition and antigenicity of peptide products
through RP-HPLC where one peak indicates a distinct peptide, and between the two subsets. My supervisor performed a 51Cr release as-
the integral below the peak is proportional to the amount of peptide. say on the peptide products from the 5RK3 intracellular degradation
At different time points during the incubation, one can examine the assay to determine their antigenicity. She purified the small peptides
quantity of 5RK3 and its degradation products. Members of the lab from the incubated extracts and pulsed identical HLA-matched B
previously showed this system to be a good approximation of the en- cells with them (Figure 5b). Those B cells served as the targets in the
dogenous processing of intracellular proteins (Le Gall et al., 2007). release assay, and CTLs specific for HLA-A3 RK9 were the effectors.
Faster degradation of 5RK3 fragment and sustained levels of We found that products from monocytes were more antigenic than
RK9, a peptide product, in monocytes those of CD4+ T-cells (Figure 8). In fact, we achieved a maximum
Using the in vitro intracellular degradation assay described in the specific lysis of 51% from monocyte degradation products whereas
earlier section, I measured the degradation of 5RK3 and the genera- those from CD4+ T-cells could not generate greater than 3% lysis.
tion of its optimal epitope RK9 (RLRPGGKKK), which requires a Other members of the lab observed similar results after replicating
multistep cleavage of the 5 N-terminal residues (RWEKI) and 3 C- the assay twice (data not shown).
terminal residues (YKL) of 5RK3. Intracellular stability of HIV-1 epitopes
I studied the production of RK9 since it is associated with delayed The previous experiments showed how differences in peptidase
progression to AIDS through HLA-A3 restricted CTL responses in activity between CD4+ T-cells and monocytes could affect the pro-
some HIV-infected patients (Altfeld et al., 2006). Furthermore, I duction of HIV-1 epitopes. However, antigen processing is a balance
could infer that the observed differences in peptidase activity and between both the production and degradation of epitopes since intra-
kinetics affected 5RK3 degradation since this process requires the cellular peptidases are constitutively active. Differences in the deg-
proteasome, aminopeptidases, and TPPII (Le Gall et al., 2007). I radation of optimal epitopes between CD4+ T-cells and monocytes
found that the degradation of 5RK3 was faster in monocytes than in could affect epitope presentation and CTL recognition of optimal
CD4+ T-cells, and monocytes degraded all the 5RK3 by the second epitopes. Little is known about the intracellular stability of short
minute of the incubation (Figure 6a). As for the production of RK9, peptides, though aminopeptidases have been implicated in regularly
monocytes not only produced it with faster kinetics, but they also cleaving short and intermediate peptides in the cytosol (Smyth and
maintained higher levels of RK9 over time compared to CD4+ T-cells O’Cuinn, 1994). Thus, I examined whether the degradation of HIV-
(Figure 6b). epitopes, which are short peptides, was the same in both CD4+ T-
Antigenic degradation products from monocytes cells and monocytes. Other members of my lab had characterized the
Besides the degradation of the original peptide, and the produc- half-lives of many of the known HIV epitopes in PBMC and found
tion of RK9, we wanted to understand the differences among peptide that although some epitopes were highly unstable, others resisted
products of the 2 PBMC subsets. No study had shown the potential degradation over time (data not shown). We speculated that HIV
differences in peptide products from the antigen processing machin- epitopes that were stable in PBMC could have varying levels of stabil-
ery. Such a difference could eventually affect CTL recognition of ity between CD4+ T-cells and monocytes since we already saw cell
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- Figure 9. Intracellular degradation of optimal HIV-1 epitopes. A)
A3+ B cells were pulsed with these products to be used as targets in 51Cr 8 optimal HIV-1 epitopes (B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-
release assay. CTLs specific for HLA-A3+ restricted epitope RK9 were used KK11, B7-FL9, B7-TL10, and B7-FR10) were incubated in whole cell extract
as effectors. For each extract from all time points during the degradation, of PBMC and immunosorted CD4+ T-cells and monocytes at 37°C for 30
percent specific lysis was measured. Monocyte degradation products (▶) minutes. B) At 2, 10, and 30 minutes, portions of the reaction were stopped
elicited greater CTL killing, and thus, were more antigenic than that of and eluted through RP-HPLC. Peptides produce unique peaks where the
CD4+ T-cells (■). area under the peaks represents the amount of peptide in the eluate.
www.thurj.org 23
RESEARCH 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
Immunology

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-
Figure 10. Shorter half-life of HIV-1 epitopes in monocytes. 8 dation assay 3 times and averaged all values.
optimal HIV-1 epitopes (B57-YY9, Cw3-RL9, A33-ER8, B7-HA9, A3-
Different intracellular degradation of HIV-1 optimal epitopes in
KK11, B7-FL9, B7-TL10, and B7-FR10) were subjected to intracellular
CD4+ T-cells and monocytes
degradation in whole cell extracts of PBMC (circle) and immunosorted
CD4+ T-cells (square) and monocytes (triangle). Peptide products at 2, 10
The calculated differences in half-life of HIV optimal epitopes
and 30 minutes were analyzed and quantified using RP-HPLC. One-phase were not the only interesting findings revealed in our comparison of
exponential decay was used to estimate the half-lives of the epitopes. Non- intracellular epitope degradation between CD4+ T-cells and mono-
parametric Mann-Whitney test was used for comparison of the half-lives. cytes. Apart from understanding the overall pattern of optimal
Optimal epitopes showed significantly shorter half-lives in monocytes epitope degradation in PBMC, we wanted to know if the kinetics of
than in CD4+ T-cells. **p<0.01. degradation was similar across the PBMC subsets that HIV-1 infects.
In the same assay as above, I qualitatively assessed the kinetics of deg-
type differences in epitope production. To measure the intracellular radation for each optimal epitope (B57-YY9, Cw3-RL9, A33-ER8, B7-
half-life of optimal HIV epitopes, I incubated them with whole cell HA9, A3-KK11, B7-FL9, B7-TL10, and B7-FR10) between the subsets.
extracts of PBMC, immunosorted CD4+ T-cells, or monocytes over I found that there was no consistent pattern of degradation among
30 minutes (Figure 9). At 2, 10, and 30 minutes, I eluted the extract- epitopes in both cell subsets, and these differences existed regardless
peptide mixture through RP-HPLC. Like the prior RP-HPLC assay, of the epitopes protein’s origin (Figure 11). This suggested functional
each peptide produced a unique peak where its integral reflects the variations in subset-specific degradation of HIV epitopes.
amount of peptide present in the eluate allowing me to quantify the
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.

24 The Harvard Undergraduate Research Journal


Volume 2 Issue 2 | Fall 2009 RESEARCH

on identifying the levels and kinetics of peptidases associated with of the epitope is dependent on a multi-step process where each step
the production and degradation of these peptide fragments and found can affect the identity of the final presented product. This process
that there were indeed differences in activity level and kinetics, which incorporates the kinetics of peptide production, degradation, trans-

Immunology
resulted in variations in the processing of HIV-1 epitopes. Observed port, and MHC-I binding, which must be completed in a timely man-
heterogeneity in the identity and production time course of peptide ner before the proliferating virions can escape. Here, I studied one
products might have important consequences for CTL recognition of of the earlier steps, intracellular epitope processing, which did not
infected PBMC subsets and the ensuing immune response. include epitope processing in the ER, peptide transport, or MHC-I
Using the intracellular fluorescence-based hydrolytic assay for binding kinetics. Products from intracellular processing need trans-
peptidase activity, I showed how proteasome-caspase-like and port into the ER by TAP, which displays binding preferences for cer-
trypsin-like, aminopeptidase, TOP, and TPPII activity levels and tain peptide sequences to transport into the ER (Procko and Gaudet,
speed in CD4+ T-cells and monocytes differ for both healthy and 2009). Thus, it is possible to infer that TAP might also influence the
HIV infected donors. We do not know whether these differences final product presented by MHC-I. Therefore, further experiments
stem from variations in transcription or translation. Moreover, we do to discern the differences in the entire antigen processing pathway
not understand what differences in intracellular peptidase quantity between CD4+ T-cells and monocytes must be completed.
lead to observable differences in activity. Therefore, I cannot know if Moreover, my research only focused on 2 subsets that HIV infects:
variations in activity come from increased expression of the proteins CD4+ T-cells and monocytes, and did not include the entire repertoire
or from increased activity of each individual peptidase in mono- of HIV-infectible PBMC subsets, such as macrophages, natural killer
cytes. Another potential reason for the increase in peptidase activity (NK) cells, and dendritic cells (DCs), which all contribute to infec-
in monocytes might be a contamination in the cytosol of cathepsins, tion (Weiss, 2008; Valentin et al., 2002). Macrophages are monocyte-
endosomal peptidases that function in the exogenous pathway of an- derived cells that can store dormant virus in the gastrointestinal tract
tigen presentation. I corrected for this by stabilizing the pH of the during chronic infection (Smith et al., 2003). Reduced levels of NK
whole cell extracts at 7.4, which considerably inhibits the function of cells in infected patients are associated with worse prognoses (Ullum
cathepsins (Turk et al., 1999). Despite the uncertainty in the source et al., 1995). DCs activate CTLs in cell-mediated immunity, making
of these functional differences between CD4+ T-cells and monocytes, them especially important in HIV pathogenesis (Zarling et al., 1999).
the effects of these variations are of primary interest because we must Although most of the infected cells in HIV-individuals are CD4+ T-
fully understand the consequences for the immune response. cells, it is still important to understand the antigen processing capa-
One limitation of this study was that it did not measure differ- bilities of all PBMC subsets that contribute to this disease in order to
ences in non-cytosolic peptidases, such as ERAP-1 and ERAP-2, be- understand the entire scope of presented HIV-1 peptides.
tween the two PBMC subsets. Much of the literature about antigen Apart from exposing peptidase activity differences between the
processing of epitopes implicates ERAPs and not cytosolic peptidases subsets, my results introduce the concept of a balance between pro-
(York et al., 2002). York and his group showed that purified ERAP-1 duction and degradation of intracellular peptides within the infected
could trim all peptides that were longer than 10 residues and half of cell. The observations that monocytes both produce and degrade
the 9-residue peptides. Though their experiment was not performed optimal HIV-1 epitopes faster than CD4+ T-cells can be seemingly
under the typical intracellular environment of the cell, it suggested contradictory if one quickly attributes faster degradation of epitopes
that ERAP-1 was important in determining the loaded peptides for as a negative factor in antigen processing. However, one must remain
MHC-I presentation. My data on intracellular epitope processing aware of the balance between production, degradation, and transport
does not conflict with the existing research, but it adds to the litera- of epitopes in the cytosol compartment. If monocytes have a more
ture by showing that cytosolic peptidases can produce optimal HIV-1 efficient transport system into the ER, then the increased degrada-
epitopes from longer peptides. However, it is still important to study tion of optimal epitopes in monocytes might be negligible. Similarly,
the potential differences in ERAP activity between the PBMC subsets slower degradation of HIV-1 epitopes in CD4+ T-cells might allot
as those peptidases can make the final peptide trimmings in the ER more time for TAP binding and transport into the ER. With my re-
that may affect its loading on the MHC-I and CTL recognition. sults, I have reiterated the importance of kinetics in the entire antigen
Even with the exclusion of non-cytosolic peptidases, my study processing pathway and established the value of a balance between
revealed differences among intracellular peptidases. For example, intracellular production and degradation of viral peptides destined
aminopeptidase showed the greatest fold differences in average activ- to be presented to CTLs.
ity values between CD4+ T-cells and monocytes. Compared to the Additionally, I have shown that these observations of differential
proteasome, TOP, and TPPII, whose fold differences in the average epitope processing might also be applicable to HIV-infected individ-
activities between the subsets fell between 1.5 and 2.1, aminopepti- uals. Though I did not perform the intracellular degradation of an
dase activity in monocytes was about 3.5 times greater than that of HIV epitope on whole cell extracts from HIV-infected donors, the
CD4+ T-cells. This suggests that each peptidase can have unique dif- significant difference in peptidase activities between CD4+ T-cells and
ferences in activity in different subsets. Also supporting this theory monocytes of HIV-infected samples suggests similarities in epitope
is the inconclusive result on the subset-specific differences in the pro- production (Sup. Fig. C and D). However, the estimated frequency of
teasome-chymotrypsin active site. Although average activity levels of infected CD4+ T-cells and monocytes waivers between 0.01% and 1%
this active site were greater in monocytes, there was no statistically in an HIV-positive individual; therefore, the differences in peptidase
significant difference in values between the two cell types (data not activity between the subsets that I detected might stem from the un-
shown). Other active sites within the proteasome showed significant infected cells (Poznansky et al., 1991; Smith et al., 2003). Although
subset-specific differences with 8 samples in the cohort, indicating HIV epitope processing in infected cells remains unclear, a larger
non-uniform subset-specific variations in the proteasome active sites study that compares intracellular peptidase activity and epitope pro-
and perhaps, in most intracellular peptidases. cessing in HIV-infected PBMC will elucidate unknown patterns.
In addition to studying the differences in intracellular peptidase Furthermore, since HIV-1 has two main tropisms for PBMC cell
activity between the CD4+ T-cells and monocytes, it is important to subsets—T-cells and monocyte-derived cells—one can speculate that
study the entire antigen processing pathway because the presentation the untimely recognition of infected CD4+ T-cells may skew the viral
www.thurj.org 25
RESEARCH Volume 2 Issue 2 | Fall 2009

population in an infected patient to the T-cell tropic strains. Recently, consequences of subset-specific differences in antigen processing and
the prevalence of HIV-1 viral strains that preferentially infect CD4+ presentation are broad reaching. The identity of the peptide present-
T-cells was found to be significantly greater in PBMC (Verhofstede et ed might affect memory T-cell activation and CTL recognition of the
Immunology

al., 2009). With increased viral levels of T-cell-tropic strains, greater infected cell.
numbers of CD4+ T-cells might be infected, thus contributing to the Even without observations on other components of the antigen
drastic reduction of CD4+ T-cells during HIV infection. Already, the processing pathway or on other cells infected by HIV, my results dem-
period following infection before the CTL response has been identi- onstrate that differences in intracellular processing might affect the
fied as crucial for the virus to establish reservoirs in T-cells (Daven- production, degradation, identity, and antigenicity of HIV-1 epitopes
port et al., 2004). This study complements my findings to suggest that in two PBMC subsets. Monocytes showed increased activity in anti-
kinetics of antigen processing and presentation and CTL recognition gen processing peptidases, which might lead to a different repertoire
are important for disease progression. Though it might be imprecise of presented HIV-1 epitopes than CD4+ T-cells. The rapid production
to generalize laboratory findings to the disease progression, my find- and larger amounts of more antigenic peptides in monocytes may
ings provide a different way of examining the dissimilar fates of CD4+ increase the probability of transport into the ER for those peptides. If
T-cells and monocytes during HIV infection. infected monocytes present more antigenic epitopes, then these cells
This research is applicable to other viral infections, especially ones might elicit a better immune response than infected CD4+ T-cells.
with tropism towards specific cell subsets. For the cell-mediated im- Accordingly, it is important to further research on subset-specific
mune response to be effective, the infected cell must present a viral differences in antigen processing not only to better understand the
peptide that DC-primed CTLs can recognize. Since uninfected DCs host’s immune response to infection but also to develop highly effec-
can undergo cross-presentation, where extracellular peptides enter tive clinical interventions, such as vaccines.
the endogenous pathway, it is important to ensure that the peptides
presented by the infected cells are the same ones that the DCs can References
process for CTL priming. For example, one study on the DC-tropic
1. Altfeld, M., Kalife, E., Qi, Y., Streeck, H., Lichterfeld, M., Johnston, M.
human cytomegalovirus (HCMV) strain found that whole PBMC in- et al. (2006). HLA Alleles Associated with Delayed Progression to AIDS
fected with HCMV could elicit more responses than infected DCs Contribute Strongly to the Initial CD8+ T-cell Response against HIV-1.
(Gerna et al., 2005). Here, differences in viral peptide presented might PLoS Medicine, 1851-1864.
be the cause for differences in the elicited immune response. Further 2. Betts, M., Ambrozak, D., Douek, D., Bonhoeffer, S., Brenchley, J., Casazza,
research on cell subsets infected by viruses with particular tropisms J. et al. (2001). Analysis of Total Human Immunodeficiency Virus (HIV)-
Specific CD4 + and CD8+ T-Cell Responses: Relationship to Viral Load in
must be completed to understand the extent of cell-type-specific dif- Untreated HIV Infection. Journal of Virology, 11983-11991.
ferences in antigen processing. 3. Borrow, P., Lewicki, H., Hahn, B., Shaw, G., and Oldstone, M. (1994). Vi-
My results also pertain to the current literature on immunodomi- rus-specific CD8+ cytotoxic T-lymphocyte activity associated with control
nance of MHC-I restricted epitopes. For this purpose, I will define of viremia in primary human immunodeficiency virus type 1 infection.
MHC-I immunodominance as the phenomenon where CTLs against Journal of Virology, 6103-10.
4. Butz, E., and Bevan, M. (1998). Differential Presentation of the Same
a certain viral epitope are more prevalent in an infected individu-
MHC Class I Epitopes by Fibroblasts and Dendritic Cells. The Journal of
al. Mathematical models based on patient observations show that a Immunology, 2139-2144.
dominant CTL response usually arises when a pathogen is homoge- 5. Callaway, D., Ribeiro, R., and Nowak, M. (1999). Virus phenotype switch-
neous; however, this does not explain the molecular reasons for over- ing and disease progression in HIV-1 infection. Proceedings of the Royal
representation of that particular viral peptide (Nowak et al., 1995). A Society of London, 266, 2523-2530.
6. Chun, T. W., and Fauci, A. S. (1999). Latent reservoirs of HIV: obstacles
recent study on intracellular processing found that flanking sequenc-
to the eradication of virus. Proceedings of the National Academy of Sci-
es near the epitope contributed to the processing of those epitopes ences, 10958-61.
and strongly determined the intracellular peptide products (Le Gall 7. Chun, T. W., Engel, D., Berrey, M. M., Shea, T., Corey, L., and Fauci, A.
et al., 2007). If cell subsets exhibit different kinetics in the production S. (1998). Early establishment of a pool of latently infected, resting CD4 +
and identity of presented HIV-1 epitopes, then the immunodominant T-cells during primary HIV-1 infection. The Proceedings of the National
epitope might not be presented by one of the subsets. This would cre- Academy of Sciences, 8869-73.
8. Crowe, S., Turner, S., Miller, S., Roberts, A., Rappolo, A., Doherty, P. et al.
ate dire consequences for the PBMC subset that does not present this (2003). Differential antigen presentation regulates the changing patterns
dominant epitope, and clearance of infected cells in that subset might of CD8+ T-cell immunodominance in primary and secondary influenza
be compromised. virus infections. The Journal of Experimental Medicine, 399-410.
Besides understanding the immune response to infect CD4+ T- 9. Davenport, M., Riberio, R., and Perelson, A. (2004). Kinetics of virus-
cells or monocytes, my findings also add to the existing literature on specific CD8+ T-cells and the control of human immunodeficiency virus
infection. Journal of Virology, 10096-103.
HIV vaccines. Unfortunately, the search for an HIV vaccine has not
10. Douek, D. (2007). HIV disease progression: immune activation, microbes,
been very successful due to the complexity of HIV and its interac- and a leaky gut. Topics in HIV Medicine, 114-7.
tions with the immune system (Johnston and Fauci, 2007). Vaccines 11. Ellery, P., Tippett, E., Chiu, Y.-L., Paukovics, G., Cameron, P., Solomon,
function by delivering antigen to a host; the antigen activates the im- A. et al. (2007). The CD16+ monocyte subset is more permissive to infec-
mune system and induces the creation of antibody responses and an- tion and preferentially harbors HIV-1 in vivo. The Journal of Immunol-
ogy, 6581-6589.
tigen-specific memory T-cells, which persist in the host. Upon infec-
12. Frahm, N., Yusim, K., Suscovich, T. J., Adams, S., Sidney, J., Hraber, P.
tion with that same pathogen, infected cells present foreign peptides et al. (2007). Extensive HLA class I allele promiscuity among viral CTL
that optimally trigger proliferation of those memory immune cells to epitopes. The European Journal of Immunology, 2419-33.
perform their effector functions to curb the disease. An efficacious 13. Gerna, G., Percivalle, E., Lilleri, D., Lozza, L., Chiara, F., Hahn, G. et al.
vaccine necessitates memory T-cells recognizing the viral peptides (2005). Dendritic-cell infection by human cytomegalovirus is restricted to
presented by infected cells. Since HIV infects various cell types that strains carrying functional UL131-128 genes and mediates efficient viral
antigen presentation to CD8+ T-cells. Journal of General Virology, 275-
potentially have differences in their antigen processing machinery, 284.
a great candidate for a vaccine should contain a viral peptide that 14. Groothuis, T., Griekspoor, A., Neijssen, J., Herberts, C., and Neefijes, J.
all cell types can process efficiently and present to CTLs. Thus, the (2005). MHC class I alleles and their exploration of the antigen-process-

26 The Harvard Undergraduate Research Journal


Volume 2 Issue 2 | Fall 2009 RESEARCH

ing machinery. Immunological Reviews, 60-76. Janek, K. et al. (2003). An essential role for tripeptidyl peptidase in the
15. Hattori, A., and Tsujimoto, M. (2004). Processing of antigenic peptides by generation of an MHC class I epitope. Nature Immunology, 375-379.
aminopeptidase . Biological and Pharmaceutical Bulletin, 777-80. 38. Sheppard, H., Lang, W., Ascher, M., Vittinghoff, E., and Winkelstein, W.
16. Hauber, J., Perkins, A., Heimer, E. P., and Cullen, B. R. (1987). Trans-acti- (1993). The characterization of non-progressors: long-term HIV-1 infec-

Immunology
vation of human immunodeficiency virus gene expression is mediated by tion with stable CD4 + T-cell levels. AIDS, 1159-1166.
nuclear events. Proceedings of National Academy of Sciences, 6364-8. 39. Smith, P., Meng, G., Salazar-Gonzalez, J., and Shaw, G. (2003). Mac-
17. Hel, Z., McGhee, J., and Mestecky, J. (2006). HIV infection: first battle rophage HIV-1 infection and the gastrointestinal tract reservoir. Journal
decides the war. Trends in Immunology, 274-281. of Leukocyte Biology, 642-649.
18. Johnston, M., and Fauci, A. (2007). An HIV Vaccine – Evolving Concepts. 40. Smyth, M., and O’Cuinn, G. (1994). Alanine aminopeptidase of guinea-
The New England Journal of Medicine, 2073-81. pig brain: a broad specificity cytoplasmic enzyme capable of hydrolysing
19. Kopp, F., Hendil, K., Dahlmann, B., Kristensen, P., Sobek, A., and short and intermediate length peptides. International Journal of Bio-
Uerkvitz, W. (1997). Subunit arrangement in the human 20S proteasome. chemistry, 1287-97.
Proceedings of the National Academy of Sciences, 2939-2944. 41. Stevenson, M., Stanwick, T. L., Dempsey, M. P., and Lamonica, C. A.
20. Korber, B., Brander, C., Haynes, B., Koup, R., Moore, J., Walker, B. et al. (1990). HIV-1 replication is controlled at the level of T-cell activation and
(2008). HIV Molecular Immunology 2006/2007. Los Alamos, New Mexi- proviral integration. The EMBO Journal, 1551-60.
co: Los Alamos National Laboratory, Theoretical Biology and Biophysics. 42. Towne, C., York, I., Neijssen, J., Karow, M., Murphy, A., Valenzuela, D. et
21. Larsen, M. V., Lundegaard, C., Lamberth, K., Buus, S., Brunak, S., Lund, al. (2005). Leucine aminopeptidase is not essential for trimming peptides
O. et al. (2005). An integrative approach to CTL eptitope prediction: a in the cytosol or generating epitopes for MHC class I antigen presentation.
combined algorithm integrating MHC class I binding, TAP transport ef- Journal of Immunology, 6605-14.
ficiency, and proteasomal cleavage predictions. The European Journal of 43. Turk, B., Dolenc, I., Lenarcic, B., Krizaj, I., Turk, V., Bieth, J. et al. (1999).
Immunology, 2295-303. Acidic pH as a physiological regulator of human cathepsin L activity. Eu-
22. Lazaro, E., Godfrey, S. B., Stamegna, P., Ogbechie, T., Kerigan, C., Zhang, ropean Journal of Biochemistry, 926-32.
M. et al. (2009). Differential HIV Epitope Processing in Monocytes and 44. Ullum, H., Gotzsche, P., Victor, J., Dickmeiss, E., Skinhoj, P., and Peder-
CD4 T Cells Affects Cytotoxic T Lymphocyte Recognition. Journal of In- son, B. K. (1995). Defective Natural Immunity: An Early Manifestation
fectious Diseases, 236-43. of Human Immunodeficiency Virus Infection. Journal of Experimental
23. Le Gall, S., Stamegna, P., and Walker, B. (2007). Portable flanking se- Medicine, 789-799.
quences modulate CTL eptiope processing. Journal of Clinical Investiga- 45. Valentin, A., Rosati, M., Patenaude, D., Hatzakis, A., Kostrikis, L., Laza-
tion, 3563-3575. nas, M. et al. (2002). Persistent HIV-1 infection of natural killer cells in
24. McLaren, P., Mayne, M., Rosser, S., Moffatt, T., Becker, K., Plummer, F. et patients receiving highly active antiretroviral therapy. Proceedings of the
al. (2004). Antigen-Specific Gene Expression Profiles of Peripheral Blood National Academy of Sciences, 7015-7020.
Mononuclear Cells Do Not Reflect Those of T-Lymphocyte Subsets. Clini- 46. Vanhems, P., and Beaulieu, R. (1997). Primary infection by type 1 hu-
cal and Diagnostic Laboratory Immunology, 977-982. man immunodeficiency virus: diagnosis and prognosis. Postgrad Medical
25. Nowak, M., May, R., Phillips, R., Rowland-Jones, S., Lalloo, D., McAdam, Journal, 403-408.
S. et al. (1995). Antigenic oscillations and shifting immunodominance in 47. Verhofstede, C., Vanderkerckhove, L., Eygen, V., Demecheleer, E., Van-
HIV-1 infections. Nature, 606-11. denbroucke, I., Winters, B. et al. (2009). CXCR4-using HIV type I variants
26. Oliveira, V., Campos, M., Melo, R., Ferro, E., Camargo, A., Juliano, M. et are more commonly found in peripheral blood mononuclear cell DNA
al. (2001). Substrate specificity characterization of recombinant metallo than in plasma RNA. Journal of Acquired Immune Deficiency Syndrome,
oligo-peptidases thimet oligopeptidase and neurolysin. Biochemistry, 126-36.
4417-4425. 48. Weiss, R. A. (2008). Special Anniversary Review: Twenty-five years of hu-
27. Pajot, A., Schnuriger, A., Moris, A., Rodallec, A., Ojcius, D., Autran, B. et man immunodeficiency virus research: success and challenges. Clinical
al. (2007). The Th1 immune response against HIV-1 Gag p24-derived pep- and Experimental Immunology, 201-210.
tides in mice expressing HLA-A02.01 and HLA-DR1. European Journal of 49. York, I., Chang, S.-C., Saric, T., Keys, J., Favreau, J., Goldberg, A. et al.
Immunology, 2635-2644. (2002). The ER aminopeptidase ERAP I enhances or limits antigen presen-
28. Paroli, M., Propato, A., Accapezzato, D., Francavilla, V., Schiaffella, E., tation by trimming epitopes to 8-9 residues. Nature Immunology, 1177-
and Barnaba, V. (2001). The immunology of HIV-infected long-term non- 1184.
progressors---a current view. Immunology Letters, 127-129. 50. Zarling, A., Johnson, J., Hoffman, R., and Lee, D. (1999). Induction of pri-
29. Pedersen, C., Lindhart, B., Jensen, B., Lauritzen, E., Gerstoft, J., Dickmeiss, mary human CD8+ T lymphocyte responses in vitro using dendritic cells.
E. et al. (1989). Clinical couse of primary HIV infection: consequences for Journal of Immunology, 5197-204.
subsequent course of infection. British Medical Journal, 154-7.
30. Pierson, T., McArthur, J., and Siliciano, R. (2000). Reservoirs for HIV-1:
Mechanisms for Viral Persistence in the Presence of Antiviral Immune
Acknowledgements
Responses and Antiretroviral Therapy. Annual Review of Immunology, I would like to thank Sylvie Le Gall for giving me the opportunity to conduct
665-708.
research and for being a great mentor to me. Her dedication to the scientific pro-
31. Poznansky, M., Walker, B., Haseltine, W., Sodroski, J., and Langhoff, E.
(1991). A rapid method for quantitating the frequency of peripheral blood cess is a quality that I will always cherish. Furthermore, my experiments would
cells containing HIV-1 DNA. Journal of Acquired Immune Deficiency have been impossible without the amazing supervision by Estibaliz (Esti) Lazaro.
Syndrome, 368-73. She helped me grow as both a researcher and as a young adult. Furthermore, I
32. Procko, E., and Gaudet, R. (2009). Antigen processing and presentation: truly appreciate working in a world-class laboratory whose director, Bruce Walk-
TAPping into ABC transporters. Current Opinion in Immunology, E-
er, gave me encouragement and support during my time there. This project was
publishing.
33. Randolph, G., Jakubzick, C., and Qu, C. (2008). Antigen presentation by supported in part by Howard Hughes Medical Institute (HHMI), the National
monocytes and monocyte-derived cells. Current Opinion in Immunology, Institutes of Health (NIH), and Bill and Melinda Gates Foundation.
52-60. I would also like to individually thank all members of the Le Gall lab, past and
34. Saric, T., Graef, C., and Goldberg, A. (2004). Pathway for Degradation present: Sasha Blue Godfrey, Jeremy Ho, Christopher Kerrigan, Mei Zhang, Ser-
of Peptides Generated by Proteasomes. Journal of Biological Chemistry,
gio Martinez, Paul Bourgine, Shao Chong Zhang, and Jonathan Chow for sharing
46723-46732.
35. Schmitz, J., Veazey, R., Kuroda, M., Levy, D., Seth, A., Mansfield, K. et al. their projects with me, assisting me with portions of my research, and staying
(2001). Simian immunodeficiency virus (SIV)-specific cytotoxic T-lym- positive in times of stress. Professor Losick’s HHMI program also supported me
phocytes in gastrointestinal tissues of chronically SIV-infected rhesus immensely in my research.
monkeys. Blood Journal, 3757-3761. To end, my family and friends have encouraged me and kept me sane through-
36. Schubert, U., Anton, L. C., Gibbs, J., Norbury, C. C., Yewdell, J. W., and out this project. I cannot imagine completing this process without them. They
Bennink, J. R. (2000). Rapid degradation of a large fraction of newly syn-
always believed and still believe in my commitment to understanding and com-
thesized proteins by proteasomes. Nature, 770-4.
37. Seifert, U., Maranon, C., Shmueli, A., Desoutter, J.-F., Wesoloski, L., bating this destructive virus.

www.thurj.org 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 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
As the cornerstone of democracy, elections play a critical role in 2000 election, George Bush received 47.9% of the vote, and thus the
the United States political system. Every four years, star candidates Republican contract paid off $47.90 at the end of the election. In a
rise and fall, scandals emerge and disappear, and long campaigns are winner-take-all (WTA) election, traders bid on contracts for which
fiercely fought before settling on a winner. Pundits devise dozens of candidate will win the election and receive $100 per share of the win-
stories that “explain” what determined the winner, and television and ning candidate held.
radio talk shows will debate these theories for months. But to the In a system with no transaction costs and risk neutrality, Wolfers
academic empiricist, understanding an election is problematic. To and Zitzwitz (2004) show that the market price of the contract should
understand the determinants of a specific election, the sample size is equal the median market participant’s expected election outcome
only one event—so how can we know whether John McCain would weighted by trading volume. Furthermore, Berg et al. (2001) show
have fared better with a different vice-presidential candidate? To un- that the market price is a remarkably accurate short-term predictor
derstand election determinants, political economists like Ray Fair of the real election outcome, consistently outperforming polls as a
have pooled the data of many elections and constructed models of last-day prediction tool. In a follow-up study, Berg et al. (2003) find
very broad trends; yet because of the small sample size and the com- that electronic markets have exceptional long-term predictive abili-
plexity of the variables impacting elections, these models yield only ties, and greatly outperform both polls and forecasting models.
a few answers about what shapes elections in general, and much less The intuition behind these results is clear: traders are forced to
about what shapes specific elections. “put their money where their mouth is,” and they are thus incentiv-
In this paper, we propose the use of a new tool for understand- ized to be accurate. Since projection models and poll results are gen-
ing elections: electronic prediction markets. These markets have been erally publically available, that information can be incorporated into
studied at length, with a broad theoretical and empirical consensus the market price. Thus the market prediction should be at least as
that they yield reasonably unbiased, efficient estimates of the prob- accurate as any type of publicly-known prediction mechanism, if not
ability that an event occurs. Instead of focusing on their efficiency, more so. Interestingly, this assumption does not depend on traders
as most papers about electronic prediction markets have, we will in- being a random sample of the population, as the market can incorpo-
stead assume that they are efficient. In this paper, we will begin to an- rate information without all voters participating. As Berg et al. (2001)
swer the following question: given that electronic prediction markets point out, market participants are far from a random sample; they
are efficient, what can they tell us about political institutions? We will tend to be wealthy, young, and highly-educated. Furthermore, the av-
provide a case study of the potential utility of this methodology in the erage trader in the market can be biased and trade based on his own
example of coattail effects. While this paper yields interesting results, bias; empirically, there is a small set of “marginal traders” who are
our focus is primarily methodological and aims to understand how the ultimate price-makers and hunt for arbitrage opportunities—and
the existence of a continuous metric of election probabilities can be their probabilistic assumptions are quite accurate.
useful for causal analysis. With this consensus of research demonstrating that prediction
Background on electronic prediction markets markets are accurate estimators, we intend to utilize the market re-
Electronic prediction markets are futures exchanges in which the sults in this paper as a continuous set of election results­—changing
value of an asset is tied to the outcome of a particular event. In a over the course of the election only as the expected outcome changes.
vote share market, traders bid in a continuous double auction for the Thus, we have a much more complete data set, with a continuous set
28 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

of data points that can be evaluated in order to measure the impor- source of coattail effects shapes the median voter’s preferences direct-
tance of contextual events. ly in lower-level elections. The median voter, typically a swing voter
Background on coattail effects who was planning to vote and does not make a straight party-line
In the paper, we apply electronic prediction markets to understand vote, may change her lower-level preferences based on a change in
the interplay between national and local elections. While resting par- her perception of a top-level candidate; her preference for a particu-
tially on local issues and candidate personalities, local elections are lar presidential candidate informs her decision of which lower-level
often a referendum on national political figures. Local elections are candidate to support.
heavily correlated with national results, and many elections—such To gain a better understanding of the impact of coattail effects on
as the 2008 election—have the same party sweep to victory in the the election, we will consider a congressional election that is simulta-
House, the Senate, and the presidency. neous with a presidential election. Define candidate C as a congres-
Some of this correlation is due to contextual shifts, such as macro- sional candidate in the same party as presidential candidate J. The
economic changes or wars abroad. Other sources of correlation may decision of whether to vote for candidate C can be modeled as:
be due to a demographic transition, leading to partisan realignment.

Economics
A third part of the correlation comes from the presence of coattail X iC = 1 if Udif_C (Equation 1)
effects, or the effect that a top-level candidate has on a local candi- X iC = 0 if –Ū < Udif_C < Ū
date. A popular presidential candidate can register new voters, drive X iC = –1 if Udif_C < –Ū
partisan turnout and party-line voting, and change voter preferences Where Udif_C = E[UiC]–E[UiC2]
on issues, leading to coattail effects that change the outcome of con-
gressional elections. X iC: vote cast by voter i for candidate C. Not voting counts as 0 votes,
Despite the intuitive nature of coattail effects, attempts at effective- and voting against counts as a negative vote for the candidate.
ly quantifying these effects have generally floundered. Since elections UiC: utility voter i receives in the state of the world where candidate
are correlated for a wide variety of reasons, measuring basic correla- C wins
tion between elections is insufficient to establish coattail effects. The UiC2: utility voter i receives in the state of the world where candidate
methodological obstacles to measuring coattail effects were pointed C’s opponent wins
out by Miller (1955), who noted the simultaneity of decisions taking Ū: absolute utility difference threshold that causes voter i to cast a
place in elections. Mondak (1990) asserted that “a growing consensus vote.
holds that the presidential vote does exert significant influence on
congressional elections,” though he added that these analyses were Additionally, we can further expand these variables as:
set back by the “long–recognized difficulties associated with measur-
ing political coattails.” E(UiC) = E(F(ii, v iC)) (Equation 2)
Two primary obstacles exist in measuring coattail effects. First, ii = i(c, ti)
there is a highly limited sample size: there is only one presidential E(v iC) = v(c, riC, pC, siC)
election every four years from which to draw data, and there are
substantial contextual changes that occur over that time frame. To UiC: utility voter i receives in the state of the world where candidate
overcome the limited datasets, we use electronic prediction markets C wins
to assess the status of elections on a continual basis. A second ob- v iC: vector of expected agreement between voter i and candidate C’s
stacle is one of endogeneity: much of the correlation between candi- decisions
dates is due to agreement on issues and shifts in economic context, ii: vector of weights of importance that a voter gives to particular is-
rather than coattail effects? In this paper, we measure coattail effects sues
within the 2008 election by using exogenous changes in the presi- c: context of election
dential election (that do not directly affect congressional elections) as ti: voter i’s tastes
an instrument. We find substantial coattail effects in the House and pC: personal characteristics of candidate C
insignificant effects in the Senate. riC: historical correlation between voter i and candidate C’s known
The paper is organized as follows: first, we describe a theoretical beliefs
model for the existence of coattail effects; then, we propose an em- siC: signals of future agreement between voter i and candidate C
pirical methodology based on the electronic prediction market data-
set; next, we analyze the results of this methodology; and finally, we Under this model, a voter’s tastes (ti) and the election’s context (c)
provide concluding remarks and suggest areas for future research. are the same in both the presidential and congressional elections, and
are not affected by either candidate. Similarly, the personal charac-
Modeling Coattail Effects teristics of a candidate (pC) and the agreement between a voter and a
candidate’s revealed beliefs (riC) are specific to a particular candidate,
Despite the difficulties in measurement, there are intuitive rea- and we assume that they are not affected across elections. Instead,
sons why coattail effects are likely to exist. First, voter registration we model coattails as an informational phenomenon, with signaled
and turnout are often driven by grassroots campaigns and excite- beliefs (siC) as the source of coattail effects between the elections.
ment for a top-level candidate. If a presidential candidate is able to Intuitively, the model suggests that coattail effects occur as voters
register millions of first-time voters, those voters are likely to vote in use presidential candidates as partial proxies for local, less-known
lower-level elections for the same party. Similarly, excitement about candidates. Beyond any information known about the candidate per-
a top-level candidate can help drive party-line voting in some states, sonally, the candidate’s party affiliation is also visible to a voter­—a
­

as voters that are ambivalent about lower-level elections choose out strong signal of the candidate’s future decisions if elected. Mondak
of simplicity and expediency to vote for a party generally rather than (1990) finds that voters without much knowledge of a political race
evaluating individual candidates. While these effects can help shift may use their views of the presidential candidate in the same party as
votes towards a popular presidential candidate’s party, a more subtle a factor in their votes. Given the time cost of information collection
www.thurj.org 29
RESEARCH Volume 2 Issue 2 | Fall 2009

and the voter’s scarce budget of time, a typical voter spends only part tails.
of her time considering the election, and uses the presidential candi- To give a concrete example of this problem, in the 2008 election,
date as a proxy for the views of the local candidate. We formalize this negative events in the economy tended to shift voters’ views in ways
view of election signaling as a simplified model: that were favorable to Democrats in general. There was substantial
correlation between local elections and the presidential election, but
siC = s(v ij, w ic) (Equation 3) much of this relationship was due to the favorable context for Demo-
siC: signals of future agreement between voter i and candidate C cratic policies that were shared among candidates. The correlation
v ij: vector of expected agreement between voter i and presidential can- in such a case could not be attributed to coattails, since it was eco-
didate j’s decisions nomic changes—rather than changes in the presidential election—
w iC: relative strength of presidential views as a proxy for congressio- that shaped the association between the two elections.
nal candidate’s views
A New Empirical Methodology
The extent that the voter expects to agree with the presidential
Economics

candidate’s political decisions (v ij) affects the voter’s expectation of Despite the intuitive theoretical justifications for coattail effects,
how much she expects to agree with the lower-level candidate (v ic). attempts to measure the effects empirically have been hindered by
The importance of the presidential candidate as a proxy is given a both causality questions and a limited dataset. To overcome these
weight (w ic) that shows the extent to which the voter relies on the obstacles, we propose an instrumental variable approach that uses
presidential candidate as a proxy (which in turn is affected by the data from electronic prediction markets. There are three steps to our
perceived correlation between the presidential candidate and the lo- methodology: first, we will broaden the dataset by turning to elec-
cal candidate as well as the relative amount known about the views tronic prediction markets; second, we will select specific events where
of each). Assuming a candidate’s views are considered to be positively the presidential elections were exogenously affected without any di-
correlated with the presidential candidate of the same party, then rect effect on congressional elections; and finally, we will analyze the
∂sic /∂v ij > 0, implying that ∂Uic/ ∂v ij > 0. Thus, the probability that impact of these events on the congressional elections.
the voter votes for candidate c increases with the voter’s expected de- Step 1: Broadening the dataset
gree of agreement with the presidential candidate (v ij). Furthermore, In order to broaden the dataset, we use electronic prediction
the magnitude of impact of coattails on voter choice increases with market data for the 2008 election from the Iowa Electronic Markets.
weight, so ∂2Uic/ ∂v ij∂w ic > 0. We track daily closing prices for Democrats in the winner-take-all
This model outlines a mechanism for why, ceteris paribus, a candi- (WTA) market for the presidential election and the seat gain WTA
date’s popularity might be affected by a change in political popular- market for the House and Senate elections from August 26, 2008
ity of the presidential candidate from the same party. Since a voter (the date the congressional markets opened) until November 4, 2008
often knows more about a presidential candidate than about a local (Election Day). In these markets, House and Senate prices are ag-
candidate, she uses the presidential candidate as a proxy for the local gregated total probabilities of the Democrats winning seats in each
one – resulting in coattail effects. respective chamber rather than looking specifically at particular dis-
The problem with traditional measurement approaches tricts. Figures 1 and 2 show the association between weekly changes
For an observer trying to estimate the impact of coattail effects, in presidential markets and changes in the House and Senate races,
many of the variables from Equations 1 and 3 are not directly observ- respectively. As expected, the figures demonstrate a clear correlation,
able; instead, what is observable is the median voter’s decision on the from which we intend to isolate the coattail effects.
presidential election, X ij and their decision in the lower-level election Due to light trading and large bid-ask spreads, we use weekly
X ic. Previous attempts at measurement, such as the C-Correlation ap- price changes for the congressional prices instead of daily changes.
proach, have effectively regressed lower-level election results directly However, if we were to compare weekly prices for each, we would un-
on presidential election results, or X ic on X ij. Equation 3, however, intentionally include correlation that does not result from coattails
demonstrates why this is problematic. The unobserved effect of con- by expanding our time horizon too greatly (for example, economic
text is a factor in both X ic and X ij, causing correlation without coat- changes over that time period may affect both if the time horizon is

Figure 1. House and presidential election correlation. Figure 1 Figure 2. Senate and presidential election correlation. Figure 2
shows weekly changes in prediction market prices for Democrats winning shows weekly changes in prediction market prices for Democrats winning
the House (on the y-axis) against weekly changes in price for Obama win- 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 ning the election (on the x-axis). There is a general positive correlation
between these two probabilities. between these two probabilities.
30 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

too long). Thus, throughout this paper, we are generally comparing stantial effect of major presidential events on the congressional mar-
weekly congressional shifts with daily presidential shifts, assuming kets that increases average gains by a factor of eight in the Senate and
that any congressional price change that occurred in the preceding a factor of four in the House.
six days is in a random direction. We also remove from the dataset To quantify this effect, we run a two-stage least squares regression
all periods in which there was no trading volume. Finally, we restrict using the presence of a major event as an instrument. As our first
the dataset to periods on which presidential candidate Barack Obama step, we regress changes in the probability of Obama winning on the
gained vote share as a proxy for an election event being positive. Sum- presence of a major event on days where Obama gained popularity in
mary statistics are shown in Table 1. 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 Volume 2 Issue 2 | Fall 2009

Change_DemPres_WTA: daily price change for prediction market ingful as a vote in a swing state. The presidential swing states may
outcome of Obama winning presidency vary from congressional swing states. Thus, a presidential announce-
ε: error term ment that “rallies the base” may have an amplified effect in states with
close congressional elections but that are heavily leaning towards a
Through the two-stage least squares outlined in Equation 4 and particular party already in the presidential election.
Equation 5, we observe the effect of an exogenous shift in presidential An alternative hypothesis for amplification is that success is self-
preferences on Congressional prediction markets, yielding an unbi- fulfilling, as the perception of being likely to win an election can in-
ased estimate of coattail effects as β1. crease the actual likelihood of winning. Campaign contributors, key
politician endorsers, and even news editorial boards are often eager
Results and Discussion to support the winning side of an election and reap potential benefits
of early support. A company’s executives may choose to donate to
General results and discussion a campaign they expect will win in order to gain potential political
Our results from the two-stage regression in Equations 4 and 5 goodwill, and they must base their decisions on probability estimates
Economics

are reflected in Table 2. The results display strong, significant coattail made weeks or months before an election. Since these acts of sup-
effects in the House election but weaker, insignificant results in the port may directly shift the outcome of an election (and likely have
Senate. A 1% increase in Obama’s likelihood of winning is associated larger effects in elections where candidates are lesser known), success
with a 2.1% increase in the Democrats winning the House, but only may have inertia that amplifies a small probability change into strong
a 0.76% change in probability in the Senate. The coattail effect in the gains.
House election is quite strong, as an event occurring in the presiden- Case study: Minnesota
tial election has a larger effect on candidates in the House than on the Turning from the general application of our model across all con-
presidential candidates themselves. gressional and Senate elections in 2008, we now apply our model to
The general model of coattail effects in Equation 3 helps explain one specific election: the 2008 Minnesota Senate election. Because of
the difference in magnitude of the effects in the House and Senate. the perceived closeness of Minnesota’s election, the Iowa Electronic
One of the model’s core results is that a voter’s perception of the presi- Markets introduced a market with reasonable trading volume that
dential candidate matters in congressional elections only to the ex- measured the vote share in state’s senatorial election. Table 3 shows
tent that the presidential candidate’s views are a useful proxy for the the results of applying the instrumental variable regression from
congressional candidate’s views, or the magnitude of the variable w ic. Equation 5 on Minnesota’s results. A 1% change in likelihood of
In an election where the median voter knows less about the candi- Obama’s victory is associated with a 5.865% increase in the expected
date, coattail effects are expected to be stronger, since the party affili- vote share for Franken (the Democratic candidate in this election).
ation of the candidate carries more meaning. Typically, Senate candi- The magnitude of the coattail effect is strikingly large, especially
dates are better known than their House counterparts; since senators for a Senate race. The difference in effect between this election and
serve longer incumbencies, wield more personal power, spend more other Senate elections is likely due to the extreme closeness of this
on their campaigns, and have broader constituencies, they generally race – a race that was ultimately decided by just a few hundred heav-
capture a larger share of news coverage and political buzz. Thus, the ily-contested ballots. Thus the impact of any perceived political shift
median voter often knows more about the candidates in the Senate would be heavily amplified in this ultra-close election. Furthermore,
election than the candidates in the House election, and presidential Minnesota was not a heavy swing state in the presidential election,
views are more necessary as a proxy in the House election. and ultimately voted by a substantial margin for Obama. Thus, events
Amplification in the presidential election that resounded with liberal voters may
One of the more surprising results of this analysis is the amplifi- have had a stronger effect on the Senate election outcome than the
cation of effects across elections. A one percent change in the prob- general election probabilities.
ability of Obama winning is transformed into a greater than one per- Limitations and further research
cent change in the probability of Democrats winning in the House There are several limitations to these conclusions, and several
election. We propose two hypotheses for why amplification exists: questions are raised for further research. First, while this methodolo-
geographic influences and the self-fulfillment of predicted success. gy could be generalized, we only used data from the 2008 elections in
In these markets, changes in voting trends in different states are not this analysis due to the limited history of electronic prediction mar-
equally important, as a vote shift in a non-swing state is not as mean- kets for Congressional elections. As more elections occur and the use

Variable House Senate Variable Coefficient

Predicted_Change_Pres_WTA_Price 2.102 ** 0.764 Predicted_Change_Pres_WTA_Price 5.865 ***


[0.901] [0.587] [1.349]

_const -0.020 -0.005 _const -0.057


[0.021] [0.015] [0.035]

Observations 29 29 Observations 39
Table 2. 2-stage least squares results. This table shows the results of Table 3. 2-stage least squares results in Minnesota. This table
Equation 5, demonstrating the predicted effect of exogenous changes in shows the results of Equation 5, demonstrating the predicted effect of ex-
presidential market probabilities on House and Senate probabilities. The ogenous changes in presidential market probabilities on the Minnesota
House demonstrates strong, significant coattail effects, while the effects in Senate election. The election demonstrates strong, significant coattail ef-
the Senate are minimal. Heteroskedasticity-adjusted standard errors are fects, while the effects in the Senate are minimal. Heteroskedasticity-ad-
displayed in brackets. ** significant at 5% justed standard errors are displayed in brackets. *** significant at 1%
32 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

of electronic prediction markets expands, a broader analysis could widely expand the data set available for analyzing election phenom-
be performed on the impact of coattail effects on elections and the ena. Nonetheless, most literature on electronic prediction markets to
determinants of the magnitude of the effects. In particular, the 2008 date has been either theoretical in nature or an empirical measure-
election was unique in recent elections because of the relative cer- ment of the market’s predictive ability. By assuming the markets are
tainty of the eventual outcome for Senate and House control, as the in fact reasonably accurate (which most analysis supports), we use
probabilities that Democrats would control each chamber were over prediction markets as robust, real-time indicators of the status of an
90% for much of the period evaluated. Thus, an analysis performed election. Our results indicate that – beyond their predictive value –
in a year when both the presidential and congressional elections are prediction markets can also be a useful tool in isolating empirical
expected to be close may yield different results. effects in presidential elections and uncovering the determinants of
As prediction markets continue to mature, another potential ex- political success.
pansion would be to observe coattail effects on a state-by-state level.
While state-by-state election details from electronic prediction mar- References
kets were limited in the 2008 election, further expansion in market
1. Berg, J. et al., “Results from a Dozen Years of Election Futures Markets

Economics
availability and trading volume would enable a study on state-specific Research.” Working Paper (2001).
coattail effects and determinants of the magnitude of coattails. 2. Berg, J. et al., “Accuracy and Forecast Standard Error of Prediction Mar-
Finally, several assumptions were made in choosing to use elec- kets.” Working Paper (July 2003).
tronic prediction market prices as proxies for probabilities of actual 3. Erikson, Robert et al. “Was the 2000 Election Predictable?” Political Sci-
outcomes. First, we assumed that they are unbiased, reasonably accu- ence and Politics, Vol. 34, No. 4 (Dec., 2001), pp. 815-819
4. Fair, Ray. The Effect of Economic Events on Votes for President. The Re-
rate estimators, claims that are generally supported by both empiri- view of Economics and Statistics, Vol. 60, No. 2 (May 1978), pp. 159-173.
cal and theoretical literature on prediction markets. A more subtle 5. Fair, Ray. “Econometrics and Presidential Elections.” Journal of Econom-
assumption is that changes in prediction market prices are due to ic Perspectives, Vol. 10, (Summer 1996), pp. 89-102.
events impacting the election directly, rather than merely changing 6. Fair, Ray. Predicting Presidential Elections and Other Things. Stanford
perceptions of the candidates themselves by market participants. For University Press (2002).
7. Ferejohn, John and Randall Calvert. “Presidential Coattails in Historical
example, an eloquent speech by a candidate would not only boost
Perspective.” American Journal of Political Science, Vol. 28, No. 1 (Feb.,
his chances in the election directly, it might also signal to predic- 1984), pp. 127-146.
tion market participants that he will be similarly eloquent in the 8. Frey, B. and Schneider, F. “Economic and Personality Determinants of
future—and traders will factor these future events into the market Presidential Popularity.” Empirical Economics, Vol. 3, No. 2 (June, 1978),
price. This effect would likely be particularly magnified early in an pp. 79-89
9. Kaplowitz, Stan. “Using Aggregate Data to Measure Presidential Coat-
election, since market participants know less about the strategic and
Tails Effects.” Working Paper (1970).
tactical competence of particular candidates. In order to mitigate this 10. L evitt, Steven. “Using Repeat Challengers to Estimate the Effect of Cam-
effect, we selected events for this study that occurred relatively late in paign Spending on Election Outcomes in the U.S. House.” The Journal of
the election and would likely have a primary effect of directly alter- Political Economy. Vol. 102, No. 4 (Aug., 1994), pp. 777-798.
ing the election, such as the selection of a vice-presidential candidate. 11. Lewis-Beck, M. “Economic voting: an introduction.” Electoral Studies,
Furthermore, even if the market changes its perception of a candidate Volume 19, Issue 2-3 (2000), pp. 113-121
12. M iller, Warren. “Presidential Coattails: A Study in Political Myth and
and incorporates information about future expected events because Methodology.” The Public Opinion Quarterly, Vol. 19, No. 4 (Winter
of one of the events we selected, as long as those future events also 1955), pp. 353-368
generate coattails, they will also be incorporated into congressional 13. Mondak, Jeffery. “Determinants of Coattail Voting.” Political Behavior,
prediction markets, leaving our estimate unbiased. Vol. 12, No. 3 (Sep., 1990), pp. 265-288.
14. Mondak, Jeffery and Carl McCurley. “Cognitive Efficiency and the Con-
gressional Vote: The Psychology of Coattail Voting.” Political Research
Concluding Remarks Quarterly, Vol. 47, No. 1 (1994), pp. 151-175.
15. New York Times. President Map. http://elections.nytimes.com/2008/re-
Our results provide an intriguing insight into one of the major sults/president/map.html. Accessed February 2009.
determinants of United States elections outcomes. Using electronic 16. Pew Research Center. “Top Events of Campaign 2008.” November 6, 2008.
prediction market trading data, we find that coattail effects have Available at http://pewresearch.org/pubs/1025/election-news-interest
17. Shaw, Daron. “The Effect of TV Ads and Candidate Appearances on State-
a major impact on House elections, but a more limited impact on
wide Presidential Votes, 1988-96.” The American Political Science Review,
Senate elections. By using major exogenous shifts in the presidential Vol. 93, No. 2 (Jun., 1999), pp. 345-361
election as an instrument, we isolate the coattail effects in the elec- 18. Snowberg, Erik et al. “Partisan Impacts on the Economy: Evidence from
tion and overcome causality concerns intrinsic to most past studies Prediction Markets and Close Elections.” The Quarterly Journal of Eco-
of empirical effects. nomics, Vol. 122, No. 2 (2007), pp. 807-829.
While this result generally confirms our intuitive expectations 19. Washington Post. Campaign Tracker. http://projects.washingtonpost.
com/2008-presidential-candidates/tracker/candidates/barack-obama/
about coattail effects, the methodology provides an intriguing new states/oh/. Accessed in February 2009.
way to study major factors shaping elections. Similar to coattail ef- 20. Wisconsin Ad Project. “Pres. TV advertising spending continues to
fects, many factors that intuitively have large effects on elections are grow; Over $28 million spent from September 28-October 4.” Univer-
difficult to show empirically, since elections occur only once every sity of Wisconsin. Available at http://wiscadproject.wisc.edu/wiscads_
few years and substantial differences in context occur from election release_100808.pdf.
21. Wolfers, Justin and Eric Zitzewitz. “Interpreting Prediction Market Prices
to election that complicate any attempts to hold multiple factors con-
as Probabilities.” CEPR Discussion Paper No 5676 (May 2006).
stant. By using market prices of a particular outcome as a proxy for 22. Wolfers, Justin. “Best Bet for Next President: Prediction Markets.” Wall
the status of an election, electronic prediction markets promise to Street Journal. December 31, 2007.

www.thurj.org 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- Figure 1. Children unprotected from IDD (2000-2006).
tal and physical health can significantly impede worker productivity
and the economy at large. Clearly, eliminating iodine deficiency has a Methodology
significant impact on the world’s poor.
To combat IDD, the WHO and UNICEF recommended in 1994 Datasets
the use of iodized salt as a safe, cost-effective and sustainable strategy The primary dataset used in this study was from India’s second
to ensure sufficient intake of iodine by all individuals. However, de- National Family and Health Survey (NFHS-2), which was conducted
spite pushing a Universal Salt Iodization program, progress has been from 1998-1999 by the International Institute for Population Sciences
limited. In India, which is a major salt-producing country, only 51% (IIPS) in Mumbai (IIPS and Macro 2000).1 This dataset was ideal
of households were using iodized salt in 2005. This is a particular for two reasons. First, it captured key socioeconomic, cultural, and
problem since out of 38 million newborns in developing countries health information about over 91,880 households, 90,303 women and
every year that remain unprotected from the lifelong consequences 33,132 children in India. Second, it provided district-level identifiers
of IDD-related brain damage, a large percentage live in South Asia, for each household, which was not available in the most recent 2005
as shown in Figure 1 (UNICEF 2008). NFHS-3 because of privacy issues. This was crucial because it allowed
However, to justify efforts that the Indian government is making for the use of within-state variation by district in order to estimate
to improve the system, it is important to understand what effect the causality, which is particularly helpful for assisting policymakers
lack of iodized salt coverage has had on public health. Specifically, it in planning and implementing strategies for improving population
is necessary to determine whether IDD affects the physical as well as health and nutrition programs.
mental capacities of children in India. Thus, identifying the causal Six secondary datasets were superimposed on a map of India and
link between iodine deficiency and child survival and growth will linked to the NFHS-2 through district-level household identifiers to
more effectively direct the appropriate resources toward its correc- create a series of district-level control and instrumental variables.
tion. In doing so, this study also aims to contribute to the method- Measurements were made using the ArcGIS9 (ArcMap Version 9.3)
ological literature on measuring intervention impacts. 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 RESEARCH

software. First, MLInfoMap: 1991 Census provided map data on The first two outcomes, children’s height-for-age and weight-
district borders, area and population. Second, the Global Precipita- for-height were evaluated relative to the median height-for-age and
tion and Climatology Center’s Full Data Reanalysis Project provided weight-for-height of the 1997 U.S. National Center for Health Sta-
information on average annual rainfall for each district. Third, the tistics (NCHS) reference population, recommended by the World
Shuttle Radar Topography Mission provided 90-meter by 90-meter Health Organization. Measures greater than two standard deviations
raster images of elevation. Fourth, the Global Self-consistent, Hier- below the reference median considered stunted (height-for-age) or
archical, High-resolution Shoreline (GSHHS) Database Version 1.3 wasted (weight-for-height). Stunting is a sign of chronic, long-term
provided information on distances to the closest coast. Fifth, Collins under-nutrition, whereas wasting is a sign of acute, short-term un-
Bartholomew World Premium 2007 provided information about the der-nutrition. It was therefore hypothesized that access to household
main and secondary railway lines, as well as the road networks in iodine would lead to improved height-for-weight outcomes and have
India. Sixth, the Government of India Salt Department’s 2005-2005 no effect on weight-for-height outcomes. Such a result would support
Annual Report provided information on how much salt was trans- the fetal origins of disease hypothesis.
ported to each state by railroad, road, or sea. On average, children in India were borderline stunted at 1.91
Variables standard deviations below the reference population. Those children
The explanatory variable of interest in this paper was the iodine living in households with salt iodine content of 0, 7, 15 and 30ppm
level of the salt used in a household. In NFHS-2, interviewers mea- were 2.06, 2.07, 1.98 and 1.67 standard deviations below the median,
sured the iodine content of cooking salt in each interviewed house- respectively. Thus, there is a clear corresponding trend between io-
hold using a rapid-test kit and recorded the iodine level as 7, 15, or 30 dine concentration and height-for-age outcome. On the other hand,
parts per million (ppm) (IIPS and Macro 2000). though children were on average 0.78 standard deviations below the
Overall, half of households used cooking salt that was iodized at reference median, they were not low enough to be considered wasted.
the recommended level of 15 ppm or more, one-quarter of house- However, unlike the prediction, there seemed to be a similar rising
holds used salt that was not iodized at all, and 21 percent used salt trend of weight-for-height (0.96, 0.89, 0.75 and 0.6 standard devia-

and Medicine
Public Health
that was inadequately iodized (less than 15 ppm). The use of iodized tions below the reference), which corresponded with rising iodine
salt varied dramatically from one state to another (as shown in Figure concentrations (0, 7, 15, and 30ppm, respectively). Such a pattern pos-
2). The variations could be due to a number of factors, including the sibly indicates that there are other factors such as standard of living
scale of salt production, transportation requirements, enforcement that may be driving these similar trends.
efforts, pricing structure, and storage patterns. In particular, salt io- The third and fourth outcomes, likelihood of being born small
dization was likely to be more common in states where salt was trans- and large, were chosen because small newborns generally face sub-
ported exclusively by railways, partly because the Salt Department stantially higher risks of dying than do newborns of normal or large
monitored the iodine content of salt shipped by railways. size. The average birth weight of the small, average and large children
The five child health outcomes that this paper examined were was 2.2 kg, 2.8 kg and 3.4 kg, respectively. According to mothers’ esti-
height-for-age, weight-for-height, likelihood of being born small, mates, 25 percent were small, 60 percent were average, and 14 percent
likelihood of being born large, and overall child survival. These out- were large. As the salt iodine concentration went up, there was also
comes were based on NFHS-2 anthropometric data of 18,521 chil- a corresponding decrease in fraction of children born small and in-
dren ages 0-5 years, and NFHS-2 birth history of 21,388 children of crease in fraction of children born large.
all ages. 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,
Figure 2. Iodine concentration. jaundice, asthma, and malaria). β3 is the vector coefficient for H, the
www.thurj.org 35
RESEARCH Volume 2 Issue 2 | Fall 2009

Figure 3 Figure 5 Figure 6

Figure 4
and Medicine
Public Health

Figure 3. Railroad. Equation 2 corresponds with the first stage of the 2SLS estimate. Î
Figure 4. Road length. is the predicted value of household salt iodine. Unlike equation 1, β1
Figure 5. Precipitation. in equation 2 is the vector coefficient for Z, the instruments. These in-
Figure 6. Elevation. clude both institutional and geographic instruments. The institution-
Figure 7. Coast. al instruments include distance to any railroad (Figure 3), distance to
the nearest main railroad, fraction of salt transported to the state by
household-level controls. These railroad, and total road length in the district (Figure 4), which pre-
include characteristics such as dict access to iodized salt. The geographic instruments include aver-
the standard of living index, age district precipitation (Figure 5), elevation (Figure 6), and distance
Figure 7 urban environment, and reli- to the nearest coastline (Figure 7), which predict baseline endemic
gion of household head. β4 is iodine deficiency.
the vector coefficient for D, the Equation 3 corresponds with the second stage of the 2SLS esti-
district-level controls. These mate. Here, Y is the child health outcome of interest. β1 is the coef-
include district population ficient for Î, the iodine level predicted by the instruments. As before,
density and area. β5 is the vec- the remaining coefficients correspond with mother-, household-, dis-
tor coefficient for S, the state- trict- and state-level controls.
fixed effects. Finally, ε is the The 2SLS regressions were also broken down to capture heteroge-
error term. neous treatment effects on male versus female children and on urban
The disadvantage with OLS versus rural households to determine whether the effect of iodized
is that there are several unob- salt had a greater impact on certain groups. Finally, three robustness
servable characteristics that checks were carried out to assess how sensitive the results were to the
cannot be controlled for which econometric specification.
may affect both the outcome
and explanatory variable. Al- Results
though OLS helps identify pat-
terns, it is difficult to use an When district-, household-, and mother-level observable charac-
OLS estimation to determine teristics were controlled for in the OLS regression (Column 1, Table
causality. Therefore, a two-stage least squares (2SLS) methodology 5), it was found that increasing the iodine concentration by one level
is used. The 2SLS technique employs instrumental variables, which (i.e. 0 to 7ppm, 7 to 15ppm, or 15 to 30ppm) correlated with a 0.0375
are supposed to be highly correlated with the outcome variable only standard deviation increase in height for age (p<0.01). The 2SLS esti-
through a correlation with the explanatory variable. Below, the em- mate (Column 2, Table 5) revealed that increasing iodine concentra-
pirical specification for the 2SLS regressions run in this analysis is tion by one level led to a 1.168 standard deviation increase in height
presented: for age. Looked at another way, increasing the salt iodization con-
centration by two levels, from 0 to 15ppm (the concentration recom-
Î = α+β 1(Z)+β 2 (M)+β 3 (H)+β 4 (D)+β 5 (S)+ε (Equation 2) 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
Y = α+β 1(Î)+β 2 (M)+β 3 (H)+β 4 (D)+β 5 (S)+ε (Equation 3) (who on average are at 1.91 standard deviations below the median) at
36 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Height-for-Age Weight-for-Height Small Child Large Child Child Survival


1 2 3 4 5 6 7 8 9 10
OLS 2SLS OLS 2SLS OLS 2SLS OLS 2SLS OLS 2SLS

Iodine 0.0375*** 1.168*** 0.0256** -0.0341 -0.00798** -0.159*** 0.00222 0.186*** 0.00167* 0.0281*
(0.0131) (0.311) (0.0107) (0.190) (0.00314) (0.0556) (0.00250) (0.0517) (0.000999) (0.0163)
n 20328 18521 20328 18521 23441 21388 23441 21388 23450 21396

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. children living in rural settings. The magnitude of the effect is sig-
Contrary to the prediction, in the OLS estimate (Column 3, Table nificant since increasing iodine concentration from 0 to 15 ppm
5), an increased iodine level correlated with a 0.0265 standard de- seemed to drive the average height-for-age standard deviation from
viation increase in weight-for-height outcomes for children (p<0,05). -1.91 (borderline stunted) to nearly +0.43 standard deviations above
However, as expected, the significance of this estimate became nil in the NCHS standard. Such an effect can have a significant effect on
the 2SLS (Column 4, Table 5). the future productivity of the nation’s children. However, iodized
Increasing the iodine concentration by one level led to a statisti- salt might not be the answer to the problem of childhood wasting.

and Medicine
Public Health
cally significant (p<0.05) but small (0.008 percent) decrease in the This is not a huge concern since the average weight-for-height was
likelihood of being born small in the OLS estimate (Column 5, Table not close to 2 standard deviations below the NCSH standard, which
5). This value became much larger and more significant in the 2SLS. defines someone as wasted. In addition, the results suggest that use
As shown in Column 6, Table 5, increasing iodine concentration level of iodized salt by mothers might reduce the likelihood of being born
led to a 16% drop in the likelihood of being born small (p<0.01). The small and increase the likelihood of being born large, especially for
correlation between iodine concentration level and likelihood of be- girls and children born in rural areas. Finally, there seemed to be a
ing born large was insignificant in the OLS regression (Column 7, positive effect of iodine on child survival, especially for males.
Table 5). However, iodine concentration seemed to have a large and It is important to note that only two of the five outcomes—height-
significant effect in the 2SLS (Column 8, Table 5). An increase in io- for-age and likelihood of being born large—passed the greatest num-
dine level led to an 18.6% increase in likelihood of being born large ber of robustness checks. This indicates that we can only begin to
(p<0.01). extend the fetal origins of disease hypothesis to these two outcomes.
The OLS and 2SLS estimates (Columns 9 and 10, Table 5) showed However, since even these two outcomes failed to pass the most strin-
that an increase in salt iodine concentration level led to a 0.22 per- gent robustness check, conclusions can only be made with some de-
cent and a 2.8 percent increase in child survival rates, respectively gree of caution.
(p<0.1). There are several measurement issues that this analysis faces. In
When the sample was broken down by gender and place of resi- this sample of the NFHS-2, the reference population used in calcu-
dence, there were significantly greater effects of iodized salt on fe- lating anthropometric outcomes was the 1997 U.S. National Center
male children and rural households. for Health Statistics (NCHS) standard. More recently however, a new
international reference population was released by the WHO in April
Discussion 2006 and accepted by the Government of India, which may better as-
sess children regardless of ethnicity, socioeconomic status and type
The goal of this study was to establish a causal link between access of feeding. In addition, the set of child health outcome variables fo-
to iodized salt and child health outcomes. Showing the direct relation cusing on size of the newborn are especially prone to selection and
between iodine deficiency and child survival and growth would help reporting biases, since mothers tend to self-report their children as
motivate researchers and policy-makers to identify the barriers that being bigger than they actually are, since size is considered a measure
prevent India from reaching more widespread use of iodized salt. In of health. Finally, the way that I calculated child survival was simply
addition, illuminating the differential effects of access to iodized salt the number of children living over the total number of children born.
on subpopulations would help direct resources more appropriately. It is unclear whether such a measurement can really capture the in-
This economic analysis is unique because it utilized Geographic tricacies of child mortality early in life.
Information Systems to measure precise distance and geographic For the explanatory variables, an assumption was made that if
information and connected this information with the district codes a household had iodized salt at the time of the survey, it probably
of households in this sample. Used together as instruments, the GIS has had access to that level of iodized salt throughout the universal
data served to identify causal effects where a controlled experiment salt iodization program, which became more active starting in 1992.
was not possible. In addition, because the analysis used the 1998 However, this assumption may not necessarily hold. The practices of
NFHS, it covered a representative sample of the Indian population as households in 1998 do not necessarily have to reflect the practices of
a whole. The timing of the survey captured the effect of five years of the household five years before.
universal salt iodization program in India and might have captured The instruments used in this analysis showed high relevance in
the ban on non-iodized salt that had been instated in 1998. the first-stage regression, yielding high F-stats and significant cor-
The results of this study suggest that access to iodized salt posi- relations with the explanatory variable. However, the exogeneity re-
tively impacted children’s height-for-age outcomes, especially for quirement was less convincing. Though precipitation and elevation
www.thurj.org 37
RESEARCH Volume 2 Issue 2 | Fall 2009

seem to be less of a problem, distance to the coast and distance to rail- Med 23(4): 367-72.
Pelletier, D. L. (1994). “The potentiating effects of malnutrition on child mor-
roads were arguably endogenous. Presumably the closer a household tality: epidemiologic evidence and policy implications.” Nutr Rev 52(12):
is to a railroad or ocean, the more resources the household can access 409-15.
and therefore the higher standard of living it may experience. Pharoah, P. and K. Connolly (1994). Iodine Deficiency in Papua, New Guinea.
The robustness check limiting the set of instruments to only dis- The damaged brain of iodine deficiency. S. Stanbury. New York, Cogni-
tance to railroad revealed insignificant results, showing that the main zant Communication: 299-305
Sarkar, S., B. Mohanty, et al. (2007). “Iodine deficiency in school going chil-
findings might be sensitive to the instruments chosen. Moreover this
dren of Pondicherry.” Indian J Pediatr 74(8): 731-4.
dataset was limited to district level data. Therefore, it is possible that Thilly, C., R. Lagasse, et al. (1980). Impaired fetal and postnatal development
the instrument used in the most stringent specification did not have and high perinatal death-rate in a severe iodine deficient area. Thyroid
enough variation to predict access to iodized salt accurately. Having research VIII. J. Stockigt, S. Nagataki, E. Meldrum, J. Barlow and P. Hard-
exact coordinates of households would enable a more robust analy- ing. Canberra, Austrailian Academy of Science: 20-23.
sis. UNICEF (2008). “Sustainable Elimination of Iodine Deficiency: Progress
since the 1990 World Summit for Children.” 52.
Whitney, E. N., C. B. Cataldo, et al. (2002). Understanding Normal and Clini-
Conclusion cal Nutrition. Australia, Canada Wadsworth Group, Thomson Learning.
WHO, UNICEF, et al. (1999). Progress towards the elimination of iodine de-
This paper revealed that an increase in salt iodine levels led to pos- ficinecy disorders (IDD). Geneva, WHO.
itive and statistically significant child health outcomes—namely, a
1.168 standard deviation increase in height-for-age, a 15% decreased Acknowledgements
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 I would like to thank the following people for their tremendous support
provided evidence that access to iodized salt had stronger effects on throughout this research process. Without their help and encouragement, this
female children and on children living in rural settings. Therefore, original work would not have been possible. Erica Field, Assistant Professor of
and Medicine
Public Health

targeting certain populations could potentially have a more power- Economics, Harvard University, for being a wonderful thesis advisor and contin-
ful impact on child health. This study is innovative in that it utilized ually guiding me in the right direction. Winnie Fung, PhD candidate in Econom-
unique instruments constructed through GIS data to predict access ics, Harvard University, for being my thesis tutorial leader and helping me clarify
to iodized salt and isolate its causal effects on childhood health. Us- my questions and methodology. Konstantin Styrin, PhD candidate in Economics
ing this estimation strategy, the paper shows that the Indian govern- and STATA Teaching Fellow, Harvard University, for answering my countless
ment would do well to continue promoting access to iodized salt in STATA coding questions. Sebastian Linnemyer, PhD candidate in Economics
order to secure the health and well-being of its children. and Teaching Fellow, Harvard University, for his advice in the Ec980 Junior Tu-
torial, “Development, Education, and Health.” Scott Walker, Digital Cartogra-
References 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-
Allen, L. and S. Gillespie (2001). What Works? A review of efficacy and effec-
tiveness of nutrition interventions, UN (ACC/SN). nie Burns, GIS Specialist, Harvard Map Collection, for helping me identify the
Bleichrodt, N. and M. Born (1994). A Meta analysis of research on iodine and 1991 India Census Data. Fred Arnold and Bridgette James, DHS/NFHS Archives,
its relationship to cognitive development. The damaged brain of iodine for providing me with district coding for the NFHS data. Joan Curhan, Debbie
deficiency. J. Stanbury. New York, Cognizant Communication: 195-200 Whitney, Suzanne Scudder, for supporting me in the Certificate in Health Policy
Cobra, C., Muhilal, et al. (1997). “Infant survival is improved by oral iodine Program. The Cordeiro Family, for providing me with a generous grant to travel
supplementation.” J Nutr 127(4): 574-8.
to India to continue on-the-ground research in July 2008. Darpana Academy of
Dunn, J. T. and F. Delange (2001). “Damaged reproduction: the most impor-
tant consequence of iodine deficiency.” J Clin Endocrinol Metab 86(6): Performing Arts, for giving me the opportunity during my Fall 2007 semester
2360-3. in India, to travel to Valsad (pictured above), a rural district in Gujarat, and be-
Field, E., O. Robles, et al. (2007). The Cognitive Link Between Geography and come involved in a UNICEF development project that inspired this thesis. Dr.
Development: Iodine Deficiency and Schooling Attainment in Tanzania. Chandrakant S. Pandav and Dr. Arijith Chakrabarty, International Council for
NBER Working Paper Series. Cambridge, MA, National Bureau of Eco-
Control of Iodine Deficiency Disorders (ICCIDD), All India Institute of Medical
nomic Research: 64.
Garrow, J. S., A. Ralph, et al. (2000). Human Nutrition and Dietetics. New Sciences, New Delhi, for spending time sharing their insight with me on the most
York, Elsevier Health Sciences. recent accomplishments and challenges of the Universal Salt Iodization Program
Hetzel, B. S. (1989). The Story of Iodine Deficiency; An International Chal- in India. Dr.Rajan Sankar, Regional Manager for the South Asia branch of Global
lenge in Nutrition. Oxford, Oxford University Press. Alliance for Improved Nutrition, New Delhi, for providing me with extensive
IIPS and O. Macro (2000). National Family Health Survey (NFHS-2), 1998-
literature on the history of iodine deficiency. Mr. S. Sunderesan, Salt Commis-
1999: India. Mumbai, IIPS.
Kapil, U., R. S. Raghuvanshi, et al. (1999). “Utility of spot testing kit in the sioner, and Mr. M.A. Ansari, Deputy Salt Commissioner at the Ministry of Salt
assessment of iodine content of salt--A mutlicentric study.” Indian Pedi- in Jaipur, Rajasthan, for communicating with me via email regarding questions
atrics 37: 182-186. about the transportation and production of salt throughout the country. Tracy
Lamberg, B. A. (1991). “Endemic goitre--iodine deficiency disorders.” Ann 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.

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

Neuroscience
memories? How do our different mental and physical states affect and directly. M/T cells form dendrodendritic synapses with granule
how we respond to odor stimulation? cells in the external plexiform layer, and can inhibit other M/T cells
Sensing an odor begins with the olfactory receptor neurons through granule cells (Wilson and Mainen, 2006). M/T cells may
(ORN) located on the nasal epithelium (Wilson and Mainen, 2006). also directly influence their neighbors through a process known as
A single mouse possesses approximately 44 million ORNs (Albeanu, spillover. When the M/T cells release glutamate through their den-
2008, Mombaerts, 2006, Wachowiak, 2004), with each ORN contain- drites, some of the glutamate excites dendrites of neighboring M/T
ing G-protein coupled receptors known as the olfactory receptors cells (Isaacson, 1999, Wilson and Mainen, 2006).
(OR). In order to remain functionally distinct, a single ORN only Finally, the olfactory system is influenced by centrifugal inputs
expresses one or perhaps a small number of different types of ORs from the cortex and other areas of the brain (Isaacson, 1999). These
(Albeanu, 2008, Mombaerts et al., 1996). Every OR can detect mul- inputs may be affected by the behavior of the mouse, genetic predis-
tiple odors, and many types of ORs detect each odor. However, each positions, and other factors. Centrifugal influences can be separated
odor is sensed by a unique combination of ORs that allows it to be into three major categories. First, odor intake is affected by areas of
distinguished from other odors (Malnic et al., 1999). The axons of the brain outside of the olfactory bulb. Passive sniffing rate is deter-
the stimulated ORN transmit the sensory signal to approximately mined by central pattern generators in the medulla (Ramirez and
two locations in a single olfactory bulb known as glomeruli, which Richter, 1996, Wilson and Mainen, 2006) while active respiration
are simply the synapses joining the ORN axons with the dendrites is controlled by the forebrain (Wilson and Mainen, 2006). Second,
of mitral/tufted (M/T) cells, which output the signal to the olfactory the bulb receives reciprocal feedback from output regions such as the
cortex (Albeanu, 2008, Mombearts et al. 1996). Since the glomeruli olfactory cortex. Finally, changing levels of neuromodulators such
are spatially defined functional clusters, the glomerular layer can be as norepinephrine, acetylcholine, and serotonin can affect odor re-
viewed as an odor map for which each odor is encoded by a unique sponses (Wilson and Mainen, 2006).
combination of glomerular activation (Mombaerts et al., 1996). Once The complexity of the olfactory system, designed to accomplish
the signal reaches the glomerular layer, it is passed to the mitral cell what may seem like a simple task, is astounding. This intricacy makes
layer, which contains the M/T cells (Wilson and Mainen, 2006). the system susceptible to outside influences such as alterations in be-
The structure of the olfactory system, however, is not a simple re- havior. Most in vivo imaging research on the olfactory system exam-
lay. Rather, the system is subject to lateral modulation from regulato- ines the bulb while an animal is in an anesthetized state. However,
ry cells. Each glomerulus is surrounded by different types of regula- anesthesia greatly limits the scope of the field because it makes it im-
tory juxtaglomerular (JG) cells, including periglomerular (PG) cells possible to study the effect of behavior on olfactory response.
and short axon (SA) cells (Albeanu, 2008, Wachowiak and Shipley, One way in which an animal’s behavioral state may affect its re-
2006). Periglomerular cells, the more populous of the interneurons sponse to odors is by influencing the internal bulb activity. Recently,
in the glomerular layer, are inhibitory regulators (Albeanu, 2008, a team headed by Kenasku Mori studied the dendro-dendritic activ-
www.thurj.org 39
RESEARCH Volume 2 Issue 2 | Fall 2009

ity in the external plexiform layer (Tsuno et al., 2008). They found good way to image freely behaving animals.
that mitral-granule activity was strongest in anesthetized animals The second question of this study is whether behavior has an effect
and was progressively weaker in lightly sleeping, awake and immo- on the pre-synaptic activity of glomeruli. As discussed earlier, there
bile, and awake and mobile animals. are several ways that behavior could influence ORN output. Thus,
In addition to direct effects upon the olfactory bulb, behavior may it seems likely that behavior will cause some change in glomerular
affect centrifugal inputs. For instance, while the sniffing rate of an activity, although attributing observed changes to specific individual
anesthetized animal is passively controlled by the brainstem, a be- behaviors as well as parsing out the causes is beyond the scope of
having animal has the ability to actively control this rate. Further- this study. In order to test this hypothesis, the aforementioned fiber
more, the levels of norepinephrine, acetylcholine, and serotonin can optic technique was used to image transgenic mice expressing syn-
be altered by different behavioral states (Wilson and Mainen, 2006). aptopHluorin in the glomeruli. In addition, a customized odor rack
As an example, Shea et al. (2008) found that norepinephrine release, was employed to expose the mice to different odors while monitoring
when coupled with odor presentation, acts in the olfactory bulb to the fluorescence response in real-time. The same mice were imaged
cause suppression of paired odor responses. in both an awake and an anesthetized state, and the responses were
To more fully understand the effects of different behaviors on compared.
odor responses, the olfactory bulb needs to be monitored while the Fundamentally, the goal of this study is simply to open the doors
animal is awake and behaving. While electrical activity in behaving to a burgeoning field of new imaging techniques while answering
animals has been extensively studied, they are limited to single cell some questions about how behavior can affect olfactory sensory ac-
readouts as opposed to imaging studies, which allow for large-scale tivity. First, this project attempts to determine whether a fiber op-
population readouts. Sub-glomerular resolution imaging studies of tic imaging bundle provides a way to image the olfactory bulb of an
the olfactory bulb in awake animals have been performed on head- awake, freely-moving animal. Second, this study uses the bundle to
fixed animals (Carey et al., 2004), but are limited. Furthermore, to track olfactory receptor neuron pre-synaptic activity in anesthetized
date, sub-glomerular resolution imaging of the olfactory bulb in and freely-behaving mice to find similarities and differences based
freely behaving animals has not yet been accomplished. The present on behavioral state.
study attempts to address this issue by offering a way to image the
bulb in a freely behaving mouse. Methods
Two important advances have made it possible to explore odor
responses in freely behaving mice. The first was the creation of trans- Subjects and surgery
genic mice expressing synaptopHluorin in the ORNs, which allowed The subjects used were transgenic adult (postnatal days 60-100)
the visualization of pre-synaptic glomerular activity in the olfactory synaptopHluorin mice, including both heterozygous and homozy-
bulb. pHluorins is a mutant form of GFP that was sensitive to pH and gous as well as male and female mice (Bozza et al., 2004). Mice were
would fluoresce in a neutral environment but not in an acidic envi- anesthetized for surgery with a cocktail of ketamine and xylazine
ronment. SynaptopHluorin (spH) was then made by fusing pHluorin (ketamine - 100 mg/kg, IP, Fort Dodge Animal Health #440761; 10
Neuroscience

to synaptobrevin (Miesenböck et al., 2000). Synaptobrevin is a vesicle mg/kg xylazine, IP, Phoenix Pharmaceutical, Inc. #4111505). The
protein required for the release of neurotransmitters into the syn- mice were mounted on a stereotaxic frame and the skin was cut to
apse. The idea is to take advantage of the acidic environment of the expose the skull. A small hole, approximately 1.5-2 mm in diameter,
neurotransmitter vesicles (pH ~5.7). When inside the vesicles, spH was opened over the right olfactory bulb by thinning the bone in a
would not fluoresce, but once a vesicle binds with the cellular mem- circle and removing the piece. A flexible fiber bundle (Schott Inc.,
brane to release neurotransmitters into the neutral pH of synaptic #1137189) with a 1.45 mm outer diameter was lowered onto the brain.
space (~7.4), spH would fluoresce (Miesenböck et al., 1998). This laid The other end of the bundle was placed in the focal plane of a wide-
the groundwork for the use of synaptopHluorin as a genetically en- field fluorescence microscope fitted with a 10x objective and a high
coded molecular probe that would allow detection of neural activity speed imaging camera (SensiCam, Cooke Corp.). The bundle was
using simple fluorescence microscopy. lowered using an XYZ translator until glomeruli became clear. The
Though there are many other ways to image ORN synaptic activ- fiber bundle provided a continuous two-way light path from the mi-
ity such as nasal calcium dye injections, bulk calcium dye loading, croscope to the glomeruli and back to the microscope, allowing the
and intrinsic signaling (Toga and Mazziotta, 2002, Albeanu, 2008), mouse to move around away from the microscope while still keep-
synaptopHluorin was used because of three significant advantages. ing an image of the glomeruli within the focal plane of the micro-
First, it is genetically encoded unlike methods involving calcium scope. Once the bundle was in the proper place on the brain, it was
dyes, which need to be exogenously loaded. Second, the signal is cemented onto the skull with RelyX Luting Plus (3M ESPE, #3525).
mostly specific to pre-synaptic activity. Finally, because it is localized The mouse was allowed to recover for several hours before the analge-
to the axon termini, spH makes it possible to more easily distinguish sic, Buprenorphine HCl (0.5 mg/kg, BD, IP, Bedford Labs, #1208141),
individual glomeruli. was given. Anesthetized imaging occurred immediately after surgery
The second necessary development for this study was the intro- while the mouse was still anesthetized. Behavioral imaging was per-
duction of an imaging technique using a flexible fiber optic imag- formed after recovery.
ing bundle attached to the skull. This method was chosen over other Imaging fiber optic bundle
methods such as the head-fixed method (Carey et al., 2004) specifi- There are two issues regarding the properties of an imaging fiber
cally because it allows the mouse to freely move around while keep- bundle that need to be addressed. The first is the pixelation of the
ing the fluorescence image in the focal plane of the microscope. image. The bundle was composed of thousands of microscopic fibers,
The first fundamental question that this study attempts to answer each 8 microns in diameter. This led to pixelation that was visible un-
is whether the fiber technique is a viable way to image freely behav- der 10x magnification. However, since the pixels were much smaller
ing animals. This technology is still in its very early stages, and has than the glomeruli (approximately 90 microns), the pixelation did
never been used to image the olfactory bulb. However, after adapting not significantly interfere with the overall quality of the image. The
and optimizing the procedure, this technique will presumably offer a second issue is the lack of focusing optics in the bundle itself. Ide-
40 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

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. During the cleaning phase, the cleaning valve was opened to allow
Odor delivery apparatus fresh air to flow through the system. In addition, the air valve was
In order to systematically present odors, a computer-controlled open, providing fresh air to the animal. During air presentation 1, the
olfactometer (Figure 1) was designed and constructed. The apparatus air valve remained open and images were taken to obtain an average
contained tygon tubing (McMaster-Carr 5046K11) and two main sets baseline fluorescence image for comparison to odor images. During
of solenoid valves (ASCO Scientific AL4124). The first set consisted the odor phase, the exhaust and air valves were closed, and the odor
of ten valves, each associated with a single odor. When a valve was valve was opened. The odor flowed through the second set of valves
opened, air would pass through the valve, going into a test tube con- to the animal. Finally, during air presentation 2, the odor valve was
taining the odor and out via another path, carrying the odor with it. closed, and the air valve was re-opened, allowing the fluorescence to
The odor would then travel to a second set of valves, which included return to baseline while images were taken to track the return.
an exhaust valve, an odor valve, and an air valve. The odor could ei- Software and experimental design
ther go through the odor valve or the exhaust valve. If the odor valve The odor delivery control and image acquisition was performed
was open, the odor would be presented to the animal. Alternatively, if by a software program written in LabView (National Instruments)
the exhaust valve was open, the odor would leave the system through adapted by Tomokazu Sato from a similar program by Edward Soucy.
a ventilation system. The air valve was connected to an air line, allow- Each experiment could be altered by changing the settings of the pro-
ing clean air to be presented to the animal. Aside from the two main gram. During the course of the experiment, the mouse was placed in

Neuroscience
sets of valves, there was a cleaning valve. a small chamber to minimize movement during awake imaging and
Each trial involved four different phases that were repeated: clean- to maximize and homogenize odor exposure. The air in the cham-
ing, air presentation 1, odor presentation, and air presentation 2. 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.
www.thurj.org 41
RESEARCH Volume 2 Issue 2 | Fall 2009

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.
not cover the entire bulb, so even strong odors may only stimulate
Results and Discussion 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
Efficacy of fiber bundle imaging limited responsiveness, the fiber was moved and retested until either
Using a fiber optic bundle to image the brain of an awake, behav- responses were seen or it was determined that the mouse would not
ing animal is an underdeveloped technique. Hence, there are several respond, in which case it was euthanized.
potential complications that may arise in its application to this study. The final step was to determine whether the fiber technique was
First, the fiber method has never been used to image glomeruli. It is suitable for imaging awake, freely behaving animals. Strong responses
hard to predict whether individual glomeruli will be distinguishable similar to those seen in anesthetized animals were observed. Howev-
because of their proximity to each other and because of variable opti- er, there were several shortcomings, as predicted. One major problem
cal properties of the imaging fiber. Secondly, due to less constraining is the small size and high curvature of the olfactory bulb, which cre-
structural support, the olfactory bulb may be more subject to move- ates problems with maintaining focus. The olfactory bulb has space
Neuroscience

ment when the mouse moves its head. If the bulb were constantly to move around, and during the awake trials, images often contain
moving relative to the fiber, it would be difficult to maintain clear motion artifacts due to brain movement relative to the fiber (Figure
images throughout a chronic experiment. Furthermore, movement 3A,B). There were two types of motion artifacts observed. The most
of the brain during the course of imaging may introduce motion ar- common was caused by horizontal movement of the brain. When
tifacts, interfering with results. A third potential problem with the the brain shifted horizontally, so too did the glomeruli in the field of
fiber technique is that the stressful nature of the surgery may cause view. The movement was manifested in ratio images as a pattern of
the mice to experience stress, pain, or fear upon recovery. This may very dark and bright spots (Figure 3A). Another type of movement
cause them to alter their behavior in response, which could affect not artifact was caused by vertical movement of the brain. Vertical move-
only odor intake, but also neural response. While these are interest- ment caused glomeruli to come in and out of focus. If a glomerulus
ing behaviors that can be studied, too high a level of stress, pain, or came into focus, the ratio image would show a dark spot at the new
fear may limit other behaviors such as natural exploration. position (Figure 3B).
The first goal in the development of the technique was to repro- Another focusing problem that was caused by the size and shape
duce detailed images comparable to simple wide-field images. The of the bulb was simply maintaining the focus over a long time period.
concerns about pixelation and lack of optics mentioned earlier would Given the mobility of the bulb, it was hard to keep glomeruli in focus
affect the quality of the images. However, the fiber technique was suc- even for a day. By the time a mouse fully recovers, the clarity of an
cessful in this respect, and consistently produced clear images, often image may be lost (Figure 3C,D). While several glomeruli may still
showing clear glomeruli (Figure 2A). be visible, others will be lost. This is one reason to keep the surgery
The next important step was to be able to consistently see glom- and recovery as short as possible. Usually, an entire experiment can
erular odor responses. (For the rest of this paper, mentions of “glom- be finished within one day, preventing problems such as this.
erular responses” means a change in ORN pre-synaptic activity as a Glomerular pre-synaptic activity in anesthetized and behaving
result of odor stimulation.) This was also successful (Figure 2B-E), mice
but not as much as simply achieving clear images. While several ani- As discussed earlier, one of the fundamental concerns of this
mals showed clear responses, others did not. Often individual ani- study is to determine whether behavior has an effect on ORN output.
mals or even litters do not respond very well to odor stimulation for a To answer this question, images from three mice were analyzed. The
multitude of possible reasons such as sickness, obstruction of the na- images from these three mice were chosen because they were the only
sal passages, or other variables. Furthermore, the surgery to implant ones that matched a set of three criteria necessary to enhance the
the fiber bundle is very invasive, and often damage to the dura matter probability of getting reliable results. First, the images remained clear
of the brain could cause a failure to respond. This could be due to ac- in both the behaving and anesthetized trials. Second, clear glomeru-
tual damaging of the glomeruli or, more likely, due to decreased im- lar responses were observed. Finally, the animals were behaving rela-
age quality caused by dura matter damage. Finally, the bundle does tively normally after recovery and did not display signs of excessive
42 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 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
stress or pain. odor. Hence, this difference cannot be attributed to animal differenc-
The first characteristic of glomerular response studied was the pat- es or odor differences. Nevertheless, the differences between the two
tern of the responses. Glomerular activation patterns for individual conditions in both animals are strikingly large. Interestingly, it seems

Neuroscience
odors were compared in the same animal to determine if the patterns that the slopes all fall around 0.05 for both animals in the anesthe-
were similar in anesthetized and behaving animals. It was predicted tized conditions. By contrast, it seems that the variance comes mostly
that the patterns would be the same. While behavior may affect the from the behaving condition, which has a slope of only 0.025 in ani-
feedback loops that can modulate the signal, it seemed unlikely that mal 2 but approximately 0.1 in animal 3. There are several possible
behavior would alter odor-ORN binding affinities or the wiring of explanations for this. One possibility is that animal 3 may have had a
the ORN. In all three mice it was found that the patterns were con- higher sniffing rate than animal 2. It is possible that animal 3 found
served in behaving animals (Figure 4). isopropyl tiglate more pleasing than animal 2 found ethyl valerate.
The next property studied was whether or not the signal strength Animal 3 could have also been more exploratory or aware than ani-
was significantly different when the animal was awake as compared to mal 2, which may have not only increased its sniffing rate but also its
when the animal was anesthetized. It was predicted that there would mental awareness. A further study could test this theory by monitor-
be a difference, although whether it would be stronger or weaker was ing sniffing rate and comparing it to the slope of the response.
unknown. ΔF/F values were obtained for several responding glom- The final characteristic examined was rate of recovery from odor
eruli in each of the three animals and averaged over 10 trials. Then, stimulation. Only animal 2 was analyzed because the data from ani-
the mean values from the anesthetized and behaving conditions were mals 1 and 3 had too much noise or large motion artifacts during re-
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.
www.thurj.org 43
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


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

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.


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

Neuroscience
unchanged by the behavioral state. As discussed earlier, one of the not only increase the image quality by reducing cross-contamination
main reasons for looking at ORN synaptic output was because ORNs of light, but it would also allow the fiber to be placed above the bulb as
are more upstream than the rest of the olfactory system. Knowing opposed to directly on it. In addition, adding a mechanism by which
that odor responses do not appear to be globally modulated at the the fiber can be focused after a surgery is performed would decrease
level of the ORN, it is now possible to study the mitral cells to see if the number of experiments that fail because of bulb movement.
a global change occurs in that layer. If a difference is observed, one The preliminary results of this study inspire several future stud-
can more reliably attribute the change to mitral cell modulation as ies that can further explore some of the findings presented. Since the
opposed to a modification in ORN activity. One way to explore this results indicate that exploratory behavior, possibly sniffing rate, may
possibility would be to use transgenic mice expressing the calcium- 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 Volume 2 Issue 2 | Fall 2009

sniffing rate and glomerular response could shed some light on this Bouret, S., and Sarah, S.J. (2004). Reward expectation, orientation of atten-
tion and locus coeruleus-medial frontal cortex interplay during learning.
variation. One potential outcome is that sniffing rate may match European Journal of Neuroscience 20, 791-802.
closely with the level of fluorescence observed, which would explain Bozza, T., McGann, J.P., Mombaerts, P., and Wachowiak, M. (2004). In Vivo
why different mice had different odor responses while freely behav- Imaging of Neuronal Activity by Targeted Expression of a Genetically En-
ing. To further explore this, one could use calcium dyes to more coded Probe in the Mouse. Neuron 42, 9-21.
clearly pair sniffing with ORN output. While synaptopHluorin is a Carey, R.M., Verhagen, J.V., Wesson, D.W., Pirez, N., and Wachowiak, M.
(2009). Temporal structure of receptor neuron input to the olfactory bulb
good indicator of overall ORN activity, it cannot be used to image
imaged in behaving rats. Jorunal of Neurophysiology 101, 1073-1088.
individual ORN action potentials. By contrast, calcium dyes react Ferezou, I., Bolea, S., and Peterson, C.C.H. (2006). Visualizing the Cortical
quickly to ORN activity, and one can visualize events on a much Representation of Whisker Touch: Voltage-Sensitive Dye Imaging in Free-
shorter time scale, including individual action potentials. ly Moving Mice. Neuron 50, 617–629.
As discussed earlier, the biggest difference between the head-fixed Flusberg, B.A., Nimmerjahn, A., Cocker, E., Mukamel, E.A., Baretto, R.P.J.,
technique and the fiber bundle technique is that with a fiber bundle, Ko, T.H., Burns, L.D., Jung, J.C., and Schnitzer, M.J. (2008). High-speed,
miniaturized fluorescence microscopy in freely moving mice. Nature
mice can freely move and behave. Thus, it would be interesting to ex- Methods 5, 935-938.
amine whether the two techniques produce differences in glomerular Guerin, D., Peace, S.T., Didier, A,., Linster C., and Cleland, T.A. (2008). No-
response. Hypothetically, any significant differences can be attribut- radrenergic neuromodulation in the olfactory bulb modulates odor ha-
ed to exploratory behavior, and this may help to resolve whether the bituation and spontaneous discrimination. Behavioral Neuroscience 122,
differences seen in this study are attributable simply to being awake 816-826.
Issacson, J. (1999) Glutamate Spillover Mediates Excitatory Transmission in
or rather to being able to move around and freely explore the envi- the Rat Olfactory Bulb. Neuron 23, 377-384.
ronment. Isaacson, J., and Strowbridge, B.W. (1998). Olfactory Reciprocal Synapses:
Finally, one could explore the effect of training on odor responses. Dendritic Signaling in the CNS. Neuron 20, 749-761.
A basic study could compare mice that are previously exposed to Malnic, B., Junzo, H., Sato, T., and Buck, L. (1999). Combinatorial Receptor
the experimentation setup to naïve mice. It is conceivable that mice Codes for Odors. Cell 96, 713-723.
Miesenböck, G., DeAngelis, D.A., and Rothman, J.E. (1998). Visualizing se-
that trained in this fashion may reduce their exploratory behavior
cretion and synaptic transmission with pH-sensitive green fluorescent
because the environment they are experiencing is not a novel one. proteins. Nature 394, 192-195.
Another study could compare mice trained to a certain odor to mice Mombaerts, P. (2006). Axonal Wiring in the Mouse Olfactory System. Annual
that are just trained to general odor stimulation or even naïve mice. Review of Cell and Developmental Biology 22, 713-737.
This might also have an effect on how a mouse approaches and ex- Mombaerts, P., Wang, F., Dulac, C., Chao, S., Nemes, A., Mondelsohn, M.,
plores the different odors presented. Edmondson, J., and Axel, R. (1996). Visualizing an Olfactory Sensory
Map. Cell 87, 675-686.
This project is the first step towards understanding the effect of Ramirez, J., and Richter, D.W. (1996). The neuronal mechanisms of respira-
behavior on the olfactory bulb’s response to odor stimulation. While tory rhythm generation. Current Opinion in Neurobiology 6, 817-825.
the field is new and there is a large amount of unexplored territory, Rinberg, D., Koulakov, A., and Gelperin, A. (2006). Sparse Odor Coding in
hopefully this study will lay the groundwork for similar future stud- Awake Behaving Mice. Journal of Neuroscience 26, 8857-8865.
Neuroscience

ies. Given the promising results of this project, the fiber technique Shea S.D., Katz L.C., and Mooney R. (2008). Noradrenergic induction of
odor-specific neural habituation and olfactory memories. Journal of Neu-
has the potential to offer a way to understand the olfactory bulb in the roscience 28, 10711-10719.
larger context of the whole brain and even the entire body. Toga, A.W., and Mazziotta, J.C. (2002). Brain Mapping: The Methods. San
Diego: Academic Press.
References Tsuno, Y., Kashiwadani, H., and Mori, K. (2008) Behavioral State Regulation
of Dendrodendritic Synaptic Inhibition in the Olfactory Bulb. Journal of
Adrian, E.D. (1950). The Electrical Activity of the Mammalian Olfactory Neuroscience 28, 9227-9238.
Bulb. Electroencephalography and Clinical Neurophysiology 2, 377-388. Wachowiak, M., and Shipley, M.T. (2006). Coding and synaptic processing of
Albeanu, D.F. (2008) Precision and diversity in an odor map on the olfactory sensory information in the glomerular layer of the olfactory bulb. Semi-
bulb [Thesis]. Harvard University. 96pp. nars in Cell & Developmental Biology 17, 411-423.
Aungst, J.L., Heyward, P.M., Puche, A.C., Karnup, S.V., Hayar, A., Szabo, G., Wachowiak, M., Denk, W., and Friedrich, R.W. (2004). Functional organiza-
and Shipley, M.T. (2003). Center–surround inhibition among olfactory tion of sensory input to the olfactory bulb glomerulus analyzed by two-
bulb glomeruli. Nature 426, 623-629. photon calcium imaging. PNAS 101, 9097-9102.
Belluscio, L., and Cummings, D.M. (2008) Charting Plasticity in the Regen- Wilson, R. and Mainen, Z., (2006). Early Events in Olfactory Processing. An-
erating Maps of the Mammalian Olfactory Bulb. Neuroscientist 14, 251- nual Review of Neuroscience 29, 163-201.
263.

46 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


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

Neuroscience
ily have been found to perform crucial regulatory roles in nervous nal cord was observed in Bhlhb5 mutants when compared to that of
system development in a broad array of cellular types (Massari and wildtype controls (Figure 2). In addition, the corticospinal tract was
Murre, 2000). A particular BHLH gene of interest is Bhlhb5, which missing in Bhlhb5 mutants (Ross et al., unpublished data) (Figure
has been found to be transiently expressed in subpopulations of post- 3). The corticospinal tract (CST) consists of the bundle of neuronal
mitotic neurons throughout the central nervous system (Ross et al., fibers extending from the motor cortex of the brain into the spinal
unpublished data). These findings suggest that the Bhlhb5 transcrip- cord, and is therefore a key factor in regulating motor coordination
tion factor may have a regulatory role in cellular differentiation and in animals. The distinct absence of the CST in mutants thus indicates
maturation during neuronal development of key pathways necessary a potential role of Bhlhb5 in the formation of motor circuits in de-
for proper movement and sensory mechanisms. velopment. To identify the genes that are mis-expressed in Bhlhb5
Bhlhb5 mutants have phenotypes indicating deficits in the ner- mutants, Affymetrix array analysis examining RNA levels in Bhlhb5
vous system knockout versus wildtype was performed. Prdm8 was the most dra-
Investigators in the Greenberg Lab examined the potential role of matically misregulated gene (Sup. Figure 1) (Ross et al., unpublished
Bhlhb5 in neural development through the generation of a Bhlhb5 data). In fact, Prdm8 was found to be up-regulated significantly in
knockout mouse (Bhlhb5 -/-). They found that Bhlhb5 mutants ex- Bhlhb5 mutants (p<0.05) (Ross et al., unpublished data), thus sug-
hibited a series of physical and behavioral deficits (Figure 1: Supple- gesting that Bhlhb5 may directly repress the transcription of Prdm8
mentary Figures). Behavioral phenotypes observed include self injury (Figure 4: Supplementary Figures).
due to excessive spot-grooming and the subsequent development of Similarity of phenotypes in Bhlhb5 and Prdm8 mutants
open sores, or skin lesions (Figure 1A). Moreover, in behavioral tests The Affymetrix RNA array results from the Bhlhb5 mutants sug-
that evaluated sensitivity of Bhlhb5 mutants to chemical stimulation gested that Prdm8 may be an important transcriptional regulator in
through exposure to capsaicin, a natural irritant found in chili pep- the same neuronal pathways as that of Bhlhb5. A putative transcrip-
pers, Bhlhb5 mutants were found to demonstrate decreased response tion factor that is a member of the PR Domain family known for its
(measured by paw licking) to the harmful stimulant. Similarly, Bhl- histone methyl-transferase activity, Prdm8 has not been researched
hb5 mutants also exhibited decreased sensitivity to thermal stimu- broadly and little is known about its role in the central nervous sys-
lation as indicated by the decreased rate of paw withdrawal when tem.
placed on a hot plate (Ross et al., unpublished data). However, Prdm8 analysis in the retina was being explored by in-
An unusual behavioral phenotype observed in the Bhlhb5 mu- vestigators at the McInnes Lab; intriguingly, Prdm8 knockout mice
tants was a “handstand” behavior in which the mice walked forward exhibited similar behavioral phenotypes and deficits when compared
on their forelimbs, while retracting their hindlimbs to display a tran- to that of Bhlhb5 mutants (Sup. Figure 2). Like Bhlhb5 knockout
sient handstand posture (Figure 1C). Bhlhb5 mutants also displayed mice, the Prdm8 mutants also displayed the unique “handstand”
lack of motor coordination when compared to wildtype controls. posture during movement. Furthermore, Prdm8 mutants also devel-
www.thurj.org 47
RESEARCH Volume 2 Issue 2 | Fall 2009

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 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.
Figure 3. Bhlhb5 mutants are missing the corticospinal tract. A)
Neuroscience

PKC-gamma immunostaining in BHLHB5 wt mice marks the CST (indi- 3) To design a Prdm8 fusion protein construct for the develop-
cated by white arrow). B) PKC-gamma immunostaining in BHLHB5 mut ment of an antibody that accurately recognizes Prdm8 protein in
mice exhibit no expression of PKC-gamma, thus demonstrating loss of the
immunhistochemistry and immunoprecipitation experiments and
CST(idicated by white arrow). (Ross et al., unpublished data)
allows for visualization of endogenous Prdm8 protein in fixed tissue
oped extensive skin lesions like the Bhlhb5 mutants. The combina- and neural lysates.
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- Materials and Methods1
mon pathway with Bhlhb5 in the development of the central nervous
system in mice. Immunoprecipitation of Bhlhb5 or Prdm8 in transfected 293T
What is the mechanism through which Bhlhb5 and Prdm8 regu- cells
late transcriptional pathways such that removal of either factor in- Supernatant of 293T cell lysate was incubated with primary Bhl-
duces the same phenotypic deficits in knockout mice? I propose three hb5 (rat) antibody (or anti-Myc epitope (mouse)) antibody. Blocked
models of interaction to answer this question (Figure 4). Protein-A agarose beads were added to the cell lysate supernatant,
Three chief objectives were addressed: 1) Which model of interac- pelleted from the cell lysate, and washed. Bhlhb5 protein (or Prdm8
tion between Bhlhb5 and Prdm8 is best supported by the observed protein) was then precipitated and bound to the beads through pull-
data? In Figure 8, several models of interaction that may characterize down.
the true collaborative function of Bhlhb5 and Prdm8 in neuronal de- Design and generation of Prdm8-directed antibody
velopment are presented. Is there evidence to support the hypothesis Multiple Prdm8 GST-fusion protein constructs were devised that
that Bhlhb5 and Prdm8 regulate the same target genes, yet do not consisted of all 6 combinations of 3 highly conserved domains in the
bind together directly in complex (Figure 4A)? Do they interact di- full length Prdm8 sequence (labeled as Domain 1, Domain 2, and
rectly with each other to regulate the same gene targets (Figure 4B)? Domain 3). Primer-targeted PCR amplification was conducted, trans-
Or, do Prdm8 and Bhlhb5 each regulate distinct groups of genes that forming constructs into competent cells, which were maxiprepped
are critical mediators of a common pathway (Figure 4C)? and sequenced. Constructs were then digested with restriction en-
2) Is there evidence to suggest a mechanism of interaction between zymes, ligated into pGEX vectors, and transformed the vectors into
Bhlhb5 and Prdm8 in the regulation of processes important neuronal Top10 Competent Cells. The researcher then cultured in high volumes
development? Do protein expression patterns of Bhlhb5 and Prdm8 the correctly transformed GST-PRDM8 constructs, maxiprepped,
in various regions of the brain and spinal cord indicate that these two and then transformed them into BL21 (E. coli) cells.
factors may be crucial in the same neurons during differentiation and 1
(see Supplementary Materials for full Materials and Methods)
development?
48 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

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. Figure 5. Western blot of immunoprecipitation conducted in
Western blot analysis of immunoprecipitation results 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
Lysates from the immunoprecipitation experiments were analyzed
is observed. Second lane displays IP for PRdm8 via its myc epitope tag,
via SDS-PAGE and western blot using Prdm8 and Bhlhb5-specific
and no co-IP of Bhlhb5 is observed. Bottom half of blot(divded by dotted
antibodies. Prdm8 rabbit antibody was used to probe the upper half line) was blotted with antibody for Bhlhb5, upper half of blot probed with
of the blot (Prdm8 protein band is approximately 72 kDa in size in antibody for Myc-epitope tag of Prdm8.
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 In order to conduct immunoprecipitation experiments on endog-
was used. Bhlhb5 rabbit antibody was used to probe the lower half of enous Bhlhb5 and Prdm8 protein from neurons, use of a Prdm8 an-
the blot (Bhlhb5 protein is approximately 35 kDa in size). tibody was required. Due to the lack of available Prdm8 antibody,
Immunohistochemistry analysis however, a series of Prdm8 GST-Fusion protein constructs were de-
To analyze the cell-specific expression patterns of Prdm8 and Bhl- signed and developed. Single constructs to be purified were selected

Neuroscience
hb5 in cortical and spinal cord tissue sections, immunohistochem- and injected into rabbits for Prdm8 antibody production.
istry analysis was performed on fixed tissue. Whole specimens were Sup. Figure 3 exhibits the design of the 6 different protein domains
frozen in blocks of optimum cutting temperature (OCT) formula, cloned into vectors with GST to create 6 individual GST-fusion pro-
and sagittal and coronal sections 20 microns thick were collected tein constructs. Sup. Figure 4 displays the induction results for each
utilizing a cryostat, and the sections were transferred onto slides. Im- of the Prdm8 GST-Fusion protein constructs in which the darkest
munostaining analysis was conducted to visualize proteins of inter- band in each lane corresponds to the appropriate sized protein band
est. 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
Results during the protein purification process. This region (termed Domain
2) was selected as the Prdm8 antigen to be injected into rabbits for
No co-immunoprecipitation of Bhlhb5 or Prdm8 in 293T cells Prdm8 antibody production.
To test the hypothesis of whether or not any evidence of protein- Designed antibody recognizes Prdm8 protein in neurons
protein interaction between Bhlhb5 and Prdm8 could be observed, To test for specificity and strength of the developed Prdm8 anti-
immunoprecipitation experiments were conducted. In this experi- body to recognize endogenous Prdm8 in neural lysate, an experimen-
ment, the researcher performed antibody-specific pull-down of a tal western blot on a wildtype whole mouse cortex and one mutant for
particular protein (Bhlhb5) via adhesion to beads and precipitation Prdm8 was conducted (Sup. Figure 5). Prdm8 antibody derived from
from the whole cell lysate. The precipitate was probed with antibod- the serum of a rabbit injected with purified antigen (derived from the
ies specific for its partner protein (Prdm8) to observe for any co-im- Prdm8 Domain 2 GST-Fusion protein construct) was used to probe
munoprecipitation. Through western blot analysis (Figure 5), no evi- for the presence of Prdm8 protein in the neural lysate. The Prdm8
dence was found of a direct interaction between Bhlhb5 and Prdm8. antibody accurately identified the Prdm8 protein in the neurons as
Antibody-specific immunoprecipitation of the Bhlhb5 protein was predicted. This finding was verified in the western blot through the
observed, and any associated Prdm8 pull down via probe identifica- observation of a ~72 kDa protein band, which corresponds to the ap-
tion of the Myc-epitope tagged to the C-terminal of the PRDM8 pro- proximate size of endogenous Prdm8 in neurons.
tein was analyzed. As seen in Figure 9, there is no observable protein No co-immunoprecipitation of Bhlhb5 or Prdm8 in neurons
band at approximately 98 kDa in the lane containing the precipitate Unlike the previous experiment performed in which 293T cells
pulled down via Bhlhb5 interaction. Conversely, immunoprecipi- transfected with Bhlhb5 and Myc-Prdm8, this immunoprecipitaton
tation of Prdm8 via its Myc-epitope tag exhibited no pull-down of experiment was performed on proteins expressed from endogenous
Bhlhb5 in the precipitate (which would have been indicated by a band genes in neural lysate drawn from the whole cortex of Prdm8 wild-
~37 kDa in size). type and mutant mice. The advantage of this protocol is that any as-
Design and development of Prdm8 GST-fusion protein constructs sociated co-factors necessary to facilitate proper Bhlhb5 and Prdm8
www.thurj.org 49
RESEARCH Volume 2 Issue 2 | Fall 2009

Figure 6. Western blot of BHLHB5 immunoprecipitation con- Figure 7. Western blot of PRDM8 immunoprecipitation conduct-
ducted in neural lysate. Neural lysate from whole cortex of both ed in neural lysate. Neural lysate from whole cortex of both Prdm8 wt
PRDm8 wt and Prdm8 mut P0 mice were utilized to IP for Bhlhb5 (both and Prdm8 mut P0 mice were utilized to IP for PRDM8. First and second
BHLhb5 wt). First and second lane display neural lysate (1% NP-40 lysis lanes display neural lysate (SDS lysis buffer) of Prdm8 wt and Prdm8 mut
buffer) of Prdm8 mut and Prdm8 wt mice, respectively. Second lane clearly mice (both Bhlhb5 wt), respetively. Presence of BHLHB5 protein in these
indicates the presence of PRDM8 protein in the neural lysate prior to the lysates is observed by the dark band at the 35kDa mark in each lane. Third
IP. Third and fourth lanes indicate IP for BHlhb5 in both Prdm8 mut and and fourth lanes indicate IP for PRDM8 in both Prdm8 wt and Prdm8
Prdm8 wt mice, respectively. No evidence of co-IP of Prdm8 is observed mut mice, respectively. No evidence of co-IP of BHLHB5 is observed in
in the Prdm8 wt neurons. the Prdm8 wt neurons.

protein interaction would also be available in the neural lysate to expressing neurons in the dorsal horn of the spinal cord.
maintain natural protein-protein interactions (such would not be To test the first hypothesis, immunohistochemistry was per-
possible in 293T cells). formed to compare Prdm8 expression in neurons in the dorsal horn
In this experiment, antibody-specific pull-down of a particular of the spinal cord between wildtype controls and Bhlhb5 knockout
protein (i.e. Bhlhb5) was conducted via adhesion to beads and pre- mice at P0. A significant loss of Prdm8-expressing neurons in the dor-
Neuroscience

cipitation from the whole cell lysate. The precipitate with the anti- sal horn of the spinal cords of Bhlhb5 mutants was detected(Figure
body specific for the partner protein (i.e. Prdm8) was then probed 8A). The mean number of Prdm8-expressing neurons located in the
to look for any co-immunoprecipitation. As indicated in Figure 6, dorsal horn of the spinal cord in Bhlhb5 mutants and controls were
there is no protein band at approximately 72 kDa (MW of Prdm8 quantified to determine if a statistically significant difference ex-
protein) in the lane that indicates the precipitate pulled down via isted. A two-tailed T-test with a confidence value of 95% (p-value =
Bhlhb5 interaction. Since Prdm8 is evident in the wildtype whole 0.0012) (Table 1) was utilized to perform the statistical analysis. A
cell lysate when compared to the Prdm8 mutant control, endogenous significant decrease in Prdm8-expressing neurons in Bhlhb5 mutant
Prdm8 is clearly present prior to immunoprecipitation. No Prdm8 mice as compared to those of Bhlhb5 wildtype controls was observed.
was co-immunoprecipitated with Bhlhb5 in neurons. Similarly, im- Moreover, the Prdm8-expressing neurons were lost to the outer-most
munoprecipiation was performed for Prdm8 and no associated pull- laminae (superficial region) of the spinal cord dorsal horn.
down of Bhlhb5 in the precipitate was found (Figure 7). Co-expression of Bhlhb5 and Prdm8 in spinal cord dorsal horn
Prdm8-expressing cells lost in the dorsal horn of spinal cord of To test the second hypothesis, immunostaining was conducted for
Bhlhb5 mutants Bhlhb5 and Prdm8 in the spinal cord of Prdm8 wildtype controls
In previous research, Bhlhb5 mutants exhibited distinct pheno- and mutants at P0. Wildtype mice exhibited high levels of Bhlhb5
types that included skin lesion development, handstand behavior, and Prdm8 co-expression in the dorsal horn region of the spinal cord
decrease in motor coordination, and decrease in sensitivity to ther- (indicated by white dotted lines in Sup Figure 6A).
mal or chemical stimulation (Ross et al., unpublished data). When Co-expression of both transcription factors (indicated in the
examined at the cellular level, Bhlhb5 mutants also exhibited a sig- “merged” panels of Sup. Figures 6A and 6B, where neurons express-
nificant loss of Bhlhb5-expressing neurons in the superficial layers ing both Bhlhb5 and Prdm8 appear purple or blue-green in color,
of the dorsal horn of the spinal cord (Ross et al., unpublished data). respectively) was detected. Interestingly, in the analysis of immunos-
Recent findings that Prdm8 mutants indicate striking similarities taining for Bhlhb5 and a GFP marker for Prdm8-expressing neurons
comparable to those of Bhlhb5 mutants (i.e. skin lesion development in Prdm8 mutants, a qualitative loss was observed of Bhlhb5 and
and handstand behavior) (McInnes, unpublished data) suggested the Prdm8 co-expressing neurons in the dorsal horn on the spinal cord
theory that Bhlhb5 and Prdm8 may function using similar regula- (Sup. Figure 6B).
tory pathways of the central nervous system. As a result, it is possible Co-expression of Bhlhb5 and Prdm8 in the frontal cortex
that these two transcription factors are expressed in the same neu- Due to the similarity in phenotypes observed in both Bhlhb5 and
rons during development. This reasoning led to the research hypoth- Prdm8 mutants (both behaviorally and at the cellular level in the spi-
esis that: 1) Bhlhb5 mutants also exhibit significant loss of Prdm8- nal cord), there is another key hypothesis. The third hypothesis is
expressing neurons in the superficial layers of the dorsal horn of the that high levels of co-expression of both Bhlhb5 and Prdm8 would
spinal cord, 2) Prdm8 mutants exhibit the loss of Prdm8 and Bhlhb5 also be observed in neurons located in the cortex where important
50 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

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.
Figure 9. PRDM8 and BHLHB5 immunostaining in cortex. A)
Immunostaining analysis of PRDM8 expression (red) and BHLHB5 ex-
neuronal components of sensory and motor circuits are derived and pression (blue) in the frontal cortex of wildtype mice (P0). High levels
send projections throughout the body. Immunostaining was con- of PRDM8 and BHLHB5 co-expression expression exhibited in the outer
ducted for Prdm8 and Bhlhb5 in sagittal sections of wildtype mice layers of the cortex (shown as purple neurons in enlarged box). B) Im-
frontal cortex, which indicated high levels of co- expression for both munostaining analysis of PRDM8-expressing neurons via GFP knockin
proteins in populations of neurons at P0 (Figure 9). Figure 9A ex- at Prdm8 locus (green) and BHLHB5 expression (blue) in the frontal cor-
hibits immunostaining for Bhlhb5 and Prdm8 in a wildtype control tex of PRDM8 mutant (-/-) mice (P0). Large population of neurons co-
brain and Figure 9B exhibits immunostaining for Bhlhb5 and Prdm8 expressing PRDM8 and BHLHB5 also exhibited in the outer layers of the
in a Prdm8 mutant cortex. Enlarged boxes of each panel in Figure 9 cortex (shown as blue-green neurons in enlarged box).
demonstrate the co-expression of both proteins in the same neurons
in wildtype and Prdm8 mutants. In the analysis of the immunostain- cospinal tract (Moreno-Flores et al., 2006), which is composed of a
ing, no qualitative difference between Bhlhb5 and Prdm8 expression bundle of fibers derived from neurons originating in the motor cor-
patterns in the cortex was observed. Nevertheless, high levels of co- tex of the brain (Purves, 2001). In the analysis of the immunostaining
expression was localized near the outermost layers of the frontal cor- experiment, the CST was found to be lacking in Prdm8 mutants (Sup.
tex of both wildtype and Prdm8 mutants. Figure 7C).
Prdm8 mutants and double heterozygotes are missing the CST The observation regarding the absence of the CST in Prdm8 mu-

Neuroscience
In previous research, the observation that both Bhlhb5 and Prdm8 tants is identical to evidence demonstrating that Bhlhb5 mutants are
mutants demonstrate similar behavioral phenotypes (i.e. develop- also missing the corticospinal tract, established through research
ment of skin lesions, handstand behavior) (Ross et al., unpublished indicating that corticospinal axons may mis-project or stop growth
data) led to the overarching theory that Bhlhb5 and Prdm8 may be prematurely and do not reach targets in the dorsal spinal cord (Ross
two transcriptional regulators involved in common regulatory pro- et al., unpublished data). Therefore, the combination of past research
cesses in neuronal development. This conceptual idea thus led to the exhibiting the absence of the corticospinal tract in Bhlhb5 mutants
reasoning that the phenotypes observed in Prdm8 mutants mirror- with this project’s findings that Prdm8 mutants may also lack the
ing that of Bhlhb5 mutants would also extend to the cellular level corticospinal tract further support the overarching hypothesis that
throughout the central nervous system. Therefore, based on the find- Prdm8 and Bhlhb5 are both crucial factors in the development of cir-
ing that Bhlhb5 mutants exhibit distinct loss of the corticospinal tract cuit formation in the central nervous system. Intriguingly, it was also
(CST) (Figure 3), Prdm8 mutants might also exhibit loss of the CST. noted that double heterozygous mice for Prdm8 and Bhlhb5 (Prdm8-
To test this hypothesis, immunohistochemistry analysis of spinal /+ Bhlhb5 -/+) also indicate loss of the CST through the absence of
cord sections of wildtype and Prdm8 mutant mice was performed PKC-gamma protein expression in spinal cord sections at P0 (Sup.
at P0. In the immunostaining analysis, the presence of the CST was Figure 7B). However, this observation regarding the loss of the CST
assessed through analyzing sectioned spinal cord tissue with a probe in heterozygous animals is not observed in animals heterozygous for
that recognizes a protein marker, Protein Kinase C-gamma (PKC- only one gene.
gamma), for the CST. PKC-gamma is highly expressed in the corti-

Table 1. PRDM8 mutants exhib-


Mean # PRDM8- Mean # PRDM8-
it signifiacnt loss of PRDM8-ex-
BHLHB5 WT BHLHB5 MUT expressing cells in expressing cells in 2-Tailed T Test P-
pressing neurons in dorsal horn
Sample Size Sample Size PRDM8 WT PRDB8 Mut Value*
of spinal cord. 2-Tailed statistical
(St. Dev) (ST. Dev)
test (*2 sample unequal variance het-
eroskedastic) comparing te number
of PRDM8-expressing neurons be-
tween Bhlhb5 mut and wt mice in-
56 Neurons 28 Neurons dicate a statistically significant differ-
N=20 N=20 (19.31608) (7.483314) 0.0011548 ence (p-value<0.05, confidence value
of 95%).

www.thurj.org 51
RESEARCH Volume 2 Issue 2 | Fall 2009

Discussion significantly lost from populations of neurons in the superficial layers


of the dorsal horn of the spinal cord (Ross et al., unpublished data),
No evidence of Bhlhb5 or Prdm8 binding in 293T cells combine to support the primary hypothesis: that Prdm8 and Bhlhb5
Results from this experiment (Figure 9) did not present any evi- may be important regulatory factors in the same pathways in central
dence of co-IP. Conversely, when immunoprecipitation for Prdm8 nervous system development. With evidence from past research that
was conducted, no presence of Bhlhb5 was observed in the precipi- Bhlhb5-expressing neurons are lost in the dorsal horn of the spinal
tate. These results suggest that Bhlhb5 and Prdm8 do not interact, cord, this finding that Prdm8-expressing neurons are also lost in the
and therefore provide evidence in support of models in the Figures same region of the spinal cord suggested that Prdm8 and Bhlhb5 may
4A and 4C. However, this experiment was conducted in transfected be highly expressed in the same neurons.
293T cells, where many endogenous factors necessary for regulato- High levels of co-expression of Prdm8 and Bhlhb5 in dorsal horn
ry processes in neurons are absent. It is possible that other proteins of spinal cord
present in neurons but absent in 293T cells are necessary co-factors to Due to the finding that a significant loss of Prdm8-expressing
facilitate the complex formation of Bhlhb5 and Prdm8. neurons was observed in the same regions of the dorsal spinal cord
The immunoprecipitation experiment was replicated in neurons where a significant loss of Bhlhb5-expressing neurons are absent in
where pull-down of endogenous Prdm8 and Bhlhb5 in their natural Bhlhb5 mutants, it was asked whether or not Bhlhb5 and Prdm8 co-
substrate environments could be observed. To conduct this experi- expression would also be observed in the dorsal horn of the spinal
ment in neurons, a direct Prdm8 antibody was required. The previous cord. Sup. Figure 6A depicts immunostaining results from a wildtype
immunoprecipitation experiment conducted in 293T cells utilized a mouse spinal cord. A high degree of co-localization of Prdm8 and
Myc-epitope tagged construct of Prdm8. Thus, immunoprecipitation Bhlhb5 protein expression in the dorsal horn of the spinal cord was
and probing for Prdm8 in 293T cells could be performed via the use observed. Furthermore, when the wildtype immunostaining results
of an anti-Myc antibody (rather than a direct antibody for Prdm8). A were compared to those from Prdm8 mutant spinal cord, a qualita-
chief obstacle in this study that arose was the lack of available Prdm8 tive loss was determined of neurons co-expressing both Prdm8 and
antibody to use in immunoprecipitation and immunohistochemistry Bhlhb5 in the dorsal horn of the spinal cord (Sup. Figure 6B). The
experiments on neurons and fixepd tissue. finding that Prdm8 and Bhlhb5 are both highly expressed in the same
Design and development of the Prdm8 antibody neurons thus provides further evidence in support of the hypothesis
To resolve the issue of the absence of a Prdm8 antibody, a series of that Bhlhb5 and Prdm8 are involved in the same pathways in nervous
Prdm8 GST-Fusion protein constructs was designed and developed system development.
(Supp. Figures 3 and 4), from which a purified Prdm8 antigen could Due to the lack of available Prdm8 mutant mice, there was not
be selected and injected into rabbits for production of an antibody. a sufficient sample size to conduct a quantitative statistical analysis
No evidence of endogenous Bhlhb5 and Prdm8 binding in neu- to determine the significance of this loss of neurons co-expressing
rons Prdm8 and Bhlhb5 in the dorsal horn of the spinal cord. Future ex-
Immunoprecipitation for Bhlhb5 in Prdm8 wildtype and mutant periments, however, should call for a quantification of the number of
Neuroscience

neurons from the whole cortex of mice demonstrated clear pull- co-expressing neurons in wildtype versus Prdm8 and Bhlhb5 mutant
down of the Bhlhb5 protein, yet no evidence of any Prdm8 was found mice, and statistical calculation to test for significance in difference
in the precipitate (Figure 6). Similarly, no co-IP of Bhlhb5 was ob- between the two groups.
served when pull-down for Prdm8 was conducted (Figure 7). Again, High levels of co-expression of Prdm8 and Bhlhb5 in frontal
these results suggested that Bhlhb5 and Prdm8 do not interact, and cortex
therefore provided evidence in support of models in the Figures 4A Due to the high levels of observed Bhlhb5 and Prdm8 co-expres-
and 4C. sion in the spinal cord, in addition to the underlying theory that
Nevertheless, it is also possible that conditions of the immunopre- these two genes are crucial in the development of the central nervous
cipitation experiment itself may have contributed to the dissociation system, Bhlhb5 and Prdm8 expression patterns in the mouse brain
of proteins in complex from one another. Considering the multiple were analyzed. Immunostaining analysis was conducted for Bhlhb5
steps involved in immunoprecipitation (i.e. washing of the beads, and Prdm8 on sagittal sections of the mouse cortex. Expression was
incubation of the antibody, and incubation with beads), it is very observed for each of these proteins in cortical neurons, and for any
plausible that the nature of the protein-protein interaction between distinguishing features between Bhlhb5 and Prdm8 expression pat-
Bhlhb5 and Prdm8 may be very sensitive to the concentration of the terns in wildtype versus Prdm8 mutant P0 mice. From the results
lysis buffer ingredients, and that such may have affected the natural (Figure 9), high levels of Prdm8 and Bhlhb5 co-expression in the cor-
affinity between Bhlhb5 and Prdm8. Future experiments to clarify tex was found. The qualitative analysis further determined that the
the interactive relationship between Bhlhb5 and Prdm8 should there- areas of greatest co-expression were the outermost layers of the fron-
fore call for a series of tests examining the effects of different lysis tal cortex. Qualitative examination of Bhlhb5 and Prdm8 expression
buffer ingredients, concentrations, and wash conditions on the im- in the frontal cortex between wildtype and Prdm8 mutant mice did
munoprecipitation and co-immunoprecipitation of proteins. not reveal any outstanding disparities in expression patterns. Nev-
Significant loss of Prdm8-expressing neurons in Bhlhb5 mutant ertheless, further analysis following the availability of more Prdm8
spinal cord mutant mice would be necessary to examine critically the differences
To further explore the relationship between Bhlhb5 and Prdm8 in in Prdm8 and Bhlhb5 expression patterns in the cortex of mutant
neural development, the expression patterns of each protein in both mice.
Bhlh5 and Prdm8 mutants were characterized. Immunohistochemis- Prdm8 mutants and Prdm8-Bhlhb5 heterozygotes exhibit loss of
try analysis was conducted on Bhlhb5 mutant mice, and a significant CST
loss of Prdm8 was observed in the dorsal horn of the spinal cord at P0. From the finding that Bhlhb5 mutants exhibit distinct loss of the
The researcher confirmed this observation through immunostaining corticospinal tract (CST) (Figure 3), it was hypothesized that Prdm8
analysis (Figure 8A) and statistical quantification (Table 1, Figure mutants would also exhibit loss of the CST. Immunostaining analy-
8B). These results, in conjunction with past research that Bhlhb5 is sis was conducted utilizing a CST marker (PKC-g) (Sup. Figure 7C),
52 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

which indicates the distinct loss of the CST in Prdm8 mutants. This neurons co-expressing Prdm8 and Bhlhb5 is observed in the dorsal
result supportes the hypothesis that Prdm8 and Bhlhb5 mutants are horn of the spinal cord, and 4) loss of the CST is observed in Prdm8
both missing the CST and further provides evidence in support of the mutants and double heterozygous mice (Bhlhb5 het, Prdm8 het). The
theory that Prdm8 and Bhlhb5 are crucial in the development of neu- combination of these four findings demonstrate that Prdm8 mutants,
rons involved in the same pathways of the central nervous system. like Bhlhb5 mutants, express similar Prdm8 and Bhlhb5 expression
More intriguingly, however, the loss of the CST in double heterozy- patterns in the brain and spinal cord. This study additionally sug-
gous mice (Bhlhb5 -/+ Prdm8 -/+) (Sup. Figure 7B) was noted. This gests that the missing subpopulation of neurons in the dorsal horn of
finding is particularly striking, since no loss of the CST was observed the spinal cord co-express both Bhlhb5 and Prdm8, thus providing
in mice heterozygous for only one gene (i.e. Bhlhb5 -/+ Prdm8 +/+ evidence in support of the hypothesis that Bhlhb5 and Prdm8 are
or Bhlhb5 +/+ Prdm8-/+). Therefore, the finding that a heterozygous crucial factors in a common pathway underlying neural differentia-
copy of both Prdm8 and Bhlhb5 could result in the absence of the tion and development.
CST, a phenotype found only in Bhlhb5 or Prdm8 full knockouts, Technical difficulties encountered included the lack of available
suggestes that Bhlhb5 and Prdm8 may have an additive impact where mice of desired Prdm8 and Bhlhb5 genotypes. With a greater number
the absence of a single copy of both Prdm8 and Bhlhb5 in mice may of Prdm8 mutants, double heterozygotes, and double mutants, more
be equivalent in phenotype to missing both copies of either gene. The immunostaining experiments could be conducted to discern for any
discovery of the absence of the CST in double heterozygous mice is nuances in phenotype that distinguish Bhlhb5 mutants from Prdm8
exceptionally intriguing and prompts the hypothesis that such mice mutants. Furthermore, the availability of more mice of desired mu-
will also exhibit other identical phenotypes at the cellular (i.e. sig- tant genotypes would allow for statistical analysis to be performed
nificant loss of Bhlhb5 or Prdm8-expressing neurons) and behavioral so that the significance of Bhlhb5 and Prdm8 co-expression may be
(i.e. skin lesion, handstand) levels. This hypothesis thus calls for fu- calculated.
ture experiments to explore the phenotypes of double heterozygous In sum, the overarching motivation that propelled this research
mice through a series of behavioral assays (i.e. rotarod balance exper- was the pursuit of a better understanding of the processes underly-
iments, observation of self-injurious behavior, thermal and chemical ing central nervous system development in mammals. The striking
stimulation assays) and examination of cellular expression patterns phenotypes exhibited by both Bhlhb5 and Prdm8 mutant mice was
(i.e. analysis of Bhlhb5 and Prdm8-expressing neurons in the spinal the first signal that a cooperative relationship may exist between the
cord and brain) and comparing such results to those of Prdm8 or two proteins, and that their function was critical in neural develop-
Bhlhb5 single gene knockout mice. ment. These results provide support for the view that a novel relation-
ship between Bhlhb5 and Prdm8 exist in the same neural pathways;
Future Directions they also help illustrate a process within the formation of the central
nervous system that have significance for the future understanding
Although results gathered in this study does not suggest direct of not only how the mammalian nervous system develops, but how it
interaction between Bhlhb5 and Prdm8, it is still not clear what the matures and ages with time.

Neuroscience
precise nature of their regulatory relationship may be. Three poten-
tial models of interaction were proposed, of which one (Figure 4B) References
was not supported by the immunoprecipitation results. However, two
Alberts, B.J., Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter,
other models (Figures 4A and 4C) are possible. Therefore, a future di- Peter, ed. (2002). Molecular Biology of the Cell (New York).
rection of this study would be to distinguish between the two mecha- Ancelin, K., Lange, U.C., Hajkova, P., Schneider, R., Bannister, A.J., Kouza-
nisms or identify other alternative models that may best describe the rides, T., and Surani, M.A. (2006). Blimp1 associates with Prmt5 and di-
relationship between Bhlhb5 and Prdm8. rects histone arginine methylation in mouse germ cells. Nature Cell Biol-
ogy 8, 623-630.
Andersson, E., Jensen, J.B., Parmar, M., Guillemot, F., and Bjorklund, A.
Conclusions (2006). Development of the mesencephalic dopaminergic neuron system is
compromised in the absence of neurogenin 2. Development 133, 507-516.
The major finding in this study was that no indication of direct Bertrand, N., Castro, D.S., and Guillemot, F.o. (2002). Proneural genes and
interaction between Bhlhb5 and Prdm8 proteins was observed. The the specification of neural cell types. Nature Reviews Neuroscience 3, 517-
models in Figures 8A and 8C are potential mechanisms of interaction 530.
Bramblett, D.E., Pennesi, M.E., Wu, S.M., and Tsai, M.-J. (2004). The Tran-
between Bhlhb5 and Prdm8 in transcriptional regulation. In these
scription Factor Bhlhb4 Is Required for Rod Bipolar Cell Maturation.
models, Bhlhb5 and Prdm8 do not bind to each other in complex, Neuron 43, 779-793.
targeting either the same genes (Figure 4A) or distinct genes in the Davis, C.A., Haberland, M., Arnold, M.A., Sutherland, L.B., McDonald,
same pathway (Figure 4C). Nevertheless, due to the nature of the O.G., Richardson, J.A., Childs, G., Harris, S., Owens, G.K., and Olson,
experimental conditions and the possible sensitivity of the proteins’ E.N. (2006). PRISM/PRDM6, a transcriptional repressor that promotes
the proliferative gene program in smooth muscle cells. Mol Cell Biol 26,
affinities for each other, it is necessary that further experiments be
2626 - 2636.
conducted before the model in which Prdm8 and Bhlhb5 interact in Derunes, C., Briknarová, K., Geng, L., Li, S., Gessner, C.R., Hewitt, K., Wu,
complex (Figure 4B) may be rejected. S., Huang, S., Woods Jr, V.I., and Ely, K.R. (2005). Characterization of the
Despite the lack of evidence identifying the precise model of inter- PR domain of RIZ1 histone methyltransferase. Biochemical and Biophysi-
action between Bhlhb5 and Prdm8, immunostaining results provide cal Research Communications 333, 925-934.
strong evidence in support of the overarching theory that Bhlhb5 and Dimou, L., Simon, C., Kirchhoff, F., Takebayashi, H., and Gotz, M. (2008).
Progeny of Olig2-Expressing Progenitors in the Gray and White Matter of
Prdm8 function as important regulatory factors in the same pathway the Adult Mouse Cerebral Cortex. J. Neurosci. 28, 10434-10442.
of neuronal development in the central nervous system. This con- Djaldetti, R., Shifrin, A., Rogowski, Z., Sprecher, E., Melamed, E., and Yar-
clusion was drawn from four critical findings: 1) Prdm8-expressing nitsky, D. (2004). Quantitative measurement of pain sensation in patients
neurons are significantly lost in the dorsal horn of the spinal cord in with Parkinson disease. Neurology 62, 2171-2175.
Bhlhb5 mutants, 2) high levels of co-expression of Prdm8 and Bhlhb5 Elworthy, S., Hargrave, M., Knight, R., Mebus, K., and Ingham, P.W. (2008).
Expression of multiple slow myosin heavy chain genes reveals a diversity
are observed in the dorsal horn of the spinal cord, 3) dramatic loss of
www.thurj.org 53
RESEARCH Volume 2 Issue 2 | Fall 2009

of zebrafish slow twitch muscle fibres with differing requirements for Marsden, C.D. (1994). Parkinson’s disease. J Neurol Neurosurg Psychiatry 57,
Hedgehog and Prdm1 activity. Development 135, 2115-2126. 672-681.
Feng, L., Xie, X., Joshi, P.S., Yang, Z., Shibasaki, K., Chow, R.L., and Gan, L. Massari, M.E., and Murre, C. (2000). Helix-Loop-Helix Proteins: Regulators
(2006). Requirement for Bhlhb5 in the specification of amacrine and cone of Transcription in Eucaryotic Organisms. Mol. Cell. Biol. 20, 429-440.
bipolar subtypes in mouse retina. Development 133, 4815-4825. Moreno-Flores, M.T., Bradbury, E.J., Martin-Bermejo, M.J., Agudo, M., Lim,
Fumasoni, I., Meani, N., Rambaldi, D., Scafetta, G., Alcalay, M., and Cicca- F., Pastrana, E., Avila, J., Diaz-Nido, J., McMahon, S.B., and Wandosell,
relli, F. (2007). Family expansion and gene rearrangements contributed to F. (2006). A Clonal Cell Line from Immortalized Olfactory Ensheathing
the functional specialization of PRDM genes in vertebrates. BMC Evolu- Glia Promotes Functional Recovery in the Injured Spinal Cord. Mol Ther
tionary Biology 7, 187. 13, 598-608.
Hamik, A., and Jain, M.K. (2008). Variety is the splice of life. Journal of Mo- Nieto, M., Schuurmans, C., Britz, O., and Guillemot, F. (2001). Neural bHLH
lecular and Cellular Cardiology 44, 44-46. Genes Control the Neuronal versus Glial Fate Decision in Cortical Pro-
Hand, R., Bortone, D., Mattar, P., Nguyen, L., Heng, J.I.-T., Guerrier, S., genitors. Neuron 29, 401-413.
Boutt, E., Peters, E., Barnes, A.P., Parras, C., et al. (2005). Phosphoryla- Ohinata, Y., Payer, B., O’Carroll, D.n., Ancelin, K., Ono, Y., Sano, M., Barton,
tion of Neurogenin2 Specifies the Migration Properties and the Dendritic S.C., Obukhanych, T., Nussenzweig, M., Tarakhovsky, A., et al. (2005).
Morphology of Pyramidal Neurons in the Neocortex. Neuron 48, 45-62. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature
Hughes, S.M. (2004). Muscle Differentiation: A Gene for Slow Muscle? Cur- 436, 207-213.
rent Biology 14, R156-R157. Purves, D., Augustine, J., Fitzpatrick, D., Katz, L. C., LaMantia, A. S., Mc-
Jiang, X., Tian, F., Du, Y., Copeland, N.G., Jenkins, N.A., Tessarollo, L., Wu, Namara, J. O., and Williams, S. M. (2001). Neuroscience, Vol 2nd Edition
X., Pan, H., Hu, X.-Z., Xu, K., et al. (2008). BHLHB2 Controls Bdnf Pro- (Sunderland, MA: Sinauer Associates).
moter 4 Activity and Neuronal Excitability. J. Neurosci. 28, 1118-1130. Ren, B., Chee, K., and Kim, T.H. (1999). PRDI-BF1/Blimp-1 repression is
Kajimura, S., Seale, P., and Tomaru, T. (2008). Regulation of the brown and mediated by corepressors of the Groucho family of proteins. Genes and
white fat gene programs through a PRDM16/CtBP transcriptional com- Development 13, 125-137.
plex. Genes and Development 22, 1397-1409. Ross, S.E., Greenberg, M.E., and Stiles, C.D. (2003). Basic Helix-Loop-Helix
Kouzarides, T. (2002). Histone methylation in transcriptional control. Cur- Factors in Cortical Development. Neuron 39, 13-25.
rent Opinion in Genetics & Development 12, 198-209. Vincent, S.D., Dunn, N.R., Sciammas, R., Shapiro-Shalef, M., Davis, M.M.,
Lu, Q.R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C.D., and Rowitch, D.H. Calame, K., Bikoff, E.K., and Robertson, E.J. (2005). The zinc finger tran-
(2002). Common Developmental Requirement for Olig Function Indicates scriptional repressor Blimp1/Prdm1 is dispensable for early axis forma-
a Motor Neuron/Oligodendrocyte Connection. Cell 109, 75-86. tion but is required for specification of primordial germ cells in the mouse.
Ma, Q., Kintner, C., and Anderson, D.J. (1996). Identification of neurogenin, Development 132, 1315-1325.
a Vertebrate Neuronal Determination Gene. Cell 87, 43-52. Völkel, P., and Angrand, P.-O. (2007). The control of histone lysine methyla-
Ma, Y.-C., Song, M.-R., Park, J.P., Henry Ho, H.-Y., Hu, L., Kurtev, M.V., Zieg, tion in epigenetic regulation. Biochimie 89, 1-20.
J., Ma, Q., Pfaff, S.L., and Greenberg, M.E. (2008). Regulation of Motor
Neuron Specification by Phosphorylation of Neurogenin 2. Neuron 58,
65-77.
Neuroscience

54 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-


ing as a glycosylase, which breaks the N-glycosidic bond to the base,
Maintaining the integrity of the genome is crucial for the sur- and a β-lyase, which nicks the DNA on the 3' side of the sugar (Boi-
vival of an organism. DNA is vulnerable to damage from a variety teux 2001). The enzyme diffuses along the DNA until it locates oxoG.
of sources, both extrinsic and intrinsic to the cell (Lindahl 1993; It induces a bend in the DNA and extrudes oxoG out of the DNA
Friedberg 2003). Unless the cell can repair this damage, it will cause helix and into its active site (Bruner 2000). The structural intermedi-
mutations in the genome. Thus, DNA repair mechanisms in the cell ates involved in flipping out the base from the DNA helix make up
are extraordinarily important (Lindahl 1993). One common kind of the base extrusion pathway. Then, hOGG1 excises oxoG, creating an
DNA damage is the oxidation of guanine to 8-oxoguanine (oxoG) by abasic site, and nicks the DNA (Dodson 1994). Other members of the
reactive oxygen species (Neeley 2006; Henle 1997), which are com- base excision repair pathway then remove the sugar and insert a new
monly produced by ionizing radiation, exposure to transition metals, guanine nucleotide, repairing the DNA (Vidal 2001).
or even as byproducts of aerobic respiration (Henle 1997; Bjelland One goal of the Verdine lab is to answer the “search problem,”
2003). Although the Watson-Crick face of oxoG is identical to that of namely, how hOGG1 distinguishes oxoG from other bases. Although
guanine and can still base pair with cytosine, it can also rotate around hOGG1 can distinguish between damaged and normal bases while
its glycosidic bond and base pair with adenine through a Hoogsteen they are within its active site through specific contacts, it is unclear
interaction (Oda 1991) (Figure 1). During replication, DNA poly- whether it is necessary to individually flip out each base into the ac-
merase preferentially adds adenine across from oxoG, which after tive site to find rare oxoG substrates hidden in the genome (Bruner
two rounds of replication, causes a G:C to T:A transversion, thereby 2000). Such a process seems unlikely since hOGG1 slides along DNA
introducing a mutation into the genome (Hsu 2004; Shibutani 2001). at a velocity approaching the upper limit for one-dimensional diffu-
Human 8-oxoguanine glycosylase 1 (hOGG1) locates and excises sion (Blainey 2006). However, when oxoG is intrahelical, there are no
oxoG from the genome as part of the base excision repair pathway 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 Volume 2 Issue 2 | Fall 2009

plexes. The first is a structure of hOGG1 containing guanine within interrogate the guanine. The crosslink forced the guanine out of the
its active site, the “G complex.” This structure was solved while we DNA helix because the crosslink physically occupied the same place
attempted to solve a structure of hOGG1 with an intrahelical gua- the intrahelical guanine would have occupied. The extruded gua-
nine. The G complex was completely unexpected because previous nine formed a noncannonical base pair with the neighboring oxoG,
studies indicated that guanine would be excluded from the active site stabilizing guanine in the major groove. Likewise, in the structure
(Banerjee 2006). In spite of being in the active site of a catalytically containing guanine in the exo site, (int. 3), the same disulfide cross-
active hOGG1, the guanine was not excised out of the DNA. Thus, linking site was used, which biased the guanine to be extrahelical
this study presents the first structure of hOGG1 with an undamaged (Banerjee 2005).
base in the wild-type active site. The second structure presented is a These two results seemed to imply that when forced out of the he-
structure of hOGG1 bound to a trans-α,β-unsaturated aldehyde, a lix, guanine should have no strong preference for any particular site
previously unreported intermediate along the catalytic pathway. extrahelical to the DNA, as both the exo site and the noncannonical
base pair do not seem offer a particularly large amount of stabiliza-
Methods 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
Because hOGG1 has no specific preference for an undamaged base, specific interactions stabilizing it. As a result, moving the crosslink
to capture a structure with guanine it is necessary to restrict hOGG1 to a different site that would not sterically displace guanine from the
to that guanine so that the protein-DNA complex would be homo- DNA helix might allow the capture of a structure containing an in-
geneous enough for crystallographic studies. We formed a disulfide trahelical guanine (Radom 2006).
bond between a mutated cystine on hOGG1 and a modified thiol- In this study, the crosslinking site was between S292C and an
containing base in the DNA, creating a disulfide crosslink that cova- adenine four bases down from the guanine. This site had previously
lently tethers the two together, restricting hOGG1 to the desired base been employed to solve the late-stage intermediate structure (int. 4)
(Banerjee 2005) (Figure 2). This disulfide crosslink was necessary to with oxoG partially in the active site of hOGG1 (Radom 2007). In
determine the structure of many of the intermediates captured thus addition, the structure of a catalytically inactive mutant of hOGG1
far (Banerjee 2005; Banerjee 2005; Radom 2007). Our lab has also crosslinked to oxoG containing DNA at the S292C site (int. 7) is
employed this strategy to determine the structure of other systems almost completely identical to the structure of the mutant without
suffering from similar problems (Huang 2000; Huang 1998). the crosslinking site (int. 5) and the structure of the mutant with
A number of intermediates along the base extrusion and catalytic the N149C crosslinking site (Bruner 2000; Radom 2007) . Thus, the
pathways have already been captured. In the base extrusion pathway, S292C crosslinking site seemed like it might not force the guanine to
crystal structures have been solved containing: guanine extruded be extrahelical. As a result, we hoped that crystallization of hOGG1
out of the helical stack, but folded back into the major groove of the crosslinked through S292C to guanine-containing DNA would allow
DNA (intermediate 1) (Banerjee 2006); oxoG (int. 2) (Radom 2007) the structural determination of a complex containing an intrahelical
and guanine (int. 3) (Banerjee 2005) in an “exo site,” with the base guanine. In reality, the structure captured was that of hOGG1 with
extrahelical, but on a part of hOGG1 adjacent to the active site; oxoG guanine in its active site. An overview of the disulfide crosslinking
entering the active site (int. 4) (Radom 2007); and oxoG fully in the strategy is presented in Figure 4.
active site (int. 5-8) (Bruner 2000; Banerjee 2005; Radom 2007; Nor- Several intermediates along the catalytic pathway have also been
man 2003). An overview of the previously captured intermediates in captured. An overview of the theorized catalytic mechanism sum-
the base extrusion pathway is summarized in Figure 3. marizing the previously captured intermediates is presented in Fig-
In the earliest intermediate so far crystallized in the pathway, ure 5. Two intermediates in the catalytic mechanism for hOGG1 have
(int. 1), guanine was extruded out of the helical stack into the major been captured using the technique of borohydride trapping (Fromme
groove (Banerjee 2006). The guanine was placed adjacent to oxoG, 2003; Radom 2006). Intermediates containing iminium ions can be
but a disulfide crosslink formed between N149C and the cytosine reduced by sodium borohydride to the amine, halting catalysis at a
opposite from the guanine was short enough to restrict hOGG1 to particular step and allowing for the characterization of a normally
Chemical
Biology

4 2
C4Cysteamine

Cysteine 292
hOgg 1 S292C 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
Crosslinking A
categorized by: the location of the interrogated base; the crosslinking site,
if there is one; and other mutations/information. PDB accession codes: 1.
Figure 2. Functionalization of adenine and crosslinking with 2I5W29, 2. 2NOF31, 3. 1YQK30, 4. 2NOZ31, 5. 1EBM15, 6. 1YQR30, 7.
hOGG1 at the S292C site 2NOL31, 8. 1N3C32.
56 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

borohydride (V) (Radom 2006).


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
Figure 4. An overview of the crosslinking strategy. A) Crystal the active site of hOGG1. Therefore, all the hOGG1 synchronously
structures of: (int. 1). K249Q hOGG1 (green) bound DNA (blue) with begins excising the oxoG. After a certain length of time, the crystal is
oxoG (red); (int. 9). S292C hOGG1 (purple) crosslinked to DNA with frozen with liquid nitrogen, and the low temperature (77K) prevents
guanine (green); (int. 3). N149C hOGG1 (yellow) crosslinked to DNA further catalysis from occurring. As crystals are usually cryoprotect-
with guanine. B) and C) A comparison of the S292C and N149C crosslink ed to protect against radiation damage and increase the diffraction
sites. quality and resolution, this method of freeze-trapping the crystals is
very convenient (Rodgers 1994; Henderson 1990). Ideally, this allows
fleeting intermediate (Zharkov 2002; Gilboa 2002). In hOGG1, the one to capture the various intermediates along the catalytic pathway
ε-amino group of Lys 249 is a nucleophile and displaces the oxoG by varying the time between photocleaving the adduct and freezing
base to create the initial Schiff base (II). This intermediate has been the crystal (Radom 2006).
captured by treatment with sodium borohydride and the structure This technique has been used to capture a late stage intermediate
determined by X-ray crystallography (III) (Fromme 2003). In ad- in the base extrusion pathway (Lee 2008). A crystal was cryoprotect-
dition, the intermediate after β-elimination – the α, β unsaturated ed immediately after photocleavage of the adduct, and the structure
Schiff base (IV) – was captured by treatment with sodium cyano- 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 RESEARCH

action with Phe 319 (Figure 7a). However, in a structure containing and is unlikely to be perturbed enough to be protonated. The other
oxoG in the active site, such as the structures captured with catalyti- structures containing oxoG in the active site have distances similar
cally inactive mutants or the structures of the catalytic intermediates, to that of (int. 5): 2.9 Å (int. 6) (Banerjee 2005), 2.8 Å (int. 7) (Radom
the α-O helix moves towards the active site into a “closed” conforma- 2007) and 2.7 Å (int. 8) (Norman 2003), indicating that the increased
tion, and His 270 instead forms a salt bridge with a phosphate of the distance to 3.2 Å may be significant. The last structure (int. 7) indi-
DNA backbone, freeing Phe 319 to form a π-stacking interaction with cates that the increase is not directly due to the crosslinker, though it
oxoG. Furthermore, Gln 315 hydrogen bonds with the Watson-Crick does not rule out a more subtle effect.
face of oxoG (Figure 7b) (Bruner 2000; Radom 2007). The salt bridge also appears to be intact in the G complex. The
In the structure of the active site G complex, the aforementioned distance between Lys 249 and Cys 253 increases slightly from 2.6 Å in
residues occupy the same positions as the residues in the “closed” (int. 5) (Bruner 2000) to 2.8 Å in the G complex, which is still consis-
conformation, with the exception of His 270, which is rotated 90°. tent with a salt bridge (Petsko 2003). Thus, it is unlikely that these re-
However, this rotation does not affect its ability to form a salt bridge pulsive interactions were completely removed, though the repulsion
with the DNA phosphate, and it functions in the same manner as the may be lessened slightly. Therefore, it is unclear as to why guanine
histidine in the “closed” conformation. would be inserted in the active site, as is seen in this structure.
Discrimination between oxoG and guanine Crosslinking bias
The previous interactions are characteristic of the “closed” con- The authors presenting (int. 2) and (int. 4), which contain oxoG in
formation and stabilize both oxoG and guanine. However, based on the exo site or partially in the active site, respectively, posed a similar
quantum mechanical calculations using (int. 3) and (int. 5), there are question (Radom 2007). When the crosslinking site was moved from
two sources of discrimination between oxoG and guanine in the ac- N149C to S292C, the oxoG moved further along the extrusion path-
tive site of hOGG1 that might destabilize guanine binding in the ac- way. It was proposed that the crystal packing of the particular crystal
tive site (Banerjee 2005). First, there is a hydrogen bond between the form of (int. 4) bends the DNA to favor an extrahelical base (Radom
Gly 42 carbonyl and the oxoG N7 that is lost with guanine. This is 2007; Radom 2006). However, this leaves unresolved why in (int. 4),
calculated to stabilize oxoG by about 3.5 kcal/mol. Second, the salt oxoG moved further along the base extrusion pathway instead of re-
bridge between Lys 249 and Cys 253 causes a dipole that interacts maining in the exo site, as presumably the crystal forms and packing
favorably with the dipole of oxoG. However, since guanine has the interactions were the same (since the crystallization conditions were
opposite dipole, this would be a repulsive interaction, favoring oxoG similar and the space groups identical).
by about 3.3 kcal/mol. Here, we propose that DNA bending is indeed the key difference
In the active site G complex, the positions of Gly 42, Lys 249 and but that the DNA is bent because of the crosslinker, not because of
Cys 253 stay roughly the same, along with the rest of the residues in crystal packing. The distance between S292 and the adenine, which
the active site, when compared to (int. 5) (Figure 8). The loop contain- is the distance a crosslinker would need span if it were placed at the
ing Gly 42 was previously found to be rigid, which may explain why it S292C site varies depending on the stage of the base extrusion path-
does not move to relieve the posited repulsion. However, the distance way (Figure 9). The structures of the early intermediates (1 and 3)
between the Cα carbonyl of Gly 42 and N7 increases slightly (2.7 Å require a larger span (10.1 Å and 9.6 Å), while the structures of later
in (int. 5) to 3.2 Å in the active site G complex) by the base shifting intermediates (5, 6, and 8) require a smaller span (8.1 Å and 8.3 Å).
down, which may relieve some repulsion. In addition, it is possible However, in the nine lowest energy conformations of the four-carbon
that the N7 of guanine is protonated, which would recreate a favor- crosslinker used at the S292C site, the largest span is 8.46 Å, which is
able interaction. This is unlikely, however, because N7 is not basic 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.
www.thurj.org 59
RESEARCH Volume 2 Issue 2 | Fall 2009

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

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 RESEARCH

it to deprotonate C2’ (Fromme 2003). Furthermore, addition of (Brunger 1998; Brunger 2007), found that the trans-α,β-unsaturated
oxoG analogues 8-bromoguanine (8-bromoG) and 8-aminoguanine aldehyde best fit the electron density (Figure 11). In addition, the R free
(8-aminoG) can accelerate strand scission of an abasic site. X-ray values after the round of refinement were 0.2602 for the trans, 0.2613
crystal structures of the initial borohydride trapped Schiff base with for the cis, and 0.2630 for the ring closed, indicating that of these
the free oxoG purified out and 8-bromoG or 8-aminoG soaked into three, the trans best described the structure. In addition, even though
the hOGG1-DNA complex show that 8-bromoG and 8-aminoG oc- the cis fit the second best, it is likely that the cis intermediate may be
cupy the same position that oxoG occupies in (III), indicating that too short lived to capture, because it can very favorably turn into the
oxoG may behave similarly to 8-bromoG and 8-aminoG (Fromme ring closed form since it is already in the conformation to do so. Thus,
2003). Taken together, this indicates that oxoG likely deprotonates the comparison is between the trans and ring closed forms, and the
C2' and causes the elimination of the 3' phosphate. trans intermediate fits the best.
Molecular modeling calculations have indicated that the depro- Thus, the bond between C2' and C3' appears to be trans, implying
tonation of the C2' proR hydrogen by oxoG is favored (80% proR that the β-elimination of the 3' phosphate occurs to give the trans
to 20% proS) (Fromme 2003). Nevertheless, it is unclear if elimina- product. Furthermore, this structure also indicates that isomeriza-
tion of the hydrogen would lead to a cis- or trans-α,β-unsaturated tion of the trans to the cis product occurs on the aldehyde and not the
Schiff base. Because the phosphate is antiperiplanar to the proR hy- Schiff base, since in this structure the Schiff base has already been
drogen, it is possible that the elimination is concerted and more E2- hydrolyzed.
like, and thus the elimination of the proR hydrogen would lead to a It is likely that this isomerization occurs through a conjugate ad-
trans-α,-β-unsaturated Schiff base. However, it is also possible that dition of a nucleophile, and then the subsequent rotation and conju-
the elimination instead forms the enamine first and then eliminates gate elimination of that nucleophile to form the cis-α,β-unsaturated
the phosphate (thus is more E1cb-like), which may lead to either the aldehyde (Figure 12B). Although there has been no direct evidence of
trans or cis product. In addition, molecular modeling found that the this isomerization, the existence of a religated product (VIII) formed
trans product is more stable than the cis product, which is consistent from the addition of 8-aminoG to a crystal containing the end prod-
with elimination reactions in general, but although this supports the uct structure (VII) implies that the phosphate is able to undergo a
trans product, it is possible that this reaction is under kinetic con- conjugate addition, albeit assisted by 8-aminoG (Figure 12A) (Chung
trol (Fromme 2003). Furthermore, although the end-product struc- 2004). Thus, it is possible that phosphate or a different nucleophile
ture (VII) contains the sugar is in the ring-closed form, and thus the such as water, may be able to undergo conjugate addition if driven
double bond between C2' and C3' must be cis eventually, this does not by an ultimate thermodynamic preference for the ring-closed struc-
preclude the formation of a trans double bond in the initial elimina- ture.
tion, since this bond can later isomerize to the cis form (Chung 2004). The “open” conformation
Unfortunately, the crystal structure containing the α,β-unsaturated Surprisingly, this structure is in the “open” conformation. The α-O
Schiff base caught by borohydride trapping (V) was similarly incon- helix is held away from the active site and His 270 is interacting with
clusive. Models containing both the cis, trans, or saturated (from Asp 322 and has an edge-face interaction with the π-system of Phe
potential overreduction by sodium cyanoborohydride) bonds fit into 319, which moves it away from the position it occupies in the “closed”
the electron density of that structure, with little difference in the R free, conformation. In addition, Gln 315 is also away from its position in
so it was not possible to distinguish between the possibilities (Radom the “closed” conformation. In contrast, the previous two catalytic in-
2006). Complicating the matter, it is also possible that hOGG1 may termediates were in the “closed” conformation (Figure 13).
produce a mixture of both the trans or cis products, which may be the At this time, it is unclear whether the “open” conformation of the
reason for the ambiguity of (V). 30 minute structure is the natural conformation or if it has been ar-
The structure presented in this study resolves this issue. In this tificially introduced by the system. Biochemical experiments done by
structure, the bond between C2' and C3' was determined to be trans. Charisse Crenshaw have indicated that the disulfide crosslink, which
The sugar of oxoG could be in one of three conformations: cis, trans, is one of the differences between this structure and the previous cata-
or ring closed. To determine which of the sugar forms was likely, we lytic intermediates, does not seem to have a significant affect on the

Chemical
Biology
modeled in the three different forms, and after performing one round conformation of the DNA, in contrast to the role it played in the G
of energy minimization and individual B factor refinement in CNS complex. However, this does not preclude bias introduced from other

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 61
RESEARCH Volume 2 Issue 2 | Fall 2009

8-aminoG accelerated
ring opening Conjugate Addition Tautomerization
VII. Ring Closed Sugar VIII. Religated Product

Conjugate Addition Rotation Conjugate Elimination Ring Closing


VI. α, β Unsaturated Aldehyde VII. Ring Closed Sugar

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 Conclusion


bias from the crosslinker.
The previous two structures obtained using this freeze trapping In the active site G complex, guanine is not cleaved out, even
technique were in the “open” conformation; however, it is unclear if though the protein is catalytically active, and the active site of the
this is because the structures were within the base extrusion pathway guanine is in the “closed” conformation and presumably ready for
or if this system biases hOGG1 to the “open” conformation. In addi- catalysis. This is surprising because excision of oxoG by hOGG1
tion, it is possible that the “closed” conformation is only necessary occurs within the 30 minute structure. The G complex and the 30
for the initial part of catalysis and the conformation may move to minute structure are in the same crystal forms and are thus presum-
the “open” form later during catalysis. Because this structure does ably affected by the same crystal packing forces. Furthermore, the
not contain oxoG in the active site, the π-stacking interaction be- S292C crosslink – at least by itself – does not prevent catalysis, as
tween Phe 319 and oxoG, which stabilizes the “closed” structure, is hOGG1 with the S292C crosslink has been shown to excise oxoG in
lost. Thus, it is possible that by this stage in the reaction, hOGG1 has biochemical studies (Crenshaw 2009). However, guanine does not
moved back into the “open” form. seem to have been excised, as the N glycosidic bond and the 3' and 5'
Moreover, although the end-product structure, which would be phosphodiester bonds are all intact. In addition, biochemical experi-
after this intermediate, does contain hOGG1 in the “closed” form ments have shown that hOGG1 with the S292C crosslink does not
(Chung 2004), this may be due to the mutation used to crystallize excise guanine.
that structure, D268E, which had previously been known to affect Thus, the G complex raises the possibility that there is a “catalytic
the nearby residues 269-271 (Norman 2003). Because the interaction checkpoint,” or a way that hOGG1 can prevent excision of guanine
between His 271 and the DNA is a key part of the “open” conforma- even in the likely rare case that guanine is placed in the active site.
tion, the mutation may be biasing the structure of the molecule to Such a catalytic checkpoint is not a ubiquitous feature of glycosylases:
the “closed” form. Again, however, it is not clear whether the “open” Uracil DNA glycosylase, (which sterically excludes thymine from en-
conformation is an accurate description of this intermediate or if it is tering the active site) when modified to allow thymine to enter the
an artifact brought on by the system used. 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
Chemical
Biology

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,
Figure 13. The 30 minute structure is in the open conformation. it would be useful to ensure that early intermediates captured using
The 30 minute structure is in blue, with the sugar from oxoG in purple. III, this methodology are in the “closed” form, like the other early inter-
a closed structure, is in green, with its oxoG and sugar in orange. Here π mediates; otherwise, it would indicate that the freeze trapping system
interactions are shown with dashes. artificially warps the structures.
62 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009 RESEARCH

Materials and Methods the Facile Structure Determination of Lesion-Containing DNA,” Struc-
ture 2008, 16, 1166-1174.
25. Lipscomb LA, et. al., X-ray structure of a DNA decamer containing 7,8-di-
For full methods, see the supplementary information online. In hydro-8-oxoguanine. Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):719-23.
brief, hOGG1 was expressed in E. coli and crosslinked to a synthe- 26. Oda, Y., et. al. 1991. NMR studies of a DNA containing 8-hydroxydeox-
sized DNA strand, yielding a hOGG1-DNA complex. This complex yguanosine. Nucleic Acids Res. 19:1407-1412.
was then crystallized and the diffraction data analyzed, yielding the 27. Banerjee, A.; Santos, W. L.; Verdine, G. L. “Structure of a DNA Glycosy-
lase Searching for Lesions,” Science 2006, 311, 1155-1157.
crystal structure of the complex.
28. Fromme, J. C.; Verdine, G. L. “Structural insights into lesion recognition
and repair by the bacterial 8-oxoguanine DNA glycosylase MutM,” Nat.
References Struct. Biol. 2002, 9, 544-552.
29. Banerjee, A.; Verdine, G. L. “A nucleobase lesion remodels the interaction
1. Lindahl, T. Instability and decay of the primary structure of DNA. Nature
of its normal neighbor in a DNA glycosylase complex,” Proc. Natl. Acad.
362, 709-715 (1993).
Sci. USA 2006, 103, 15020-15025.
2. Friedberg, E. C. DNA damage and repair. Nature 421, 436–440 (2003).
30. Banerjee, A.; Yang, W.; Karplus, M.; Verdine, G. L. “Structure of a repair
3. Lindahl, T. & Wood, R.D. Quality control by DNA repair. Science 286,
enzyme interrogating undamaged DNA elucidates recognition of dam-
1897-1905 (1993)
aged DNA,” Nature 2005, 434, 612-618.
4. Neeley, W. L. & Essigmann, J. M. Mechanisms of formation, genotoxic-
31. Radom, C.; Banerjee, A.; Verdine, G.L. “Structural characterization of
ity, and mutation of guanine oxidation products. Chem. Res. Toxicol. 19,
human 8-oxoguanine DNA glycosylase variants bearing active site muta-
491–505 (2006).
tions,” J. Biol. Chem. 2007, 282, 9182-9194.
5. Mutsuo Sekiguchi and Teruhisa Tsuzuki, Oxidative nucleotide damage:
32. Norman, D. P. G.; Chung, S. J.; Verdine, G. L. “Structural and biochemical
consequences and prevention. Oncogene (2002) 21, 8895-8904
exploration of a critical amino acid in human 8-oxoguanine glycosylase,”
6. Henle, E. S., and S. Linn. 1997. Formation, prevention, and repair of DNA
Biochemistry 2003, 42, 1564-1572.
damage by iron/hydrogen peroxide. J. Biol. Chem. 272:19095-19098.
33. Huang, H., Harrison, S. C. & Verdine, G. L. Trapping of a catalytic HIV
7. Bjelland S, Seeberg E. Mutagenicity, toxicity and repair of DNA base dam-
reverse transcriptase template:primer complex through a disulfide bond.
age induced by oxidation. Mutat Res 2003;29:37-80.
Chem. Biol. 7, 355−364 (2000)
8. Oda Y et. al. NMR studies of a DNA containing 8-hydroxydeoxyguanos-
34. Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a
ine. Nucleic Acids Res. 1991 Apr 11;19(7):1407–1412.
covalently trapped catalytic complex of HIV-1 reverse transcriptase: im-
9. Hsu, G. W., Ober, M., Carell, T. & Beese, L. S. Error-prone replication
plications for drug resistance. Science 282, 1669−1675 (1998)
of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature
35. Fromme, J. C.; Bruner, S. D.; Yang, W.; Karplus, M.; Verdine, G. L. “Prod-
431, 217–221 (2004).
uct-assisted catalysis in base-excision DNA repair,” Nat. Struct. Biol.
10. Shibutani, S., Takeshita, M. & Grollman, A. P. Insertion of specific bases
2003, 10, 204-211.
during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature
36. Radom, C.T., Structural and Biochemical Exploration of Human 8-Ox-
349, 431–434
oguanine Glycosylase I, in Chemistry and Chemical Biology. 2006, Har-
11. Crenshaw, C. M., Structures and Dynamics of DNA Repair Enzymes:
vard University: Cambridge, MA. p. 172.
Mechanistic Insights into Catalysis and Damage Targeting [Thesis], Cam-
37. Zharkov, D.O. et al. Structural analysis of an Escherichia coli endonu-
bridge MA, Harvard University, 2009, 142 pp.
clease VIII covalent reaction intermediate. EMBO J. 21, 789-800 (2002).
12. S. David, V. O'Shea, and S. Kundu, Nature 447, 941-950 (2007)
38. Gilboa, R. et al. Structure of formamidopyrimidine-DNA glycosylase co-
13. David, S. S. & Williams, S. D. Chemistry of glycosylases and endonucleas-
valently complexed to DNA. J. Biol. Chem. 277, 19811-19816 (2002).
es involved in base-excision repair. Chem. Rev. 98, 1221–1261 (1998).
39. Chung, S. J.; Verdine, G. L. “Structures of End Products Resulting from
14. Boiteux S and Radicella JP (2001) Mechanism of stimulation of the DNA
Lesion Processing by a DNA Glycoylase/lyase,” Chem. & Biol. 2004, 11,
glycosylase activity of hOGG1 by the major human AP endonuclease: by-
1643-1649.
pass of the AP lyase activity step. Nucleic Acids Research, 2001, Vol. 29,
40. Lee, S.; Radom, C. T.; Verdine, G. L. “Trapping and Structural Elucida-
No. 6 1285-1292
tion of a Very Advanced Intermediate in the Lesion-Extrusion Pathway of
15. Bruner, S. D.; Norman, D. P. G.; Verdine, G. L. “Structural Basis for Rec-
hOGG1,” J. Am. Chem. Soc. 2008, 130, 7784-7785.
ognition and Repair of the Endogenous Mutagen 8-oxoguanine in DNA,”
41. Rodgers, D.W. Cryocrystallography. Structure 2, 1135-1140 (1994).
Nature 2000, 403, 859-866.
42. Henderson, R. Cryo-protection of protein crystals against radiation dam-
16. Dodson, M.L., Michaels, M.L. & Lloyd, R.S. Unified catalytic mechanism
age in electron and x-ray diffraction. Proc. R. Soc. Lond. [Biol] 241, 6-8
for DNA glycosylases. J. Biol. Chem. 269, 32709-32712 (1994).
(1990).
17. Vidal A.E., Hickson,I.D., Boiteux,S. and Radicella,J.P. Mechanism of
43. Petsko, G. & Ringe, D. Protein Structure and Function (Sinauer Associ-

Chemical
stimulation of the DNA glycosylase activity of hOGG1 by the major hu-

Biology
ates, Sunderland, Massachusetts, USA, 2003).
man AP endonuclease: bypass of the AP lyase activity step. Nucleic Acids
44. Kavli, B., et. al., Excision of. Cytosine and Thymine from DNA by Mutants
Research, 2001, Vol. 29, No. 6 1285-1292
of Human. Uracil-DNA Glycosylase, EMBO J., 1996, vol. 15, no. 13, pp.
18. Sheehan AM, McGregor DK, Patel A, Shidham V, Fan CY, Chang CC. Ex-
3442–3447.
pression of human 8-oxoguanine DNA glycosylase (hOGG1) in follicular
45. Bennett MT, et. al. Specificity of human thymine DNA glycosylase de-
lymphoma. Modern Pathology (2005) 18, 1512–1518.
pends on N-glycosidic bond stability. J. Am. Chem. Soc. (2006) 128:12510–
19. Fan CY et. al. Frequent Allelic Imbalance and Loss of Protein Expression
12519.
of the DNA Repair Gene hOGG1 in Head and Neck Squamous Cell Carci-
46. Z. Otwinowski and W. Minor, “Processing of X-ray Diffraction Data Col-
noma. Lab Invest 2001, 81:1429–1438
lected in Oscillation Mode”, Methods in Enzymology, Volume 276: Mac-
20. O. Minowa, et. al., Mmh/Ogg1 gene inactivation results in accumulation
romolecular Crystallography, part A, p.307-326, 1997, C.W. Carter, Jr. &
of 8-hydroxyguanine in mice, Proc. Natl. Acad. Sci. U.S.A. 97 (2000), pp.
R. M. Sweet, Eds., Academic Press (New York).
4156–4161
47. A.T. Brunger, et. al.,Crystallography & NMR System (CNS), A new soft-
21. A. Klungland, et. al., Accumulation of premutagenic DNA lesions in
ware suite for macromolecular structure determination, Acta Cryst. D 54,
mice defective in removal of oxidative base damage, Proc. Natl. Acad. Sci.
905-921(1998).
U.S.A. 96 (1999), pp. 13300–13305.
48. A.T. Brunger, Version 1.2 of the Crystallography and NMR System, Na-
22. K. Sakumi, et. al. Ogg1 knockout-associated lung tumorigenesis and its
ture Protocols 2, 2728-2733 (2007).
suppression by Mth1 gene disruption, Cancer Res. 63 (2003), pp. 902–
49. Zbyszek Otwinowski, W.M., Processing of X-ray diffraction data collected
905.
in oscillation mode. Methods in Enzymology, 1997. 276: p. 307-326.
23. Blainey, P. C.; van Oijen, A. M.; Banerjee, A.; Verdine, G. L.; Xie, X. S. “A
50. Read, R.J., Improved Fourier coefficients for maps using phases from par-
base-excision DNA-repair protein finds intrahelical lesion bases by fast
tial structures with errors. Acta Crystallogr. Sect. A, 1986. 42: p. 104.
sliding in contact with DNA,” Proc. Natl. Acad. Sci. USA 2006, 103, 5752-
51. A. W. Schuettelkopf and D. M. F. van Aalten (2004). PRODRG - a tool for
5757.
high-throughput crystallography of protein-ligand complexes. Acta Crys-
24. Bowman, B. R.; Lee, S.; Wang, S.; Verdine, G. L. “Structure of the E. coli
tallogr D60, 1355--1363.
DNA Glycosylase AlkA Bound to the Ends of Duplex DNA: A System for

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