The 11th Annual DSAC Student Research Symposium
Friday, January 10
Woodruff Health Sciences Center Administration Building
(WHSCAB) Auditorium
8:30 – 9:00 Breakfast – WSHCAB Lobby
Session I: Microbial Systems
9:00 am
9:00 Phillip Zakas (MSP)
Reduced Antigenicity of Ovine Factor VIII in Human Hemophilia A Inhibitor Plasmas
9:15 Jennifer Frediani (NHS)
Plasma High-Resolution Metabolomic Profiling Reveals Upregulation of Specific Resolvins in Patients
with Pulmonary Tuberculosis
9:30 Mary Bushman (PBEE)
Within-Host Competition of Malaria Parasites in Humans and the Fitness Cost of Drug Resistance
9:45 Brigid O’Flaherty (MMG)
Viral Rta and Cellular IRF4 Synergistically Regulate MHV68 M1 Gene Expression in Plasma Cells
10:00 Emily Kuiper (BCDB)
Interrogating RNA Structural Dynamics Upon Substrate Recognition by the Thiostrepton-Resistance
Methyltransferase
10:15 – 10:30 Break
Session II: Immunity to Infection
10:30 am
10:30 Maria Georgieva (MMG)
Mycobacterium tuberculosis Hip1 is a Serine Protease that Cleaves GroEL2 and Modulates Macrophage
Responses
10:45 David Pinelli (IMP)
Rapamycin Augments the CD8 T Cell Response to a Latent Viral Infection in the Presence or Absence of
CTLA4-Ig
11:00 Victor Band (IMP)
Recognition of B. anthracis Cell Wall Polysaccharide by the Host Innate Immune System
11:15 Justin Kandler (MMG)
Neisseria gonorrhoeae Infection of Macrophages Impacts the Host Iron-Limiting Innate Immune Defense
and Upregulates Gonococcal Iron-Acquisition Genes
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [1]В В В Session III: Translational Models
11:30 am
11:30 James Cordova (MSP)
Quantitative, Unbiased Tumor Segmentation for Evaluation of Extent of Brain Tumor Resection to
Facilitate Multisite Clinical Trials
11:45 James Burkett (NS)
Neural Mechanisms of Empathy-based Behavior in Monogamous Prairie Voles
12:00 – 1:30 Poster Sessions & Lunch– WHSCAB Lobby
Poster Session
12:00 – 12:45pm Odd Numbered Poster Presentations
12:45 – 1:30pm Even Numbered Poster Presentations
1:30 – 1:45 Break
Session IV: Life of a Cell
1:45 pm
1:45 Chantel Cadwell (BCDB)
Cadherin Endocytosis, Adhesion, and Cytoskeletal Linkage Cooperatively Regulate Cell Migration
2:00 Jason Conage-Pough (CB)
Phosphorylation Influences the Preferential Binding of Bim to Anti-Apoptotic Proteins in Multiple
Myeloma
Session V: Receptors and Signaling
2:15 pm
2:15 Gina Alesi (CB)
RSK2-Mediated Phosphorylation of Stathmin Promotes Microtubule Polymerization and Provides a Proinvasive Advantage to Metastatic Cancer Cells
2:30 Jessica Konen (CB)
LKB1 Regulates a Molecular Switch that Drives #-D Lung Cancer Invasion
2:45 Ariana Mullin (NS)
NSF Regulates Synaptic Homeostasis at the Drosophila Neuromuscular Junction
3:00 George Kannarkat (IMP)
The rs3129882 Single Nucleotide Polymorphism Affects MHC-ii Expression and May Increase Risk for
Parkinson’s Disease by Heightening Immune Responses
3:15 – 3:30 Break
Session VI : DNA and Gene Expression
3:30 Will Hudson (MSP)
The Structural Basis of Direct Glucocorticoid-Mediated Transrepression
3:45 Paul Musille (MSP)
Divergent Sequence Tunes Ligand Sensitivity in Phospholipid-Regulated Hormone Receptors
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [2]В В В 3:30 pm
4:00 Kevin Van Bortle (BCDB)
A Subclass of Insulator Binding Sites Establish Dense Clusters to Delineate Topologically Associating
Domains
4:15 Mariana Mandler (BCDB)
A Cytoplasmic Quaking Isoform Regulates an hnrnpf/h-dependent Alternative Splicing Pathway in
Myelinating Glia
4:30-Leila Myrick (NS)
Pathogenesis of Novel FMR1 Mutations in Fragile X Syndrome
4:45-Jordan Morreall (GMB)
TNF-О± Inhibits DNA Repair Activity of BER Variant OGG1 Through Oxidative Stress
5:00-5:15pm Break
Keynote Address
5:15-5:45 pm
Adam M. Katz
Adam M. Katz is the Policy and Advocacy Specialist at Research!America, where he
leads a variety of advocacy initiatives to make science and medical research a higher
national priority. Prior to joining Research!America, Adam held positions at the
University of Rochester, the University of California at Irvine and the National
Institute of Mental Health. He holds a masters degree in biomedical science policy and
advocacy from Georgetown University and undergraduate degrees in brain and
cognitive sciences as well as clinical and social psychology from the University of
Rochester.
5:45pm Reception and Awards Presentation – WHSCAB Lobby
Images
Front Cover: Image Contest Winners
1st Place: Todd Deveau (NS) Page 5
2nd Place: Ana Montiero (BCDB) Page 24
3rd Place: Amanda York (BCDB) Page 58
All images were judged by the Integrated Cellular Imaging center
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [3]В В В THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [4]В В В THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [5]В В В Session I
Microbial Systems
9:00 am
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [6]В В В Phillip Zakas, MSP
9:00 am
REDUCED ANTIGENICITY OF OVINE FACTOR VIII IN HUMAN
HEMOPHILIA A INHIBITOR PLASMAS
Philip M. Zakas1, Kristine Vanijcharoenkarn2, Rebecca Markovitz3, Shannon Meeks, MD4 and Christopher B. Doering,
PhD41Graduate Program in Molecular and Systems Pharmacology, Emory University, Atlanta, GA, 2Emory University School of
Medicine, MD candidate, 3Emory University School of Medicine, MD PhD candidate, 4Aflac Cancer and Blood Disorders
Center, Emory University/Children’s Healthcare of Atlanta, Atlanta, GA
Neutralizing antibodies (inhibitors) develop in approximately 30% of severe hemophilia A patients and
present the greatest obstacle to the treatment of patients with access to care. Currently, a recombinant
porcine factor VIII (pFVIII) product (OBI-1в„ў, Baxter International) is undergoing phase III clinical trials
due to reduced cross-reactivity of anti-human FVIII (hFVIII) inhibitors to pFVIII. Herein, we investigate
the antigenicity of orthologous ovine FVIII (oFVIII), and its potential utility for inhibitor patients. Analysis
of 38 hemophilia A inhibitor patient plasmas revealed significantly reduced inhibition of oFVIII coagulant
function compared to hFVIII, with some patients showing lower titers against oFVIII than pFVIII.
Inhibitors primarily target the functionally essential A2 and C2 domains of FVIII. Characterization of A2
and C2 inhibitors within patient plasmas by competition ELISA with anti-hFVIII murine monoclonals
revealed a high prevalence of potent inhibitors overlapping with known epitopes. Using an indirect ELISA,
we demonstrate that these inhibitors do not cross-react with oFVIII with the exception of specific C2
domain targeting inhibitors. Therefore, we hypothesize that non-conserved residues within oFVIII alter the
clinically important surface epitopes and thereby diminish cross-reactivity. These data demonstrate that
oFVIII could have potential as a third line treatment in the FVIII inhibitor patient setting.
Jennifer Frediani, NHS
9:15 am
PLASMA HIGH-RESOLUTION METABOLOMIC PROFILING REVEALS
UPREGULATION OF SPECIFIC RESOLVINS IN PATIENTS WITH
PULMONARY TUBERCULOSIS
Jennifer K. Frediani1, Dean P. Jones1, Nestan Tukvadze2, Eka Sanikidze2, Maia Kipiani2, Karan Uppal1, Shaheen S. Kurani1,
Gautam Hebbar1, Romain A. Colas3, Jesmond Dalli3, Charles N. Serhan3, Vin Tangpricha1, Henry M. Blumberg1, Thomas R.
Ziegler1
1
Emory University, Atlanta, GA; 2National Center for Tuberculosis and Lung Disease, Tbilisi, Georgia; 3Harvard University,
Boston, MA
Background: Characterization of metabolites altered by tuberculosis (TB) disease may identify
pathophysiologic pathways. Resolvins are lipid mediators derived from endogenous EPA and DHA critical
for resolution of inflammation. Methods: Plasma samples from 17 patients (35В±12 yrs old) with new
pulmonary TB and 17 matched household contacts without TB (controls) were analyzed using highresolution LC-MS. False discovery rate (FDR; q=0.05) and hierarchal cluster analysis was used to
distinguish accurate mass ions (metabolites) significantly different between groups. The METLIN
Metabolite Database was used to match ions to known metabolites. Identification of specific D-series
resolvins (RvD1 and RvD2) was confirmed by LC-UV-MS/MS. Results: Over 23,000 metabolites were
detected in untargeted metabolomic analysis, of which 61 were significantly different between the two
groups by FDR. Eight metabolite clusters were evident containing several metabolites quantitatively
upregulated in patients with TB (e.g. glutamate, choline derivatives, anti-TB drug metabolites and several
lipids, including likely Mtb cell wall lipids and resolvins RvD1 and RvD2. Conclusions: This pilot study
revealed metabolites that are upregulated during newly diagnosed TB potentially involved in pathogenesis.
Upregulation of specific resolvins may mediate recovery from inflammation in TB disease.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [7]В В В Mary Bushman, PBEE
9:30 am
WITHIN-HOST COMPETITION OF MALARIA PARASITES IN HUMANS AND THE FITNESS
COST OF DRUG RESISTANCE
Mary Bushman1, Nancy O. Duah2, Neils Quashie2, Benjamin Abuaku2, Kwadwo A. Koram2, Jacobus de
Roode1, Venkatachalam Udhayakumar3
1
Emory University, Atlanta, GA, United States, 2Noguchi Memorial Institute for Medical Research,
University of Ghana, Legon, Ghana, 3Centers for Disease Control and Prevention, Atlanta, GA, United
States
Most infections with the malaria parasite Plasmodium falciparum consist of multiple strains, which
presumably occupy the same ecological niche; therefore, we expect within-host competition to occur in
mixed-strain infections. Competition may suppress transmission of drug-resistant strains, which could
significantly slow evolution and/or accelerate decline of resistance following retirement of a failing drug.
To test the hypothesis that within-host competition occurs in mixed infections, we used quantitative realtime PCR to determine parasite density of two strains of P. falciparum (one chloroquine-sensitive, one
chloroquine-resistant) in 1,423 blood samples from three African countries. We found that total parasite
density did not differ between single-strain and mixed-strain infections, while each strain exhibited lower
densities in mixed infections that those achieved in single-strain infections. These findings were replicated
in all three countries, and suggest that competition does indeed occur in mixed-strain infections. Further
investigation also revealed a significant fitness cost of resistance to chloroquine. First, chloroquineresistant parasites attained lower densities than sensitive parasites in both single and mixed infections.
Second, in one country, the frequency of resistance dropped from 78% to 43% over six years following the
retirement of chloroquine. These findings constitute a significant advance in our understanding of the
evolution of drug-resistant malaria.
Brigid O’Flaherty, MMG
9:45 am
VIRAL RTA AND CELLULAR IRF4 SYNERGISTICALLY REGULATE MHV68
M1 GENE EXPRESSION IN PLASMA CELLS
Brigid M. O’Flaherty1,2, Tanushree Soni1,2, Samuel H. Speck1,2
1
Department of Microbiology and Immunology, 2Emory Vaccine Center, Emory University School of
Medicine Atlanta, Georgia 30322
MHV68 is a murid gammaherpesvirus that infects laboratory mice and provides a small animal model for
gammaherpesvirus biology and pathogenesis. Co-evolution with their hosts has resulted in acquisition of
virus specific unique genes important in modulating infection. One such gene, M1, unique to MHV68,
encodes a secreted protein with no known homolog. Previously M1 was identified as the stimulatory ligand
responsible for expansion of cytolytic IFNпЃ§/TNFО± secreting VОІ4+ CD8+ T cells following MHV68
infection. In the absence of M1, VОІ4+ T cell expansion fails to occur and viral reactivation is poorly
controlled. However, the mechanism of action and regulation of M1 are poorly understood. Utilizing a
recombinant virus expressing M1 promoter driven YFP, we identified of splenic plasma cells as the
predominant site of M1 expression. We extended these findings to characterize the transcriptional
regulators of the M1 promoter, identifying cellular interferon regulatory factor 4 (IRF) and viral replication
and transcription activator (Rta) as synergistic partners which regulate M1 expression through proteinprotein interaction. These findings shed light on the function and mechanism of M1 mediated VОІ4+ CD8+ T
cell expansion, and suggests a conserved means of transcriptional regulation for MHV68 genes linked to
viral reactivation and plasma cell differentiation.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [8]В В В Emily Kuiper, BCDB
10:00 am
INTERROGATING RNA STRUCTURAL DYNAMICS UPON SUBSTRATE
RECOGNITION BY THE THIOSTREPTON-RESISTANCE
METHYLTRANSFERASE
Emily G. Kuiper1,2, Jennifer Hernandez3,4, Graeme L. Conn1.
1
Department of Biochemistry, 2Biochemistry, Cell and Developmental Biology (BCDB) Program, 3SURE
Program, Emory University School of Medicine, Atlanta, GA, 4Georgia State University, Atlanta, GA.
As bacteria become increasingly resistant to antibiotics in the clinic, it is critical to understand the
molecular mechanisms of resistance in order to slow its spread or develop new antimicrobial therapies.
Antibiotic-producing bacteria are a reservoir for new resistance determinants that could appear in the clinic,
as they must contain mechanisms to protect themselves from their toxic products. In the thiostrepton
producer Streptomyces azureus, the thiostrepton-resistance methyltransferase (Tsr) methylates the drug
binding site on the 23S ribosomal RNA thereby inhibiting its binding and action against the bacterial
ribosome. Resistance by this mechanism is common in drug producers and is now becoming increasingly
prevalent in the clinic, e.g. providing resistance to macrolide and aminoglycoside antibiotics. To
understand how antibiotic-resistance conferring enzymes recognize their RNA substrates we have
biochemically dissected Tsr’s substrate recognition mechanism. Using RNA structure probing, binding and
enzymatic experiments we show that the carboxyl-terminal domain (CTD) of Tsr docks on the RNA, then
engages its amino-terminal domain (NTD) to bind and unfold the RNA tertiary structure in a critical step
necessary prior to catalysis. These findings provide new insights into the intricacies of substrate
recognition for antibiotic resistance conferring and other RNA modifying enzymes that recognize the
bacterial ribosome.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [9]В В В Session II
Immunity to Infection
10:30 am
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [10]В В В Maria Georgieva, MMG
10:30 am
MYCOBACTERIUM TUBERCULOSIS HIP1 IS A SERINE PROTEASE THAT
CLEAVES GROEL2 AND MODULATES MACROPHAGE RESPONSES
Maria Georgieva1,*, Jacqueline L. Naffin-Olivos2,*, Nathan Goldfarb3, Ranjna Madan-Lala1,
Dagmar Ringe2, Ben M. Dunn3, Gregory A. Petsko2 and Jyothi Rengarajan1, 4
1
Emory Vaccine Center, Emory University, Atlanta, GA, USA, 2Rosenstiel Basic Medical Sciences
Research Center, Brandeis University, Waltham, MA, USA, 3Department of Biochemistry and Molecular
Biology, University of Florida, Gainesville, FL, USA, 4Division of Infectious Diseases, Department of
Medicine, Emory University, Atlanta, GA, USA *These authors contributed equally
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses. We have
previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, dampens macrophage
responses. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1
function. We establish that Hip1 is a serine protease and show that the Mtb GroEL2 protein is a substrate of
Hip1 protease activity. Cleavage of GroEL2 occurs between Arg12 and Gly13 and is optimal at
intraphagosomal pH conditions. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2
converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved
GroEL2 monomers into the hip1 mutant restored wild type levels of cytokine responses in infected
macrophages. Thus Hip1-dependent proteolysis is a novel regulatory mechanism that helps Mtb respond
rapidly to immune environments during infection. These findings position Hip1 as a target for developing
immunomodulatory therapeutics against Mtb.
David Pinelli, IMP
10:45 am
RAPAMYCIN AUGMENTS THE CD8 T CELL RESPONSE TO A LATENT VIRAL
INFECTION IN THE PRESENCE OR ABSENCE OF CTLA4-IG
David F Pinelli1, Maylene E Wagener1, Mandy L Ford1
1
Department of Surgery, Emory University School of Medicine
Latent viral infections are a major concern among immunosuppressed transplant patients. Treatment with
belatacept, a CTLA4-Ig fusion protein, is contraindicated in EBV- patients, due to an increased risk of
PTLD associated with primary EBV infection. Recent reports have shown that treatment with rapamycin
(rapa) paradoxically enhances CD8 T cell responses to certain pathogen infections, raising the possibility
that rapa could be used to enhance responses to EBV in belatacept-treated patients. We infected mice with
gammaherpesvirus 68 (gHV), the murine homolog of EBV, and treated with rapa +/- CTLA-4 Ig. Rapa
treatment alone increased the number of CD8 T cells response to gHV at Day 20 compared to mice that
received no treatment, and also led to an increase in IFN-Оі production. While CTLA4-Ig treatment alone
dampened the T cell response to gHV, the addition of rapamycin was able to restore the response to
baseline levels by day 20 post-infection. In addition, all rapamycin treated animals showed increased levels
of CD127 on virus-specific cells, indicating that rapa may generate higher quality memory T cells
regardless of treatment with CTLA4-Ig. These results suggest that rapamycin may be able to be used in
combination with CTLA4-Ig in order to augment protective immune responses.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [11]В В В Victor Band, IMP
11:00 am
RECOGNITION OF B.ANTHRACIS CELL WALL POLYSACCHARIDE BY THE
HOST INNATE IMMUNE SYSTEM
Victor Band1,4, Nazia Kamal2, Elke Saile2, Scott Forsberg3, Conrad Quinn2, Geert-Jans Boons3, David
Weiss4
1
Emory University Immunology and Molecular Pathogenesis, 2Centers for Disease Control and Prevention,
3
University of Georgia Carbohydrate Research Center, 4Emory Vaccine Center
B. anthracis is a Gram positive bacterium that is the causative agent of the acute disease anthrax. It
contains a protein capsule that protects it from antimicrobials and is linked to a unique cell wall
polysaccharide. We show that this cell wall polysaccharide is a novel activator of the immune system,
acting as a pathogen associated molecular pattern (PAMP). Using macrophages from knockout mice
lacking various innate immune receptors, we determined that this polysaccharide is sensed by Toll like
receptor 4 (TLR4), which also detects Gram negative lipopolysaccharides. After characterizing the
structures of the cell wall polysaccharides of various B. anthracis strains, we determined that this
polysaccharide is modified by galactose additions in virulent strains, which we show reduces TLR4
activation. Further, we discovered that an avirulent strain whose basis for attenuation is not known, does
not have these galactose additions and is much more inflammatory in vitro. These data suggest that
modification of this cell wall polysaccharide may be critical to the ability of B. anthracis to avoid immune
detection and to cause disease, with important implications for the treatment of anthrax and our
understanding of host-pathogen interactions.
Justin Kandler, MMG
11:15 am
NEISSERIA GONORRHOEAE INFECTION OF MACROPHAGES IMPACTS THE
HOST IRON-LIMITING INNATE IMMUNE DEFENSE AND UPREGULATES
GONOCOCCAL IRON-ACQUISITION GENES
Justin L. Kandler1, Susu M. Zughaier1, and William M. Shafer1,2
1
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA
30322, 2Laboratories of Microbial Pathogenesis, VA Medical Center, Decatur, GA 30033
Neisseria gonorrhoeae (the gonococcus) is the causative agent of the disease termed gonorrhea and a strict
human pathogen. Though many mechanisms of gonococcal iron-piracy are well-established, little is known
about how intracellular gonococci acquire sufficient iron within the iron-limited host to sustain viability
during infection. Due to the crucial role of macrophages in host iron homeostasis, we investigated the
impact of gonococcal infection of macrophages on host and gonococcal iron-related genes. We found that
phagocytosis of wild-type gonococci by mouse and human macrophages increased expression of host
genes encoding hepcidin, NRAMP1, and NGAL, but decreased expression of host genes encoding
ferroportin and BDH2. These changes were associated with an increased level of labile (free) iron within
host cells. Analysis of gonococcal transcripts revealed that expression of iron-responsive genes (including
iron-acquisition genes) was increased during intracellular residence within macrophages. Furthermore,
approximately 15% of the live, internalized gonococci remaining after 1 hour survived the next 4 hours of
the phagocytic killing process. Taken together, these data suggest that gonococci modulate the host ironlimiting innate immune defense and upregulate their own iron-acquisition genes to survive intracellularly.
We conclude that this mechanism contributes to gonococcal survival in the infected host.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [12]В В В Session III
Translational Models
11:30 am
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [13]В В В James Cordova, MSP
11:30 am
QUANTITATIVE, UNBIASED TUMOR SEGMENTATION FOR EVALUATION
OF EXTENT OF BRAIN TUMOR RESECTION TO FACILITATE MULTISITE
CLINICAL TRIALS
James S. Cordova 1,2, Eduard Schreibmann3, Constantinos G. Hadjipanayis4, Ying Guo,5 Tim Fox3 , Hui-Kuo Shu3,6,
Hyunsuk Shim1,6,*, and Chad A. Holder1,*
1
Dept. of Radiology, 2Medical Scientist Training Program, 3Dept. of Radiation Oncology, 4Dept. of Neurosurgery,
5
Dept. of Biostatistics, 6Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322
RATIONALE: Generating tumor volume measurements is a difficult component of neurosurgical extent-ofresection (EOR) assessment. We utilized data from an ongoing trial to develop a semi-automated method
for the generation of T1-weighted (T1W) contrast-enhancing tumor volumes pre- and postoperatively.
EXPERIMENTAL DESIGN: T1W MR images from glioblastoma cases and controls were segmented using
Otsu multi-level thresholding and Fuzzy C-means clustering for comparison to a specialist’s manual
contours. Agreement and inter-rater variability between segmentations was assessed using the concordance
correlation coefficient (CCC), and spatial accuracy was determined using the Dice similarity index (Dice)
and Hausdorff distance (HD).
RESULTS: Fuzzy C-means clustering with three classes (Fuzzy3) was the best method, generating volumes
with high agreement to manual contours pre- and postoperatively (CCCs = 0.996 and 0.986, respectively)
and between readers pre- and postoperatively (CCCs = 0.990 and 0.983, respectively). Spatial agreement
with manual contours was high preoperatively for Fuzzy3 with mean Dice and HDs of 0.916 and 0.142 mm,
respectively; and postoperatively, Fuzzy3 exhibited the highest spatial agreement of all algorithms.
CONCLUSION: The proposed segmentation method allows tumor volume measurements of magnetic
resonance images in the unbiased, reproducible fashion necessary for quantifying EOR in multicenter
clinical trials.
James Burkett, NS
11:45 am
NEURAL MECHANISMS OF EMPATHY-BASED BEHAVIOR IN
MONOGAMOUS PRAIRIE VOLES
James Burkett1,2, Daniel Curry1,2, Melissa Reyes1,2, Larry Young1,2
Emory University, Atlanta, GA, 2Center for Translational Social Neuroscience.
1
Consolation is a common human expression of sympathy for another’s pain. Consoling behavior has also
been naturally observed in several animal species, including some great apes, canids, corvids, and
elephants. However, no laboratory model has been established to study this behavior. Here I present the
first experimental data showing that male prairie voles (Microtus ochrogaster) express consoling behavior
under laboratory conditions. Males significantly increase their partner-directed allogrooming toward
stressed female partners, as compared to baseline and to unstressed controls. Promiscuous meadow voles
do not show this stress-evoked increase. Prairie males who observe their stressed partners show an increase
in self-grooming and an elevation of plasma corticosterone that is highly correlated to the corticosterone of
their female partner, suggesting that consolation is based on an empathy mechanism. Finally, injection of
an oxytocin receptor antagonist (OTA) into the cerebral ventricle prevents the stress-evoked increase in
allogrooming. We are also exploring the role of OTA in specific brain regions in modulating consolation.
These experiments are the beginning of a foundation for an animal model that will inform us about the
neurobiology of consolation and empathy.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [14]В В В Session IV
Life of a Cell
1:45 pm
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [15]В В В Chantel Cadwell, BCDB
В 1:45 pm
CADHERIN ENDOCYTOSIS, ADHESION, AND CYTOSKELETAL LINKAGE
COOPERATIVELY REGULATE CELL MIGRATION
Chantel M. Cadwell1, Benjamin A. Nanes1, and Andrew P. Kowalczyk1,2,3
Departments of Cell Biology1 and Dermatology2, and the 3Winship Cancer Institute, Emory University School of
Medicine, Atlanta, GA
Endothelial cell adhesion is dynamically regulated during angiogenesis, a process important for
development. Angiogenesis occurs by migration and proliferation of endothelial cells, activities that require
the modulation of adhesion. The adherens junction protein VE-cadherin mediates adhesion through
homophilic interactions via a conserved tryptophan (Trp2) in its extracellular domain. VE-cadherin is
linked to the cytoskeleton through cytoplasmic interactions with catenins. These interactions stabilize the
cadherin at the cell surface and increase adhesive strength. Previously, we determined that mutation of
specific amino acids in the VE-cadherin cytoplasmic tail prevents endocytosis of the cadherin and inhibits
cell migration. Interestingly, we now report that the inhibition of migration by this mutant is dependent
upon both adhesion and cytoskeletal linkage. Mutation of the critical Trp2 residue disrupted adhesion and
increased VE-cadherin endocytosis relative to wild type cadherin. Additionally, we observed that forced
lateral dimerization of VE-cadherin prevented endocytosis even in the absence of homophilic adhesion.
Importantly, dimerized VE-cadherin also inhibited migration in an adhesion dependent manner. Lastly,
inhibition of VE-cadherin endocytosis disrupts Golgi reorientation, a process that is important for directed
cell migration. These findings suggest that endocytosis, adhesion, and cytoskeletal linkages of VE-cadherin
cooperate to control endothelial cell migration by regulating endothelial cell polarity.
Jason Conage-Pough, CB
2:00 pm
PHOSPHORYLATION INFLUENCES THE PREFERENTIAL BINDING OF BIM
TO ANTI-APOPTOTIC PROTEINS IN MULTIPLE MYELOMA
Jason E Conage-Pough1,2,3, Vikas A Gupta, MD PhD1,3, Shannon M Matulis, PhD1,3 and Lawrence H
Boise, PhD1,2,3,4
1
Winship Cancer Institute, 2Graduate Program in Cancer Biology, 3Department of Hematology and
Medical Oncology, 4Department of Cell Biology, Emory University School of Medicine, Atlanta GA
Myeloma cells are strongly dependent on the anti-apoptotic protein Mcl-1 for survival. However,
studies have shown that some myeloma cells are sensitive to treatment with drugs that target Bcl-2 and
Bcl-xL, but not Mcl-1. These studies suggest that sensitive cells have more of the pro-apoptotic protein Bim
bound to Bcl-2/Bcl-xL than to Mcl-1. We have previously shown that the expression levels of pro- and antiapoptotic proteins have little influence on which anti-apoptotic protein binds Bim, leading us to further
explore what factors influence the preferential binding of Bim.
We determined that Bim is constitutively phosphorylated in unstimulated myeloma cells at multiple
unknown sites. We also found that the phosphorylation status of Bim affects its binding to anti-apoptotic
proteins, with phosphorylated species preferentially binding to Bcl-2/Bcl-xL, and not to Mcl-1. We
observed that the phosphorylated Bim species bound to Bcl-2 and Bcl-xL were present at a significantly
lower level in myeloma cells that were more dependent on Mcl-1, than in cells with greater Bcl-2/Bcl-xL
dependency. Lastly, we showed that inhibitors targeting the MAP kinase family affect the levels of
constitutive Bim phosphorylation, providing a potential avenue for influencing Bim distribution through
targeting of kinases and phosphatases, and improving therapeutic efficacy.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [16]В В В Session V
Receptors and Signaling
2:15 pm
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [17]В В В Gina Alesi, CB
2:15pm
RSK2-MEDIATED PHOSPHORYLATION OF STATHMIN PROMOTES
MICROTUBULE POLYMERIZATION AND PROVIDES A PRO-INVASIVE
ADVANTAGE TO METASTATIC CANCER CELLS
Gina Alesi,1 Dan Li,1 Lingtao Jin,1 Georgia Z. Chen,1 Dong M. Shin,1 Fadlo Khuri,1 and Sumin Kang1
1
Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322
Protein kinases have been implicated in mediating pro-metastatic signaling in human cancers. We
previously demonstrated that a serine/threonine kinase, p90 ribosomal S6 kinase 2 (RSK2) promotes cancer
cell invasion and migration, anoikis resistance, and tumor metastasis in human cancers. We investigated
the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell
invasion and tumor metastasis. SiRNA-mediated knockdown of RSK2 disrupted microtubule formation in
diverse metastatic human cancer cell lines. Stathmin (STMN) is a microtubule destabilizing protein that
regulates microtubule dynamics. We found that stable knockdown of RSK2 decreased phosphorylation of
STMN at Serine 16. Moreover, RSK2 directly phosphorylated STMN at Ser16 in an in vitro RSK2 kinase
assay. We observed that phosphorylation of STMN by RSK2 reduced STMN-mediated microtubule
depolymerization in vitro, while stable expression of phospho-deficient STMN S16A but not STMN wild
type led to decreased invasive potential of metastatic cancer cells. These data suggest STMN as a potential
novel RSK2 substrate/effector, which may contribute to RSK2-mediated pro-metastatic signaling.
Therefore, the RSK2-STMN pathway represents a promising therapeutic target in the clinical prognosis
and treatment of metastatic human cancers.
Jessica Konen, CB
2:30 pm
LKB1 REGULATES A MOLECULAR SWITCH THAT DRIVES 3-D LUNG
CANCER INVASION
Jessica Konen1, Adam I. Marcus2
1
Graduate Program in Cancer Biology, Emory University, Atlanta, GA, 2Hematology and Medical
Oncology, Emory University, Atlanta, GA
LKB1 is a tumor suppressor and serine/threonine kinase that is the third-most commonly mutated gene in
NSCLC. Additionally, a KrasG12DLkb1fl/fl knockout mouse had increased tumor burden and metastasis
when compared to other clinically-relevant mutant mice. Together, these data solidify LKB1 as an
important regulator of lung cancer metastasis; however, the mechanism by which LKB1 mutation results in
increased metastasis is still unknown. To dissect how LKB1 loss increases the metastatic potential of
cancer cells, we utilized a lung cancer cell spheroid model to analyze invasion into a 3-D
microenvironment in real-time. We found shLKB1 cells switch their invasive phenotype, performing a
mesenchymal to amoeboid transition (MAT). Quantification of live-cell imaging shows shLKB1 amoeboid
cells have longer tracks and significantly greater velocity than LKB1 wildtype amoeboid cells. Because
RhoA, and its effector kinase ROCK, have been implicated in regulating amoeboid motility, we examined
the effects of inhibiting myosin II (downstream of RhoA/ROCK signaling) on 3-D motility in control and
shLKB1 spheroids. We found inhibition of myosin II inhibits amoeboid morphology and results in a
significant decrease in shLKB1 amoeboid cell velocity. This work shows LKB1 loss results in MAT during
invasion, potentially providing LKB1-depleted cells a migratory advantage through the microenvironment.
В THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [18]В В В Ariana Mullin, NS
2:45 pm
NSF REGULATES SYNAPTIC HOMEOSTASIS AT THE DROSOPHILA
NEUROMUSCULAR JUNCTION
Ariana P. Mullin1,2 Avanti Gokhale1, Subrabhata Sanyal3, Victor Faundez1
1
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA, 2Graduate
Program in Neuroscience, Emory University, Atlanta, GA, USA
3
Biogen-Idec, 14 Cambridge Center, Cambridge, MA, USA
Synaptic homeostasis describes the ability of the presynaptic cell to adapt to changes in the postsynaptic
response. During this phenomenon, the presynaptic cell integrates feedback from the postsynaptic cell to
regulate neurotransmitter release properties, thus maintaining appropriate postsynaptic excitation. This type
of synaptic plasticity is crucial during neural development, as synaptic contacts are particularly dynamic.
Bloc-1 (Biogenesis of lysosome-related organelles complex 1) is an octameric complex involved in
synaptic membrane protein trafficking, but the role of Bloc-1 in neurotransmission is unclear. Recently,
however, it was shown that loss of function dysbindin, one of eight proteins that comprise Bloc-1, results in
deficits in synaptic homeostasis at the Drosophila neuromuscular junction (NMJ). Dysbindin has been
identified by multiple independent GWAS analyses as a schizophrenia-susceptibility factor, and loss of
function in dysbindin is consistently implicated as a risk factor across multiple neurodevelopmental
disorders. Here, we identify a novel role for N-ethylmaleimide sensitive factor (NSF) in synaptic
homeostasis at the NMJ. NSF biochemically and genetically interacts with the Bloc-1 complex to regulate
synaptic homeostasis. Our results suggest that interactions between protein complexes within a
biochemically defined network regulate complex neural phenotypes, such as synaptic homeostasis,
necessary for proper neuronal development.
George Kannarkat, IMP
3:00pm
THE RS3129882 SINGLE NUCLEOTIDE POLYMORPHISM AFFECTS MHC-II
EXPRESSION AND MAY INCREASE RISK FOR PARKINSON’S DISEASE BY
HEIGHTENING IMMUNE RESPONSES
George T. Kannarkat1, Darcie A. Cook1, Jianjun Chang1, Jaegwon Chung1, Jae-Kyung Lee1, Elaine M.
Sperin2, Stewart Factor2, Jeremy M. Boss3, and Malu G. Tansey1.
1
Department of Physiology, 2Emory Movement Disorders Clinic, and 3Department of Microbiology and
Immunology, Emory University School of Medicine, Atlanta, GA
The non-coding rs3129882 HLA-DRA gene single nucleotide polymorphism (SNP) was associated with
altered risk for Parkinson’s disease (PD) in genome-wide association studies and is part of the major
histocompatibility complex-II (MHC-II) locus that mediates antigen presentation. People homozygous for
the G (high risk) allele at this locus have a 1.7 fold higher risk for Parkinson’s disease. This association
suggests that alteration in antigen presentation is a potential mechanism to link environment and genetics in
conferring disease susceptibility. We have demonstrated that this SNP is linked to specific expression
patterns of MHC-II molecules in B cells and interferon- -stimulated monocytes from healthy controls and
PD patients. These findings suggest that the SNP is an immune biomarker that may predict susceptibility
for PD based on antigen presentation responses and engagement of adaptive immunity. Furthermore, we
observed a positive correlation between MHC-II expression on B cells and disease severity but not disease
duration suggesting a link to pathogenesis. This novel finding is the first direct evidence in humans that
antigen presentation via MHC molecules plays a role in sporadic PD risk.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [19]В В В Session VI
DNA and Gene Expression
3:30 pm
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [20]В В В Will Hudson, MSP
3:30 pm
THE STRUCTURAL BASIS OF DIRECT GLUCOCORTICOID-MEDIATED
TRANSREPRESSION
William H Hudson1, Christine Youn1, Eric A Ortlund1
1
Emory University School of Medicine, Department of Biochemistry
A newly discovered negative glucocorticoid response element (nGRE) mediates DNA-dependent
transrepression by the glucocorticoid receptor (GR) across the genome and has a major role in
immunosuppressive therapy. The nGRE differs dramatically from activating response elements, and the
mechanism driving GR binding and transrepression is unknown. To unravel the mechanism of nGREmediated transrepression by the GR, we characterized the interaction between GR and an nGRE in the
thymic stromal lymphopoietin (TSLP) promoter. We show using structural and mechanistic approaches that
nGRE binding is a new mode of sequence recognition by human GR and that nGREs prevent receptor
dimerization through a unique GR-binding orientation and strong negative cooperativity, ensuring the
presence of monomeric GR at repressive elements.
В Paul Musille, MSP
3:45pmВ DIVERGENT SEQUENCE TUNES LIGAND SENSITIVITY IN PHOSPHOLIPIDREGULATED HORMONE RECEPTORS
Paul M. Musille1, Manish Pathak1, Janelle L. Lauer2, Patrick R. Griffin2, and Eric A. Ortlund1
1
Emory University School of Medicine, Atlanta, GA 30322, USA 2 The Scripps Research Institute, Jupiter,
Florida, USA.
The NR5A subfamily family of nuclear receptors are important regulators of pluripotency, lipid and
glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1) and steroidogenic factor 1
(SF-1), have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor
understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical
targets.
In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL)
binding and regulation. Both human and mouse LRH-1 (mLRH-1) respond to newly discovered medium
chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate human LRH-1 by altering
receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila
orthologs contain divergent sequence in this region potentially altering PL-driven activation. Structural
evidence suggests that these sequence differences in mLRH-1 and Drosophila FTZ-f1 confer at least partial
ligand independence, making them poor models for human LRH-1 studies; however, the mechanisms of
ligand independence remain untested. We show using structural and biochemical methods that the recent
evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet
does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays
that FTZ-f1 is indeed a ligand independent receptor. This work provides a structural mechanism for the
differential tuning of PL-sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic
models for LRH-1-dependent diseases.
В THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [21]В В В Kevin Van Bortle, BCDB
4:00 pm
A SUBCLASS OF INSULATOR BINDING SITES ESTABLISH DENSE CLUSTERS
TO DELINEATE TOPOLOGICALLY ASSOCIATING DOMAINS
Kevin Van Bortle1, Mike Nichols, Li Li1,2, Chintong Ong1, Naomi Takenaka1, Zhaohui S. Qin2, Victor G.
Corces1.
1
Department of Biology, Emory University, Atlanta, GA 30322, USA, 2Department of Biostatistics and
Bioinformatics, Emory University, Atlanta GA 30322, USA
Chromatin insulators represent a unique class of regulatory elements proposed to play a role in
chromosome domain organization. Recent advances in 3C – based assays have led to novel insight into the
three dimensional arrangements of chromatin, and suggest that genomes are organized into tissue-invariant
sub-megabase (Mb) scale structures call Topologically Associating Domains (TADs). Insulator proteins are
enriched at the boundaries of TADs, yet a majority of insulator proteins localize within domains,
suggesting boundary-associated insulator elements represent a unique subclass. By mapping the genomewide binding profiles for several insulator factors in D. melanogaster, we demonstrate that insulator
proteins establish dense clusters analogous but distinct from HOT regions, called Architectural Cluster
Elements (ACEs). ACEs associate with the SMC-containing cohesin (as shown for HOT regions) and
condensin complexes, suggesting these factors may be necessary for the co-occupancy of several proteins.
ACEs correlate strongly with topological domain boundaries as well as the strength of domain interaction
separation, and represent open chromatin regions that are stably accessible throughout development,
consistent with the tissue invariant nature of TADs in humans. Finally, we uncover a similar relationship
between insulator occupancy and TADs in humans, suggesting a conserved role for clustered architectural
proteins in sub-Mb scale chromatin organization.
Mariana Mandler, BCDB
4:15 pmВ A CYTOPLASMIC QUAKING I ISOFORM REGULATES AN HNRNPF/HDEPENDENT ALTERNATIVE SPLICING PATHWAY IN MYELINATING GLIA
Mariana Mandler1, Li Ku1, Yue Feng1
1
Department of Pharmacology, Emory University
The selective RNA-binding protein Quaking I (QKI) plays important roles in controlling alternative
splicing (AS). Three QKI isoforms are broadly expressed, which display distinct nuclear-cytoplasmic
distribution. However, molecular mechanisms by which QKI isoforms control AS, especially in distinct
cell types, still remain elusive. The quakingviable (qkv) mutant mice carry deficiency of all QKI isoforms
in oligodendrocytes (OLs) and Schwann cells (SWCs), the myelinating glia of central and peripheral
nervous system (CNS and PNS), respectively, resulting in severe dysregulation of AS. We found that the
cytoplasmic isoform QKI-6 regulates AS of polyguanine (G-run)-containing transcripts in OLs and rescues
aberrant AS in the qkv mutant by repressing expression of two canonical splicing factors, heterogeneous
ribonuclearproteins (hnRNPs) F and H. Moreover, we identified a broad spectrum of in vivo functional
hnRNPF/H targets in OLs that contain conserved exons flanked by G-runs; many of which are
dysregulated in the qkv mutant. Surprisingly, AS targets of QKI-6 and hnRNPF/H in OLs are differentially
affected in SWCs, suggesting that additional cell-type-specific factors modulate AS during CNS and PNS
myelination. Together, our studies provide the first evidence that cytoplasmic QKI-6 acts upstream of
hnRNPF/H, forming a novel pathway that controls AS in myelinating glia.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [22]В В В Leila Myrick, NS
4:30 pm
PATHOGENESIS OF NOVEL FMR1 MUTATIONS IN FRAGILE X SYNDROME
Leila Myrick1, Mika Nakamoto-Kinoshita1, Xuekun Li1, Jeannie Visootsak1,2, Noralane Lindor4, Salman Kirmani5,
Peng Jin1, Xiaodong Chen3, Stephen Warren1,2,3
1
Department of Human Genetics, Emory University, Atlanta, GA, 2Department of Pediatrics, Emory University,
Atlanta, GA, 3Department of Biochemistry, Emory University, Atlanta, GA, 4Department of Medical Genetics, Mayo
Clinic, Rochester, MN, 5Department of Health Science Research, Mayo Clinic, Scottsdale, AZ
Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability (ID) and a leading known
cause of autism spectrum disorder. FXS is most often caused by a trinucleotide repeat expansion within the FMR1
gene. In more than the two decades since FMR1 was discovered, only a single pathological missense mutation has
been reported (I304N). Recently we identified 2 novel variants, G266E and R138Q, in males with ID who tested
negative for repeat expansion. To determine if these variants are pathological, we used lentivirus to infect Fmr1 KO
cells with either G266E-FMRP or R138Q-FMRP. We found that G266E behaves like a functional null and is unable
to rescue AMPAR trafficking, associate with polyribosomes, or bind mRNA. Crystallographic modeling of G266E
also indicates considerable steric structural disruption. Conversely, R138Q-FMRP was able to rescue all these
phenotypes. However, when neuronally expressed in transgenic dfmr1-deficient Drosophila, R138Q failed to rescue
synaptic overgrowth at the neuromuscular junction, suggesting this mutant impairs presynaptic function while
retaining its postsynaptic function. In conclusion, G266E is a pathological null mutation, identified in a patient with
classic FXS phenotype. R138Q retains postsynaptic FMRP function but may exhibit presynaptic specific defects.
These data demonstrate both the investigational and clinical utility of screening for conventional FMR1 mutations in
individuals with ID.
Jordan Morreall
4:45 pm
TNF-О± INHIBITS DNA REPAIR ACTIVITY OF BER VARIANT OGG1 THROUGH
OXIDATIVE STRESS
Jordan Morreall1,2, Clayton Sheppard4, Erica Werner1, Yoke Wah Kow4, Paul W. Doetsch1,3,4,5
1
Department of Biochemistry, 2Graduate Program in Genetics and Molecular Biology, 3Emory Winship
Cancer Institute, and 4Department of Radiation Oncology and 5Hematology and Medical Oncology, Emory
University School of Medicine, Atlanta, GA
8-oxoguanine (8OG) is a common, mutagenic, oxidative DNA lesion. 8OG is repaired by the base
excision repair (BER) pathway, initiated by 8-oxoguanine glycosylase 1 (Ogg1). A common Ogg1 allelic
variant is S326C-Ogg1, associated with cancer, and inhibited by oxidative stress. Oxidative stress arises
during inflammation, for which a key mediator is tumor necrosis factor alpha (TNF-О±), a cytokine driving
oxidative stress and contributing to all stages of cancer. However, inflammatory cytokines’ role in
modulating BER glycosylase variant phenotypes is poorly understood. We hypothesized that TNF-О± drives
oxidative stress causing DNA damage and inhibits S326C-Ogg1 glycosylase activity through cysteine 326
oxidation.
To measure Ogg1 activity, we incubated whole-cell lysates with 32P-labeled 8OG
oligonucleotides, quantifying intact vs. cleaved oligonucleotide fragments. In vitro, S326C-Ogg1 activity
is inhibited by oxidative stress caused by treatment with H2O2 or TNF-О±, but not after treatment with the
antioxidant N-acetylcysteine. Our in vivo model of S326C-Ogg1 activity uses the comet assay, measuring
the migration of cellular DNA via electrophoresis after Ogg1 treatment. In vivo, TNF-О± drives oxidative
stress that impairs S326C-Ogg1 activity. Our experiments suggest that TNF-О± may promote tumorigenesis
in S326C-Ogg1 backgrounds by promoting oxidative stress that inhibits S326C-Ogg1 activity, while
possibly driving mutagenic oxidative DNA damage.
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [23]В В В THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [24]В В В Poster PresentersВ Poster
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Presenter
Allen, Megan
Alter, Shawn
Barnes, Dawn
Barnhart, Katelyn
Bauer, Nicholas
Bizzell, Erica
Blanchard, Kristen
Braykov, Nikolay
Brown, Harrison
Brown, Nicole
Byun, Phil
Calderon, Brenda
Carter, Richard
Christopher, Michael
Cutler, Alicia
Deveau, Todd
Deymier, Martin
Donlin-Asp, Paul
Dunn, Amy
Gaudette, Brian
Gavile, Catherine
Harrell, Constance
Huang, Brenda
Hunter, Emily
Hwang, Juyeon
Infield, Daniel
Kohn, Jordan
Limpose, Kristin
Lohr, Kelly
Meng, Jia
Program
BCDB
MSP
BCDB
CB
BCDB
MMG
GMB
PBEE
MSP
MSP
GMB
BCDB
GMB
GMB
BCDB
NS
MMG
BCDB
NS
IMP
IMP
NS
GMB
BCDB
BCDB
MSP
NS
CB
NS
MMG
Poster
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Presenter
Miller-Kleinhenz, Jasmine
Morris, Kevin
Noble, Donald
Peake, Bridgette
Perez Diaz, Maylen
Perszyk, Riley
Reding, Katherine
Rha, Jennifer
Rose, Samuel
Ruppersburg, Chelsey
Rye, Emily
Salgueiro, Alessandra
Sanabria-Figueroa,
Eduardo
Schmidt, Karl
Schureck, Marc
Seladi-Schulman, Jill
Shah, Shardule
Sia, Jonathan
Stout, Kristen
Torres, E. Shannon
Umstead, MaKendra
Vachon, Virginia
Vinal, Kellie
Wasson, Jadiel
Weir, David
Wendt, Amanda
Wilkinson, Scott
Williams, Kathryn
Woodworth-Hobbs, Myra
Program
CB
BCDB
NS
MSP
NS
MSP
NS
BCDB
NS
BCDB
BCDB
CB
MSP
NS
BCDB
MMG
IMP
IMP
MSP
GMB
CB
MMG
MMG
BCDB
CB
NHS
CB
BCDB
NHS
POSTER SESSION: Odd Numbers, 12:00 – 12:45PM & Even Numbers, 12:45 – 1:30PM
GDBBS Programs
Biochemistry, Cell & Developmental Biology( BCDB)
Cancer Biology (CB)
Genetics & Molecular Biology (GMB)
Immunology & Molecular Pathogenesis (IMP)
Microbiology & Molecular Genetics (MMG)
Molecular & Systems Pharmacology (MSP)
Neuroscience (NS)
Nutrition & Health Sciences (NHS)В Population Biology, Ecology & Evolution (PBEE)
THEВ ELEVENTHВ ANNUALВ DSACВ STUDENTВ RESEARCHВ SYMPOSIUMВ [25]В В В Megan Allen, BCDB
Poster #1
POST-TRANSCRITIONAL REGULATION OF CDK5 ACTIVITY IN BRAIN NEURONS BY THE
RNA BINDING PROTEIN HUD
Megan Allen, Wenqi Li, Guanglu Liu, Andrew Bankston, Wei Feng, Yue Feng
Department of Pharmacology, Emory University, Atlanta, GA 30322
Activity of Cyclin dependent kinase 5 (Cdk5) is essential for normal brain function and is dysregulated in
neurological diseases. The abundance of p35 and p39, either of which can bind and activate Cdk5, controls
Cdk5 activity. In neurons, the role of p35 is well-characterized, while p39 function remains unknown. We
demonstrate that up-regulation of p39 expression is responsible for Cdk5 activity during neuronal
development. Additionally, we show that the neuronal RNA-binding protein, HuD, binds p39 mRNA and
regulates abundance of the p39 transcript through AU-Rich elements in the p39 3’UTR. In mice which harbor
elevated hippocampal HuD expression (HuDtg+), p39 mRNA and protein are significantly up-regulated and
we observe increased Cdk5 activity. Consistent with a role for Cdk5 in promoting axonal growth, mossy
fibers of the hippocampus are over-projected in HuDtg+ mice, a phenomenon commonly observed in epilepsy
models. We found that HuDtg+ mice respond more severely to kainic acid induced seizure than WT
littermates. Importantly, elimination of p39 rescues mossy fiber and seizure phenotypes in HuDtg+ mice.
These studies identify a functional role for p39 and a novel mechanism controlling Cdk5 activity in neurons,
providing clues for future studies investigating Cdk5 in the normal brain and in neurological disease.
Shawn Alter, MSP
Poster #2
SEROTONERGIC DISRUPTIONS IN THE VMAT2-DEFICIENT MOUSE MODEL OF
PARKINSON'S DISEASE
Shawn Alter, Tonya N. Taylor, Minzheng Wang, and Gary Miller
Department of Environmental Health, Emory University, Atlanta, GA
Serotonergic denervation and loss of the raphe nuclei occur in the progression of Parkinson’s disease (PD).
The selective vulnerability of monoaminergic neurons in PD is in part thought to be due to their susceptibility
to oxidative stress and the instability of their neurotransmitters in the cytosol. Mice with very low expression
(5%) of the vesicular monoamine transporter 2 (VMAT2) undergo progressive nigrostriatal degeneration,
have decreased monoamines, increased monoamine turnover, and exhibit both motor and nonmotor symptoms
of PD. Neurochemically, VMAT2- deficient mice have reductions in tissue serotonin in the striatum (85%),
hippocampus (90%), and cortex (80%), and increased turnover to 5-hydroxyindoleacetic acid, as measured by
HPLC. Here we report the effects of VMAT2 hypomorphy on the integrity of the serotonergic system.
Immunohistochemical analyses of serotonergic neurons in the dorsal and median raphe nuclei indicate normal
morphological development.
We will present evaluation of serotonin transporter expression in efferent regions throughout the brain.
Additionally, we will show that VMAT2-deficient mice have increased behavioral indices of depressive
behavior in the forced swim and tail suspension tests that are responsive to the SSRI fluoxetine. This work
demonstrates the critical role of vesicular sequestration to the viability of monoaminergic neurons.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [26]В Dawn Barnes, BCDB
Poster #3
COMPLEXITY OF TROPOMYOSIN ISOFORMS IN C. ELEGANS GENERATED BY
ALTERNATIVE SPLICING OF THE LEV-11 GENE
Dawn Barnes1,2, Shoichiro Ono1,2
1
Pathology, Emory University; 2Cellular Biology, Emory University, Atlanta, GA, USA
Tropomyosin is a coiled-coil dimer that lies along actin filaments in both muscle and non-muscle cells.
Dimers can connect to form an elongated polymer. Tropomyosin is involved in cellular processes including
cell motility, cell division, and muscle contraction. In mammals, more than 40 isoforms of tropomyosin are
expressed from 4 genes through extensive alternative splicing. We utilized Caenorhabditis elegans to study
functional diversity of tropomyosin isoforms. In C. elegans, previous studies reported 4 isoforms are
expressed from a single gene, lev-11, by alternative promotors and splicing. Two high-molecular-weight
(HMW) isoforms are expressed from the upstream promoter and encoded by 9 exons; whereas 2 lowmolecular-weight (LMW) isoforms are expressed from the downstream promoter and encoded by 7 exons.
Through analysis of Expressed Sequence Tag sequences and RT-PCR, we identified a novel exon (Exon 7a)
that is alternative to Exon 7b and HMW transcript containing Exon 7a. Additional RT-PCR analysis suggests
that Exon 7a is also utilized in a transcript driven by the downstream promoter to produce an LMW isoform.
To characterize biochemical properties, we established a system for the expression and purification of
recombinant tropomyosin isoforms. Two HMW isoforms containing Exon 7b bound actin filaments with
similar affinity.
Katelyn Barnhart, CB
Poster #4
DUAL INHIBITION OF MCL-1 BY THE COMBINATION OF CARFILZOMIB AND TG02 IN
MULTIPLE MYELOMA
Katelyn G. Barnhart1*, Shannon M. Matulis, Ph.D.2*, Sadae Hitosugi, MS3*, Cathy Sharp, R.N.4*, Francis
Burrows, PhD5*, Ajay K. Nooka, MD, MPH6, Jonathan L. Kaufman, MD6, Sagar Lonial, M.D.6 and
Lawrence H. Boise, PhD7
1
Cancer Biology Graduate Program, Emory University, Atlanta, GA; 2Hematology and Medical Oncology
and Winship Cancer Institute, Emory University, Atlanta, GA; 3Hematology and Medical Oncology, Emory
University, Atlanta, GA;4Winship Cancer Institute of Emory University, Atlanta, GA; 5Tragara
Pharmaceuticals, San Diego, CA; 6Winship Cancer Institute, Emory University, Atlanta, GA; 7Hematology
and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA
Carfilzomib (KyprolisВ®) is an FDA-approved proteasome inhibitor for single-agent use in
relapsed/refractory multiple myeloma (MM). We combined carfilzomib with TG02, a multi-kinase inhibitor.
Both independently target Mcl-1 and MM cells are dependent on it for survival.
Four different human myeloma cell lines were treated for 24 hours with carfilzomib +/- TG02. Cotreatment resulted in additive apoptosis in all lines. Carfilzomib pulse dosing was performed to mimic
pharmacokinetics. Similar activity was observed with continuous and pulse dosing. Activity of the
combination was also observed in freshly isolated samples from relapsed/refractory patients. Co-culture with
Hs-5 cells protected against combination treatment in three lines, while addition of Hs-5 conditioned medium
was protective in RPMI-8226.
We then determined the molecular basis for increased apoptosis. Treated cells were isolated for western
blot and RT-qPCR analysis to determine Bcl-2 family protein levels. Carfilzomib treatment caused an
increase in NOXA mRNA. TG02 treatment resulted in decreased Mcl-1 protein but not mRNA expression.
Mcl-1 loss at the protein level occurs in the presence of carfilzomib, therefore, the effect of TG02 is unlikely
due to increased degradation. Mcl-1 translational regulation is a likely mechanism. These data suggest Mcl-1
dual inhibition is active in myeloma and warrants further preclinical testing.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [29]В Nicholas Bauer, BCDB
Poster #5
AUTOMATED QUANTIFICATION OF THE SUBCELLULAR LOCALIZATION OF MULTICOMPARTMENT BASE EXCISION REPAIR PROTEINS VIA Q-SCAN
Nicholas C. Bauer1,2, Anita H. Corbett1,3, Paul W. Doetsch1,3,4,5
1
Department of Biochemistry, 2Graduate Program in Biochemistry, Cell and Developmental Biology,
3
Winship Cancer Institute, 4Department of Radiation Oncology, 5Department of Hematology and Medical
Oncology, Emory University School of Medicine, Atlanta, GA
Defects in various components of base excision repair (BER) lead to increased mutation frequencies and
genetic/genomic instability. Although the biochemical mechanism of BER is well-characterized, few studies
have addressed how this critical pathway is regulated. Many BER proteins localize to both the nucleus and
mitochondria, and changes in localization have been observed in Saccharomyces cerevisiae Ntg1 in response
to oxidative stress. We hypothesize that dynamic localization is a general mode of regulation for BER.
However, quantifying the extent of DNA repair protein localization is a major barrier to elucidating this
potential regulatory mechanism. To address this issue, we developed a novel technique termed Quantitative
Subcellular Compartmentalization Analysis (Q-SCAn). This analysis technique uses markers to define
subcellular compartments and then quantifies the relative amount of target protein in each compartment of
each cell. To validate Q-SCAn, we analyzed relocalization of the transcription factor Yap1 following
oxidative stress and then extended the approach to multi-compartment localization by examining two DNA
repair proteins critical for the base excision repair pathway, Ntg1 and Ung1. Our findings demonstrate the
utility of Q-SCAn for quantitative analysis of the subcellular distribution of multi-compartment proteins, and
has revealed new information about the regulation of Yap1, Ntg1 and Ung1.
Erica Bizzell, MMG
Poster #6
CHARACTERIZATION OF THE
MYCOBACTERIUM TUBERCULOSIS.
SECRETED
SERINE
PROTEASE,
RV2223C
OF
Erica Bizzell,1,2, Maria Georgieva1,2 and Jyothi Rengarajan1,3
Emory Vaccine Center. 2Microbiology and Molecular Genetics Program, Graduate Division of Biological
and Biomedical Sciences, Emory University. 3Division of Infectious Diseases, Emory University.
1
Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to evade host immune defenses. Several
pathogenic bacteria use extracellularly secreted proteases to regulate processes ranging from repression of
cytokine production to degradation of surface-associated host proteins. Mtb encodes several putative secreted
proteases whose functions are poorly understood. Our lab is characterizing the predicted Mtb protease,
Rv2223c. Rv2223c is located in a predicted operon with cell wall-associated serine protease, Hip1
(Rv2224c), with which it shares 52% amino acid identity. Hip1 is involved in modification of the Mtb cell
wall during infection, and functions by dampening normal immune responses to Mtb infection. Using the
model system Mycobacterium smegmatis expressing Mtb Rv2223c, we found that Rv2223c is secreted from
mycobacterial cells in a signal-sequence dependent manner and undergoes autoproteolytic cleavage upon
secretion. Additionally, we have observed that Rv2223c interacts with the Hip1 physiological substrate,
GroEL2, suggesting potentially overlapping or cooperative functions of these proteases. We hypothesize that
Rv2223c is a secreted protease that functions by cleaving Mtb substrates and/or host cell substrates to modify
host responses during Mtb infection. Further work is currently being conducted to determine the enzymatic
activity of Rv2223c and to identify novel bacterial and host interactors and/or substrates.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [30]В Kristen Blanchard, GMB
Poster #7
ENZYME EVOLUTION: EXAPTATION IN THE E. COLI GENOME
Kristen Blanchard, Ichiro Matsumura
Department of Biochemistry, Emory University
How do new catalytic functions evolve? I hypothesize that enzymes often evolve through exaptation proteins that have evolved towards one catalytic goal can be co-opted for a secondary purpose. Our lab has
previously demonstrated that many proteins in E. coli have weak secondary activity that can be revealed
through expression at high levels on multicopy plasmids. When these promiscuous enzymes are expressed at
very high levels, their secondary activity is sufficient to rescue a lethal deficiency. I hypothesize that these
promiscuous proteins can also be exploited in their native state on the E. coli chromosome. To this end, I have
mutagenized E. coli conditional auxotrophs and selected for rescues of the lethal phenotype. By discovering
which mutations rescue the auxotrophs, it should be possible to discover new examples of proteins with
secondary activity. This work should demonstrate that the E. coli proteome contains ample candidates for
exaptation; many proteins exist which have evolved towards one primary function, but under selective
conditions may be evolved to perform a second function. Such a finding would aid in the understanding of
how new catalytic functions emerge, and would also help change the way proteins are evolved in the lab.
Nikolay Braykov, PBEE
Poster #8
IMPACT OF ANTIBIOTIC EXPOSURE AND OTHER MANAGEMENT PRACTICES ON THE
PHENOTYPIC AND GENOTYPIC COMPOSITION OF DRUG-RESISTANT E. COLI IN BROILER
CHICKENS
Nikolay Braykov1, Lixin Zhang2, William Cevallos2, Natalia Burbano, Nadia LГіpez, Carl Marrs2, Betsy
Foxman2, Gabriel Trueba, Joseph Eisenberg2, Karen Levy4
1
Emory University Dept. of Biology, Atlanta, GA, United States, 2 University of Michigan Dept. of
Epidemiology, Ann Arbor, MI, United States, 3 Universidad San Francisco de Quito, Quito, Ecuador, 4 Emory
University Dept. of Environmental Health, Atlanta, GA, United States
Non-therapeutic use of antibiotics in animal husbandry promotes the development of antibiotic
resistance (AR), contributing to an environmental reservoir of resistance genes and potentially threatening
human health. A factorial experiment was conducted in a chicken farm in Northern Ecuador to describe the
emergence, phenotypic and genotypic diversity of AR-Escherichia coli in broilers raised under different
conditions. Seventy-two newly hatched chickens raised in single, 6-chicken and 34-chicken cages were
administered tetracycline via water on days 9 and 24, and sampled on days 1, 19-21 and 38. Susceptibility to
twelve antibiotics was categorized using disk diffusion. Isolates were fully genotyped on a microarray
platform. In preliminary data from 559 isolates, we found that >80% of samples were resistant to at least one
antibiotic on day 1, suggesting chickens arrived at the facility pre-colonized. Prevalence of AR decreased over
time without statistically significant difference between treatment and controls. There was a drastic increase
in the number of unique genotypes after day 1. Time was more important than selection pressure or number of
chickens/cage in explaining the phenotypic and genotypic community composition of samples, suggesting
that physiological factors or external environmental colonization play a bigger role in shaping the ecology of
AR E. coli.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [31]В Harrison Brown, MSP
Poster #9
BIOENGINEERED COAGULATION FACTOR VIII ENABLES LONG-TERM CORRECTION OF
MURINE HEMOPHILIA A FOLLOWING LIVER-DIRECTED ADENO-ASSOCIATED VIRAL
VECTOR DELIVERY
Harrison C. Brown, J. Fraser Wright, Shangzhen Zhou, Jordan E. Shields H. Trent Spencer, Christopher B.
Doering
Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy
applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of
obstacles impede the development of a similar approach for hemophilia A. These include 1) the large
transgene size of factor VIII (fVIII), 2) humoral immune responses to the transgene product, 3) inefficient
biosynthesis of human coagulation factor VIII, and 4) dose limitations imposed by capsid mediated cytotoxic
immunity. To address the latter two of these concerns, our laboratory has developed a bioengineered fVIII
molecule, designated ET3, which demonstrates 10-100 fold improvement in fVIII biosynthesis in
recombinant expression systems. When constructed within an oversized AAV vector expression cassette, the
ET3-AAV genomes packaged into viral particles primarily as partial genome fragments. Despite the potential
reduction in potency caused by genome fragmentation, a single peripheral vein injection the oversized ET3AAV vector into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at
vector doses lower than those of smaller, previously reported fVIII constructs, thus demonstrating that ET3
improves vector potency thereby mitigating two of the critical barriers remaining to AAV gene therapy for
hemophilia A.
Nicole Brown, MSP
Poster #10
INTEGRATION OF H-RAS AND GО±i SIGNALING PATHWAYS BY REGULATOR OF G
PROTEIN SIGNALING 14 (RGS14)
Nicole E. Brown1, Christopher P. Vellano1, Devrishi Goswami2, Patrick R. Griffin2, and John R. Hepler1
1
Department of Pharmacology, Emory University 2Department of Molecular Therapeutics, the Scripps
Research Institute – Scripps Florida
Regulator of G protein signaling 14 (RGS14) is a scaffolding protein that integrates G protein and H-Ras
signaling pathways. RGS14 possesses an RGS domain that binds active GО±i/o-GTP subunits to promote GTP
hydrolysis and a G protein regulatory (GPR) motif that binds inactive GО±i1/3-GDP subunits to form a stable
complex at cellular membranes. RGS14 also contains two Ras-binding domains (RBDs), the first of which
(RBD1) binds activated H-Ras. Here we show RGS14 interactions with H-Ras-GTP are regulated by inactive
GО±i1-GDP. Using bioluminescence resonance energy transfer (BRET), we show RGS14-Luc and active HRas(G/V)-Venus exhibit a robust BRET signal that is enhanced in the presence of inactive GО±i1-GDP, but not
active GО±i1-GTP. Moreover, experiments using hydrogen deuterium exchange (HDX) indicate binding of
inactive GО±i1-GDP through the GPR motif stabilizes RBD1, potentially regulating interactions with activated
H-Ras-GTP. HDX also indicated changes in the RGS domain upon binding GО±i1-GDP suggesting RGS
activity may be regulated by interactions with GО±i. Further BRET studies examining the RGS domain using
Luc-RGS14 and aluminum fluoride (AlF4-) activated GО±i1-YFP demonstrate a robust BRET signal that is
decreased in the presence of activated H-Ras(G/V). Together, these studies highlight the interdomain
regulation of RGS14 by its binding partners, GО± and H-Ras.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [32]В Phil Byun, GMB
Poster #11
GENETIC ANALYSIS OF TISSUE INVASION DRIVEN BY THE DROSOPHILA NCOA-1,2,3
HOMOLOG TAIMAN
Phil K. Byun1, Kenneth H. Moberg1
Emory University
1
Cancer cells spread from the primary tumor and seed new tumors in other organs and tissues through a
process called metastasis. While the gross morphologic changes associated with metastasis are well defined,
specific pathways that trigger these �motogenic’ phenotypes in vivo are not. Model systems, such as
Drosophila melanogaster, have been critical in providing powerful genetics tools to define such pathways. We
have discovered that ectopic expression of the Drosophila steroid-receptor transcriptional coactivator protein
Taiman (Tai) is sufficient to drive non-invasive pupal wing disc cells to invade into adjacent thoracic tissue.
Tai normally supports invasive and motile behavior of ovarian border cells during the oocyte maturation but
little is known about the transcriptional targets or interacting cofactors involved in this process. We find that
Tai binds directly to the Hippo pathway target and transcriptional co-activator Yorkie, whose vertebrate
homolog Yap1 promotes metastasis in mouse models of breast cancer. In light of these data, we hypothesize
that Tai functions with specific factors such as Yorkie to promote the invasion of wing cells, and that a
genetic screen for modifiers of this phenotype will define pathway(s) through which Tai controls cell motility.
Brenda Calderon, BCDB
Poster #12
STRUCTURAL ANALYSIS OF NC886, A CELLULAR NON-CODING RNA REGULATOR OF THE
ANTIVIRAL KINASE PKR
Brenda M. Calderon1,2, and Graeme L. Conn2
1
Biochemistry, Cell and Developmental Biology Program, Emory University, Atlanta, GA 2Department of
Biochemistry, Emory University, Atlanta, GA
The double-stranded (ds)RNA-activated protein kinase (PKR) senses dsRNA produced during viral
replication and acts as the first line of defense in the innate immune response to viral infection. Activated
PKR phosphorylates the eukaryotic initiation factor 2 (eIF2), halting protein synthesis and viral replication.
How PKR basal activity is regulated in the absence of viral infection is unknown. Recently, the cellular noncoding RNA 886 (nc886) was discovered and proposed to act as a novel, endogenous regulator of PKR. We
hypothesize that nc886 regulates PKR by binding to the monomeric form and preventing the dimerization
step of activation. This maintains PKR in an inactive state that is poised for a rapid response to viral infection.
Our goal is to define the molecular mechanism of nc886-mediated PKR inhibition using RNA structure
probing and mutational analyses combined with binding and activity assays. We show that nc886 RNA exists
as two stable, structurally and functionally distinct conformations. One conformation binds and inhibits PKR
autophosphorylation while the other lacks both activities. By delineating the structural basis for PKR
interaction with the active nc886 conformer, we will uncover the structural motifs that control PKR inhibition
and determine how PKR is poised to act in the antiviral response.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [33]В Richard Carter, GMB
Poster #13
NEURAL PROGENITOR CELLS DERIVED FROM TRANSGENIC HUNTINGTON’S DISEASE
MONKEY INDUCED PLURIPOTENT STEM CELLS DEVELOP DISEASE CELLULAR
PHENOTYPES
Richard L. Carter1,2,3 , Chen YJ2 , Kunkanjanawan T3,4, Chan AWS1,2,3*
1
Genetics and Molecular Biology Program, Graduate Division of Biological and Biomedical Sciences, Emory
University 2 Department of Human Genetics, Emory University School of Medicine 3 Division of
Neuropharmacology and Neurologic Diseases, Yerkes National Primate Research Center Emory University,
Atlanta, GA 30329, USA. 4 Embryo Technology and Stem Cell Research Center, School of Biotechnology,
Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of
CAG repeats in the coding region of the Huntingtin (HTT) gene. A major focus of HD research is on
understanding and modeling mechanisms contributing to the degeneration of neuronal populations.
Pluripotent cellular models are emerging as a powerful tool to understand disease related changes as neurons
develop, therefore we describe the use of neural progenitor cells (NPCs) derived from transgenic HD
monkeys as a cellular platform for pathological and therapeutic study in HD. We have derived NPCs from
iPSCs of an HD monkey (rHD-NPCs). rHD-NPCs were capable to differentiate to GABA positive neurons in
vitro and in vivo. rHD-NPCs showed disease associated susceptibility to cell stress demonstrated by increased
apoptosis. Upon differentiation to neurons, HD neural cells developed cellular features of HD including the
formation of nuclear inclusions and accumulation of oligomeric mutant HTT aggregates. The cell death
phenotype and mutant protein aggregation was rescued upon siRNA mediated reduction of HTT transcript,
and after treatment with memantine in culture. These findings highlight the utility of iPSCs derived NPCs
from transgenic HD monkeys in providing a unique non-human primate platform for studying HD
pathogenesis and therapeutic strategies.
Michael Christopher, GMB
Poster #14
THE HISTONE DEMETHYLASE LSD1/KDM1A IS A MASTER REGULATOR OF THE STEM
CELL FATE
Michael A. Christopher1,3, Dexter A. Myrick2,3, and David J. Katz3
1
Genetics and Molecular Biology, 2PREP, 3Department of Cell Biology
During transcription, stem cell genes acquire the histone modification dimethylation of lysine 4 on histone H3
(H3K4me2). It has previously been shown that LSD1 is necessary to repress stem during stem cell
differentiation by removing H3K4me2. Based on this, we investigated the role of LSD1 during neural stem
cell (NSC) differentiation in the mouse. Deletion of Lsd1 in NSCs results in perinatal lethality within 24
hours of birth. We find that cultured motor neurons from these mutants continue to express the stem cell
genes Nestin and Sox2. These findings suggest that LSD1 is generally required to repress the stem cell
network during neural stem cell differentiation. To determine if LSD1 continues to represses the stem cell
network in differentiated cells, we inducibly deleted LSD1 throughout the adult mouse. Surprisingly, loss of
LSD1 results in paralysis and death in as little as 4 weeks with massive neuronal cell death in the
hippocampus and surrounding cortex. Additionally, immunohistochemistry demonstrated the reactivation of
the master pluripotency gene Oct4 exclusively in these dying neurons. These data suggest that LSD1 is
continually required to repress the stem cell network in differentiated neurons. As a result, we conclude that
LSD1 is a master regulator of the stem cell fate.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [34]В Alicia Cutler, BCDB
Poster #15
NUCLEAR IMPORT AND PERMEABILITY IN MULTINUCLEATED SKELETAL MUSCLE
CELLS
Alicia Cutler1, Jennifer Jackson1, Justin Ho1, and Grace Pavlath1
1
Department of Pharmacology, Emory University
Skeletal muscle is critical for survival and quality of life. Proper gene expression is essential for myogenesis
and maintenance of skeletal muscle. Proteins that regulate gene expression are synthesized in the cytoplasm
and cross the nuclear pore to access their targets in the nucleus. The intact nuclear pore allows free diffusion
of small molecules between the nucleus and cytoplasm but excludes molecules larger >40kDa. To cross the
nuclear pore, larger proteins must be bound to a nuclear transport receptor, which recognizes a nuclear
localization signal (NLS) within the cargo protein. During myogenesis, myoblasts differentiate into myocytes,
which fuse to form mature myotubes containing thousands of nuclei. Our results demonstrate that nuclear
properties vary during myogenesis and aging. During myogenesis the ability of nuclei to import a protein
containing the classical NLS (cNLS), changes with differentiation. Almost all myoblast nuclei are cNLS
import-competent; however, only 38% of myocyte nuclei are cNLS import-competent. As myocytes develop
into mature myotubes, cNLS import-competence is restored. Additionally, as skeletal muscle ages, the
percentage of myonuclei permeable to >40kDa molecules not bound to nuclear transport receptors increases.
These results suggest nuclear characteristics are connected to stages of muscle development and aging
potentially affecting gene expression.
Todd Deveau, NS
Poster #16
INTRANASAL ADMINISTRATION OF INDUCED-PLURIPOTENT STEM CELL DERIVED
NEURAL PROGENITORS IMPROVES FUNCTIONAL RECOVERY FOLLOWING ISCHEMIC
STROKE IN MICE
Todd C. Deveau1, Katharine Yuengling1, Xiaohuan Gu1, Shan Ping Yu1, and Ling Wei1
1
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA 30322
Induced Pluripotent Stem (iPS) cells are currently under extensive examination in cellular biology and disease
treatment investigations. iPS cells can differentiate into neurons and non-neuronal cells in vitro and after
transplantation into the ischemic brain of experimental stroke animal models. The present study investigates
intranasal administration (iNA) of induced pluripotent stem cell-derived neural progenitors (iNA-iPS-NPCs)
to improve functional recovery following ischemic stroke. Adult male mice were subjected to a focal
ischemic insult primarily in the barrel cortex. Vehicle or iPS-NPCs were administered into the nasal passages
of mice 1-3 hours after stroke. At day 3 and 7 after transplant, there was a trend in local cerebral blood flow
(LCBF) increase in the ischemic region of animals that received iNA- iPS-NPCs compared to vehicle.
Animals who received iNA- iPS-NPC administration showed significant improvements in functional recovery
compared to vehicle at day 7. Current experiments are examining functional recovery at even delayed time
points after stroke. Further analysis will examine gene regulation and signaling pathways using WB, RTPCR, and histological techniques to gain insight into the mechanisms behind iNA-iPS-NPC treatment. Our
available results suggest that intranasal administration of iPS-derived neural progenitors is a viable treatment
approach for stroke and other CNS disorders.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [35]В Martin Deymier, MMG
Poster #17
GENETIC & PHENOTYPIC CHARACTERISTICS OF SUBTYPE C FULL LENGTH GENOME
HIV-1 FROM LINKED HETEROSEXUAL TRANSMISSION PAIRS
Martin J. Deymier1, Zachary S. Ende1, Daniel T. Claiborne1, William Kilembe2, Susan Allen1,2, and Eric
Hunter1,2.
1
Emory University, Atlanta, GA, USA, 2Zambia Emory HIV Research Project, Lusaka, Zambia2
In 80-90% of heterosexual transmissions of HIV, a genetic bottleneck occurs, in which an individual, with a
diverse viral quasispecies, transmits a single viral variant, the Transmitted/Founder (TF), to a naГЇve host.
Previous genetic studies of the HIV envelope glycoprotein have suggested a non-stochastic process for
transmission and evidence for selection of a specific TF variant. Although some genetic signatures have been
found, no biological correlate for TF variants have thus far been elucidated. We have performed HIV near
full-length single genome amplification followed by direct sequencing, from plasma of six subtype C acutely
infected individuals and each of their chronically infected linked partners from the Zambia-Emory HIV
Research Project. We provide genetic evidence for the selection of TF variants that more closely resembles
the Most Recent Common Ancestor from the donor quasispecies than the median of the donor variants
(p=0.0313). The generation of full-length infectious molecular clones from these viruses has provided us with
the unique observation, for one transmission pair, that the TF virus is not necessarily the highest replicating
virus from the donor quasispecies. The genetic bottleneck thus provides an opportunity for understanding the
unique properties of TF viruses and may provide potential targets for intervention.
Paul Donlin-Asp, BCDB
Poster #18
THE SURVIVAL OF MOTOR NEURON PROTEIN IN mRNP COMPLEX ASSEMBLY
Paul G. Donlin-Asp1,2, Claudia Fallini2,4, Gary J. Bassell2,3, Wilfried Rossoll2.
1
Biochemistry, Cell and Developmental Biology Graduate Program, Emory University Laney Graduate
School, 2Dep. of Cell Biology and Center for Neurodegenerative Disease, 3Dep. of Neurology, Emory
University School of Medicine, Atlanta, GA, 4Dep. of Neurology, University of Massachusetts Medical
School, Worcester, MA
Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by a specific degeneration of motor
neurons. SMA results from a reduction in the survival of motor neuron (SMN) protein, which is ubiquitously
expressed with a well characterized role in promoting the assembly of spliceosomal small nuclear
ribonucleoproteins (snRNPs). While underlying defects in splicing have been observed in SMA models, these
defects are not unique to motor neurons, leaving their role in motor neuron degeneration unclear. We and
others have demonstrated that SMN colocalizes with various mRNA binding proteins known to play vital
roles in mRNA transport. Additionally, a defect in localization of mRNA and mRNA-binding proteins in the
axon terminus in motor neurons from the SMA mouse model has been observed. These and other data suggest
that SMN plays a non-canonical role in mediating the assembly and delivery of mRNA and protein associated
complexes (mRNPs). We have optimized trimolecular fluorescence complementation (TriFC) as a novel
approach to visualize the assembly of mRNPs in cultured motor neurons derived from control and SMA
embryos. Our results show a marked reduction in TriFC fluorescent signal in SMA motor neurons, suggestive
that SMA motor neurons are deficient in mRNP complex assembly.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [36]В Amy Dunn, NS
Poster #19
GENETIC MANIPULATION OF VESICULAR FUNCTION AS A POTENTIAL MEDIATOR OF
DOPAMINERGIC NEURO-TOXICANT VULNERABILITY
Amy R. Dunn1,3, Kelly M. Lohr1,3, Kristen A. Stout2,3, Alison I. Bernstein3, Thomas S. Guillot3, Minzheng
Wang3, Yingjie Li3, Ali Salahpour4, Gary W. Miller3,4.1Neuroscience Graduate Program, Emory University,
2
Molecular and Systems Pharmacology Graduate Program, Emory University, 3Department of Environmental
Health, Emory University, 4Center for Neurodegenerative Disease, Emory University, 5Department of
Pharmacology and Toxicology, University of Toronto.
Proper vesicle function is critical for dopamine neuron health and contributes to neuron integrity after toxicant
exposure. Dopaminergic vesicles have two important roles: packaging transmitter for synaptic transmission,
and sequestering toxicants to prevent cell damage. Various proteins maintain vesicular function, and the
current study focuses on two (vesicular monoamine transporter 2 (VMAT2) and the synaptic vesicle
glycoprotein 2C (SV2C)). Our lab has previously shown that impaired vesicular function causes dopamine
mishandling and cell death, but the converse—enhanced vesicular function—had been previously unexplored.
The current investigation explores the consequences of overexpressing VMAT2 in a transgenic mouse model
(VMAT2-HIs). Analysis of vesicular morphology by electron microscopy indicates that VMAT2
overexpression increases vesicular volume by a third, which likely underlies neurochemical changes and
resistance to toxicants. A novel target, SV2C, is a pharmacologically-targetable, undercharacterized protein
thought to be involved in vesicular storage and release. It is restricted to dopaminergic brain regions, and
SV2C genotype mediates the protective effect of nicotine against Parkinson’s disease. Here, we describe and
characterize our SV2C-KO mouse. VMAT2 overexpression and SV2C-KO are excellent tools with which we
will investigate the effect of altered vesicular function on the vulnerability of dopaminergic degeneration after
toxicant exposure.
Brian Gaudette, IMP
Poster #20
HETEROGENEOUS BCL-2 FAMILY EXPRESSION IN WALDENSTRГ–M
MACROGLOBULINEMIA DETERMINES RESPONSE TO AGENTS OF INTRINSIC APOPTOSIS.
Brian T. Gaudette1,3,4, Kasyapa S. Chitta, Ph.D.5, StГ©phanie Poulain6, Bhakti Dwivedi, Ph.D.3, Doris Powell2,3, Kelvin P.
Lee, M.D.7, Asher A. Chanan-Khan, M.D.8, Stephen M. Ansell, M.D., Ph.D.9, Xavier Leleu, M.D.10, Ph.D., Jeanne
Kowalski, Ph.D.3, Paula Vertino, Ph.D.2,3, Sagar Lonial, M.D.1,3, Lawrence H. Boise, Ph.D.1,3,4
1
Departments of Hematology and Medical Oncology and Cell Biology, 2Department of Radiation Oncology, 3Winship
Cancer Institute, 4Graduate Program in Immunology and Molecular Pathogenesis - Emory University, Atlanta, GA
30322; 5Cancer Biology, Mayo Clinic, Jacksonville, FL; 6Service d'HГ©matologie Immunologie CytogГ©nГ©tique, Hopital
de Valenciennes, Valenciennes, France; 7Immunology, Roswell Park Cancer Institute, Buffalo, NY; 8Hematology and
Oncology, Mayo Clinic, Jacksonville, FL; 9Hematology and Oncology, Mayo Clinic, Rochester, MN; 10Service des
Maladies du Sang, Hopital Claude Huriez, CHRU Lille, Lille, France
Waldenström Macroglobulinemia (WM) is a disorder of lymphoplasmacytoid cells that inhabit lymph nodes
and the bone marrow. 91% of WM cases carry an activating mutation of MyD88 (L265P). Having observed
previously that plasmablasts driven to differentiate using the TLR4 ligand LPS enter a Bcl-xL-dependent state,
we sought to determine if WM cells were Bcl-xL-dependent. We treated three WM cell lines all harboring the
MyD88 (L265P) mutation with ABT-737, bortezomib and arsenic trioxide (ATO) all agents that induce
apoptosis through the intrinsic pathway. While all three lines were insensitive to ABT-737, one line was
insensitive to all three agents. Further examination of the Bcl-2 family proteins showed dysregultion of the
same key step in the intrinsic apoptosis pathway with downregulation of Bim in BCWM.1 and MWCL-1 cells
and loss of Bax and Bak in RPCI-WM1 cells. Furthermore, we examined expression data from WM patients
and determined that WM cells expressed lower levels of pro-apoptotic BH3 proteins than MM cells. These
WM cell lines demonstrate that sensitivity to agents that kill through the intrinsic apoptotic pathway may vary
within a disease that is characterized by a single activating mutation and suggests that additional
heterogeneous events regulate the expression of Bcl-2 family proteins.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [37]В Catherine Gavile, IMP
Poster #21
CD28 AND CD86 REGULATE INTEGRIN SURFACE EXPRESSION IN MYELOMA CELL LINES
Catherine M. Gavile1, David Egas1, Gregory Doho1, Sagar Lonial1, Kelvin P. Lee2, Lawrence H. Boise1
1
Departments of Hematology and Medical Oncology and Winship Cancer Institute, Emory University,
Atlanta GA 30322 2Departments of Immunology and Medicine, Roswell Park Cancer Institute, Buffalo, NY
14263
CD28 and CD86 are better known for their roles in T-cell costimulation. Recent reports have shown that this
signaling module also plays an important role in the maintenance of normal long-lived plasma cells (LLPCs).
As myeloma cells retain most of the physiological characteristics of LLPCs, our lab is currently investigating
the role of CD28 and CD86 in myeloma. Previously, we have shown that CD28 and CD86 are necessary for
myeloma cell survival, as knockdown of expression of either CD28 or CD86 leads to cell death in 3 myeloma
cell lines. We have also shown that blocking the ability of these molecules to interact using a chimeric soluble
receptor CTLA4-Ig (Abatacept) also leads to cell death. We are currently investigating what downstream
signals are mediated upon CD28-CD86 signaling. Based on results obtained via RNA-Seq, our data indicate
that CD28-CD86 signaling plays a role in regulation of integrin expression in myeloma cell lines.
Constance Harrell, NS
Poster #22
CONSUMPTION OF A HIGH-FRUCTOSE DIET DURING ADOLESCENCE INCREASES
DEPRESSIVE-LIKE BEHAVIORS, ALTERS HPA AXIS FUNCTION, AND REMODELS
HYPOTHALAMIC GENE EXPRESSION
Constance S. Harrell1,2, Zachary P. Johnson3,4, Jillybeth Burgado5, Gretchen N. Neigh1,6
1
Department of Physiology; 2Molecules to Mankind Doctoral Pathway; 3Nonhuman Primate Genomics Lab,
4
Yerkes National Primate Center, 5NET/Work Program, Center for Behavioral Neuroscience; 6Department of
Psychiatry and Behavioral Sciences
The 25% increase in fructose consumption over the past thirty years has been linked to obesity.
Fructose is implicated in dyslipidemia and insulin resistance but little is known about its impact on behavior
and the brain. Given that adolescents are the highest consumers of fructose and in a “critical period” of
development, we examined the effects of an adolescent high-fructose diet on behavior, the HPA axis, and
whole-transcriptome hypothalamic expression.
Male rats were assigned to either standard chow or a 55% fructose diet from weaning to adulthood.
Weight, blood glucose and consumption were measured throughout while behavior and serum corticosterone
were assessed in adulthood. Hypothalamic RNA was extracted for RNA-Sequencing.
Fructose consumption resulted in elevated baseline corticosterone yet a blunted response to acute
stress. Fructose also reduced struggle and increased immobility in the forced swim test. Moreover, fructose
impacted hypothalamic gene expression, altering 966 genes, or 5.6% of those tested. Principal Components
Analysis demonstrated clustering by diet type. Both the dopamine transporter and tyrosine hydroxylase were
upregulated by fructose and highly ranked in the first component. Several pathways involved in dopamine
signaling were significantly enriched by differentially expressed genes, suggesting a potential link among
hypothalamic dopaminergic expression, consumption, and behavior.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [38]В Brenda Huang, GMB
Poster #23
OLIGODENDROCYTE DYSFUNCTION IN HUNTINGTON’S DISEASE MICE
Brenda Huang1,2, Cindy Wei1, Marta Gaertig1, Xiao-Jiang Li1, Shihua Li1
1
Department of Human Genetics, Emory University, Atlanta, GA 303222 Graduate Program in Genetics and
Molecular Biology
Huntington’s disease is a neurodegenerative disorder that is caused by an expanded polyglutamine tract in the
N-terminal region of huntingtin. It is characterized by cognitive and behavioral deficits, as well as movement
disorders. While the effect of mutant huntingtin on neuronal function has been extensively studied, its effect
on the function of glial cells remains to be fully investigated. There is evidence of white matter abnormalities
in both HD patients and HD mouse models, suggesting that mutant huntingtin might affect oligodendrocyte
function. We hypothesize that mutant huntingtin causes oligodendrocyte dysfunction, which contributes to
the pathogenesis of the disease. To this end, we have generated transgenic mice that express an N-terminal
fragment of mutant huntingtin exclusively in oligodendrocytes. These mice develop a tremor by 2 months of
age and have locomotor and behavioral deficits, as well as body weight loss and reduced lifespan compared to
control mice. Reduced myelination is present in the striatum of mutant mice, and preliminary data suggest
that there is a dysregulation of myelin gene expression. These results suggest that the expression of mutant
huntingtin in oligodendrocytes plays an important role in HD pathogenesis.
Emily Hunter, BCDB
Poster #24
MECHANISMS OF CILIARY ASSEMBLY
Emily L. Hunter1, Lea M. Alford1, Winfield S. Sale1*
1
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA
Cilia, found on most human cells, play an essential role in development, cell signaling, and organ function.
Defective ciliary assembly results in diseases collectively known as ciliopathies. The goal of our research is
to determine mechanisms of ciliary assembly, focusing on the assembly of the axonemal radial spoke
structure that is essential for regulating ciliary motility. The radial spoke assembles into a large precursor
complex before entry into the cilium and subsequent transport to the distal tip for final assembly. To further
assess radial spoke transport, we analyzed the Chlamydomonas mutant pf27 that is deficient in radial spokes.
We determined the pf27 radial spokes that dock to the microtubule core are fully assembled and localize to
the proximal end of the cilium. We postulate the radial spoke deficiency is caused by a defect in Intraflagellar
Transport (IFT) of the radial spoke precursor. Consistent with this hypothesis, assembly of radial spokes
occurs asynchronously from other axonemal components in pf27 and radial spoke proteins are greatly reduced
in the pf27 membrane-matrix fraction of the cilium where IFT occurs. We are now testing the hypothesis that
PF27p is required for normal radial spoke transport.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [39]В Juyeon Hwang, BCDB
Poster #25
CONTROL OF PP2A FAMILY PHOSPHATASE METHYLATION BY LCMT-1 AND PME-1
Juyeon Hwang1,2, Jocelyn A. Lee1,2, and David C. Pallas1,2
1
Department of Biochemistry and Winship Cancer Institute, Emory University School of Medicine, Atlanta,
Georgia 30322, USA, 2Biochemistry, Cell, Developmental Biology Graduate Program, Emory University
School of Medicine, Atlanta, Georgia 30322, USA.
Protein phosphatase 2A (PP2A) and the PP2A-related protein phosphatases, PP4 and PP6, are multifunctional
serine/threonine protein phosphatases involved in various cellular processes including cell cycle control,
apoptosis, and development. Methylation of the PP2A catalytic (C) subunit is a reversible process tightly
regulated by leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1
(PME-1). The site of methylation on PP2A is conserved in PP4 and PP6, but the identities of the PP4 and PP6
methyltransferase and methylesterase enzymes are not known. We now show that LCMT-1 is the major
methyltransferase for both PP4 and PP6 and that PME-1 is responsible for PP4 and PP6 demethylation.
Analysis of PP2A-related protein phosphatase complex formation by blue native gel electrophoresis suggests
that loss of LCMT-1 affects complex formation. Consistent with these results, loss of LCMT-1 leads to a
reduction in the amount of PP2A BпЃЎ regulatory subunit and a decrease in association of PP2A C subunit with
the remaining BпЃЎ subunit. Lastly, loss of LCMT-1 increased protein stability of PP4 C subunit without
affecting translation initiation. Collectively, our data suggest that LCMT-1 and PME-1 regulate the
methylation of PP2A-related phosphatases, and consequently, protein phosphatase complex formation and
stability of individual subunits.
Daniel Infield, MSP
Poster #26
USE OF FUNCTIONAL CROSSLINKERS TO STUDY CONFORMATIONAL CHANGES
ASSOCIATED WITH CFTR GATING
Daniel T. Infield1,2,, Guiying Cui, MD Ph.D.2, Christopher Kuang2, and Nael A McCarty, Ph.D.1,2,3
Emory University Molecular and Systems Pharmacology Graduate Program, 2 Departmentof Pediatrics,
Emory University School of Medicine, 3Childrens Healthcare of Atlanta
1
The chloride ion channel CFTR is a member of the ABC transporter family. However, it is unknown whether,
like transporters, the transmembrane domains of CFTR undergo an inward-to-outward facing transition upon
NBD dimerization. To test this, we made cysteine substitutions in CFTR in extracellular loops (ECL) 1 and
4, which appose each other in inward-facing but not outward-facing structures of ABC transporters. We found
the macroscopic current of D110C/K892C-CFTR channels (but not corresponding single mutants) was
increased in the presence of DTT. Multichannel patch recordings revealed that DTT increased number of
open double cysteine mutant channels, indicating that they were locked into closed state by a spontaneous
disulfide bond prior to DTT. To further understand the kinetics of the interaction, we applied Cd2+ and found
that D110C is transiently bound by Cd2+, causing a rapidly reversible inhibition of current. Under identical
cumulative exposure, Cd2+ inhibited D110C/K892C-CFTR irreversibly. K892C and E115C-CFTR were only
slightly functionally modified (<2%) by Cd2+, though E115C-CFTR was labeled by MTS-TAMRA,
indicating accessibility. Neither D112C/K892C nor E115C/K892C CFTR were affected by DTT, and
D112C/K892C was not affected by Cd2+. The data suggest ECL1 and ECL4 come into close proximity in a
closed state of CFTR.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [40]В Jordan Kohn, NS
Poster #27
THE IMPACT OF ACUTE PSYCHOSOCIAL STRESS ON GENOME-WIDE INFLAMMATORY
SIGNALING IN THE FACE OF CHRONIC SOCIAL ADVERSITY.
Jordan N. Kohn1,2, Steve W. Cole3,4, Mark E. Wilson1, and Zach P. Johnson1
1
Division of Developmental and Cognitive Neuroscience, Yerkes National Primate Research Center, Atlanta,
GA, 2Graduate Program in Neuroscience, Emory University, Atlanta, GA, 3University of California Los
Angeles Molecular Biology Institute, 4The Norman Cousins Center, University of California, Los Angeles,
CA.
Chronic social stress leads to chronic inflammation, a well-established risk factor for disease and all-cause
mortality. Understanding the relationship between stress and inflammation is therefore an important priority
for improving human health. Studies suggest that chronic stress disrupts glucocorticoid (GC) signaling at
multiple levels of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis, leading to amplified inflammatory
gene expression by immune cells. Chronic stress results from repeated exposure to acute stressors, so one may
gain insight into the mechanisms of stress-induced inflammation by studying the immune response. In this
study, we used a well-validated translational model of social subordination stress in female rhesus macaques
to examine social status-dependent differences in the genome-wide inflammatory response to psychosocial
stress challenge. Total mRNAs were sequenced from mononuclear cells, and promoter-based bioinformatics
analysis of differentially expressed genes revealed core promoter enrichment for proinflammatory
transcription factors, nuclear factor kappa B (NF-ОєB) and AP1, at all timepoints in social subordinates.
Interestingly, there was a trend toward promoter enrichment of glucocorticoid receptor (GR)-binding motifs
in up-regulated genes, in the absence of plasma cortisol or interleukin-6 differences. This finding suggests a
unique mechanism by which GR-mediated transcriptional responses during acute stress are enhanced in
chronic social adversity to dampen concurrent hyperinflammatory signaling.
Kristin Limpose, CB
Poster #28
REGULATION OF BASE EXCISION REPAIR IN GENOME MAINTENANCE: IMPLICATIONS
IN CANCER
Kristin Limpose1, Erica Werner2, Anita H. Corbett2, Paul W. Doetsch1,2,3,4
Department of Cancer Biology, Emory University School of Medicine, Atlanta, GA, USA; 2 Department of
Biochemistry, Emory University School of Medicine, Atlanta, GA, USA; 3 Department of Hematology and
Medical Oncology, Emory University, Atlanta, GA, USA; 4 Department of Radiation Oncology, Emory
University, Atlanta, GA, USA
1
Dysregulation of DNA repair and maintenance pathways contribute to cancer by increasing levels of DNA
damage. Ultimately, unrepaired DNA damage results in the accrual of mutations and genomic instability. A
common cause of DNA damage is reactive oxygen species (ROS) that are generated from normal cellular
metabolism, inflammation, or through exogenous sources such as radiation. Importantly, tumor cells
experience an increased ROS burden from these sources, potentially facilitating more mutations. Base
excision repair (BER) is the main pathway for repairing ROS-induced DNA damage, and is initiated by the Nglycosylase proteins. How BER dysregulation contributes to cancer development is largely unknown. To
investigate this question, the NTHL1 glycosylase will be used as a model to address the mechanisms of BER
regulation and the role of BER in preventing cancer. Possible modes for NTHL1 regulation are compartmentspecific localization and/or posttranslational modification(s) in response to ROS-induced DNA damage. The
comet assay, immunoprecipitation, mass spectrometry, and cleavage assays are being employed to interrogate
NTHL1 regulation in a panel of colon cancer cell lines. BER dysregulation is being tested through
biochemical and cellular assays. This project will provide insight into BER mechanisms that contribute to
genome integrity, and how BER dysregulation contributes to tumorigenesis.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [41]В Kelly Lohr, NS
Poster #29
ENHANCED IN VIVO NEUROTRANSMISSION VIA INCREASED VESICULAR TRANSPORT:
VMAT2 OVEREXPRESSION IN A MOUSE
Kelly M. Lohr1, Alison I. Bernstein1, Kristen A. Stout1, Amy R. Dunn1, Xueliang Fan3, Ellen J. Hess3,4, Hong
Yi5, David S. Goldstein6, Thomas S. Guillot1, Ali Salahpour7, Gary W. Miller1,2,3,4
1
Dept, of Environmental Health, Rollins School of Public Health, 2Center for Neurodegenerative Diseases,
3
Dept. of Pharmacology, 4Dept. of Neurology, 5Robert P. Apkarian Integrated Electron Microscopy Core,
Emory University,6 National Institute of Neurological Disorders and Stroke, 7Dept. of Pharmacology and
Toxicology, University of Toronto
Several therapeutic strategies have been employed to enhance signaling of the monoamine neurotransmitters
(dopamine, serotonin, and norepinephrine): administration of precursors to increase synthesis, inhibition of
metabolic enzymes to prevent break down, inhibition of plasma membrane transporters to increase the
synaptic lifespan, and administration of receptor agonists to directly activate postsynaptic targets. However,
many of these interventions have deleterious side effects or lose effectiveness due to desensitization or altered
regulation of receptors over time. These undesirable consequences likely occur because these strategies
disrupt the temporal nature of regulated neurotransmitter release and uptake. We demonstrate that monoamine
neurotransmission can be amplified by increasing vesicular packaging of neurotransmitter via overexpression
of the vesicular monoamine transporter 2 (VMAT2). Mice with elevated VMAT2 display increased vesicular
filling and size, higher stimulated release and extracellular levels of dopamine, and enhanced spontaneous and
amphetamine-induced locomotion. This unexpected plasticity of the vesicle reveals a new therapeutic
approach for treating neurological and neuropsychiatric disorders that involve impaired monoaminergic
signaling. In support of this, VMAT2-HI mice also show both improved outcomes on measures of anxiety and
depressive-like behavior and protection from nigral cell loss by the dopaminergic toxicant MPTP.
Jia Meng, MMG
Poster #30
FUNCTIONAL ANALYSIS OF THE RSV STRAIN A2001/2-20 ATTACHMENT GLYCOPROTEIN
Jia Meng1,2, Michael, G Currier1, 2, Kate L Stokes1, 2, 3, Anne L Hotard1,2, and Martin L Moore1,2
1
Department of Pediatrics, Emory University, 2 Children’s Healthcare of Atlanta, Atlanta, GA, 3 Currently St.
Jude Children’s Research Hospital
RSV strain A2001/2-20 (2-20) induced more airway epithelial damage and pulmonary mucus expression in
BALB/c mice than the laboratory A2 strain [Stokes et al. J. Virol. 2011; 85: 5782.]. We hypothesize that
increased fusion caused by the fusion (F) and attachment (G) glycoproteins in infected cells is a determining
factor for virulence. We transfected cells with RSV glycoprotein expression plasmids and used a dual-split
protein fusion assay to quantify cell-cell fusion. There was no difference in fusion activity between A2 F and
2-20 F. Co-expression of 2-20 F and 2-20 G resulted in higher fusion activity than co-expression of A2 F and
A2 G. We generated viruses that either express G and F or F only: A2-A2GA2F, A2-GstopA2F, A2-2-20G220F, and A2-Gstop2-20F. We determined infectivity and entry kinetics in BEAS-2B, CHO-K1, and pgsD-677
cell lines. In all these cell lines, A2-A2GA2F had higher infectivity and entry kinetics than A2-2-20G2-20F.
Expression of G increased infectivity and entry more for 2-20F than for A2F-containing virus. Furthermore,
viruses with both G and F produced larger plaques than viruses with F only. Taken together, the attachment
glycoprotein G of RSV increased infectivity, entry kinetics and plaque size, depending on the strain its F
protein is from.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [42]В Jasmine Miller-Kleinhenz, CB
Poster #31
DECREASED EXPRESSION OF IGF-1R AND KI67 USING UPAR-TARGETED THERANOSTIC
NANOPARTICLES IN DRUG-RESISTANT TRIPLE-NEGATIVE BREAST CANCER PATIENTDERIVED XENOGRAFT
Jasmine Miller-Kleinhenz1, Hongyu Zhou1, Weiping Qian1, Ruth O’Regan2, Amelia Zelnak2, Toncred
Styblo1, Lily Yang1
1
Winship Cancer Institute, Department of Surgery, Emory University School of Medicine, Atlanta, GA,
2
Winship Cancer Institute, Department of Hematology and Medical Oncology, Emory University School of
Medicine, Atlanta, GA
Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease with a poor clinical
prognosis. While the primary therapeutic option for TNBC patients is pre-operative neo-adjuvant
chemotherapy followed by surgical resection, about 60% of patients develop drug resistance and have a very
high incidence of tumor recurrence and poorer prognosis. Given its poor survival and lack of available
targeted therapeutics, the development of novel treatment options for chemo-resistant TNBC is critical. Our
lab has developed urokinase plasminogen activator receptor (uPAR) targeted theranostic magnetic iron oxide
nanoparticles (IONPs) carrying therapeutic agents that are clinically beneficial to TNBC patients such as
doxorubicin (Dox). uPAR is an ideal target due to its high expression in malignant tumors including
aggressive breast cancer tissues and tumor stromal cells that are enriched in TNBC tissues. Using a patient
derived xenograft model of TNBC we investigated uPAR-targeted IONP-Dox effect on tumor growth
inhibition. We also investigated the effect the nanoparticle treatment had on relevant biomarkers such as
insulin-like growth factor 1 receptor (IGF-1R) and Ki67. Results of our study demonstrated that uPARtargeted theranostic IONPs decrease expression of IGF-1R and Ki67.These results support
further investigation of theranostic nanoparticles as an approach to overcome drug resistance in TNBC.
Kevin Morris, BCDB
Poster #32
DEFINING THE NEURONAL FUNCTION OF THE POLYADENOSINE RNA-BINDING PROTEIN
ZC3H14
Kevin Morris1, Jennifer Rha1, Sara Leung1, Gary Bassell2, Kenneth Moberg2, and Anita Corbett1.
Biochemistry, Cell and Developmental Biology Graduate Program, Emory University Laney Graduate
School, Dept. of Biochemistry1, Dept. of Cell Biology2, Emory University School of Medicine, Atlanta, GA.
RNA-binding proteins are tasked with the major responsibility of regulating the co- and posttranscriptional
events of gene expression. Impairments to this process can result in human disease including intellectual
disability (formerly termed mental retardation). Recently, our group identified inactivating mutations in the
ZC3H14 gene, which encodes an evolutionarily conserved, RNA-binding protein, Zinc finger Cys3His
domain-containing protein number 14 (ZC3H14) that lead to non-syndromic intellectual disability. ZC3H14
is an RNA-binding protein that binds with high affinity to polyadenosine RNA. Although ZC3H14 is
ubiquitously expressed, ZC3H14 mutant patients only display brain specific phenotypes. While the steadystate localization of ZC3H14 is primarily nuclear, our recent studies reveal that a population of ZC3H14 is
present in the axons of primary hippocampal neurons. In addition, ZC3H14 associates with 80S ribosomes.
Notably, Drosophila studies of the ZC3H14 fly ortholog, reveal functional interactions with translation
regulators. Therefore, we hypothesize that ZC3H14 works cooperatively with RNA regulatory factors to
modulate translation. Currently, we are exploiting a recently created ZC3H14 knockout mouse to assess the
requirement for ZC3H14 in translation (Aim 1) and to define the spectrum of ZC3H14-interacting RNA
regulatory factors (Aim 2). Ultimately, the information gained from the proposed research could provide
insight into the molecular role of ZC3H14 and how ZC3H14 affects proper higher order brain function.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [43]В Donald Noble, NS
Poster #33
CONDITIONING SLOWED RESPIRATORY RATE FOR STRESS REDUCTION IN THE RAT
Donald Noble1, William N. Goolsby2, Shawn Hochman1.
1
Department of Physiology, 2Department of Cell Biology, Emory University, Atlanta, GA
Meditation and yoga techniques involving slowed respiratory rate (SRR) have shown promise in treating a
variety of cardiorespiratory and stress-related disorders. SRR exercises improve sympathetic-parasympathetic
balance, inducing physical and emotional relaxation. We have developed an animal model of the relaxation
response using strobe light as negative reinforcement to operantly condition SRR. During daily two-hour
sessions, paired rats (“SRR” and “yoked”) were placed in dual chambers and respiration was continuously
and noninvasively monitored using whole body plethysmography. Real-time respiratory rate (RR) was
compared to a user-defined target and a closed-loop feedback control system provided time-varying light
intensity cues. Only SRR rats had control over the stimulus, while yoked animals received responseindependent reinforcement. SRR rats slowed their breathing by 16 breaths/minute over the course of 20
sessions and spent a greater percentage of each session under the target RR, whereas yoked controls did not.
Results support the conclusion that feedback-based light intensity control can lead to a selective reduction in
respiratory rate. SRR animals also showed a trend toward maintained RR reduction following conditioning,
and decreased anxiety-like behavior in an open field. Further studies are aimed at verifying these preliminary
findings and identifying mechanisms that link SRR to stress circuit deactivation.
Bridgette Peake, MSP
Poster #34
GROWTH DIFFERENTIATION FACTOR 15 AND EPITHELIAL-MESENCHYMAL TRANSITION
IN BREAST CANCER
Bridgette F. Peake1, Siobhan M. Donnelly2 and Rita Nahta1,2,3,5,*
Molecular & Systems Pharmacology Program, Graduate Division of Biological and Biomedical Sciences
2
Departments of Pharmacology and 3Hematology & Medical Oncology, School of Medicine;; 4Biostatistics,
School of Public Health; 5Winship Cancer Institute; Emory University, Atlanta, GA 30322
1
HER2 amplification occurs in approximately 25% of breast cancers. An additional 15% of breast cancers are
called triple-negative (TNBC) due to the absence of estrogen and progesterone receptors and the lack of
HER2 overexpression. We previously found that drug-resistant HER2-positive cells express increased levels
of the cytokine growth differentiation factor 15 (GDF15). Similarly, TNBCs expressed relatively high levels
of GDF15. Immunohistochemical analysis of a tumor tissue array representing almost 600 breast cancer
patients indicated significant correlations between high GDF15 staining and ER-negativity, HER2-positivity,
high tumor grade, and reduced patient survival. In vivo studies showed increased tumor growth and increased
HER2 phosphorylation in GDF15-overexpressing xenografts compared to empty vector control xenografts.
Furthermore, GDF15 overexpression induced epithelial-mesenchymal transition (EMT), as demonstrated by
changes in cell morphology and increased expression of mesenchymal markers. Importantly, HER2overexpressing GDF15 stable cell lines showed up-regulation of FOXM1 in comparison to empty vector
control cells. We previously published that GDF15 induces MEK signaling, which is an established activator
of FOXM1. We hypothesize that GDF15 may promote EMT and tumor growth via MEK-FOXM1 signaling.
The current study supports further investigation into the role of GDF15-mediated EMT in both TNBC and
HER2-overexpressing breast cancers.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [44]В Maylen, Perez Diaz, NS
Poster #35
THE HIGHLY SELECTIVE 5-HT2C AGONIST WAY-163909 ELIMINATES
METHAMPHETAMINE SELF-ADMINISTRATION IN RHESUS MONKEYS
Maylen Perez Diaz1, Kevin S. Murnane1, and Leonard L. Howell1,2
Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA2Department of Psychiatry
and Behavioral Sciences, Emory University, Atlanta, GA, USA
1
Growing evidence indicates that serotonin (5-HT) neurotransmission can modulate the neurochemical and
behavioral effects of abused stimulants. Studies have shown that selective antagonists of the 5-HT2A receptor
can attenuate cocaine reinstatement, but not self-administration (SA), in nonhuman primates. In contrast, 5HT2C receptor agonists can partially block cocaine SA in rhesus monkeys. In the present study, we
determined if a novel, highly selective 5-HT2C agonist could fully attenuate methamphetamine (METH) SA.
Four rhesus macaques with a previous history of drug SA self-administered 0.01mg/kg/injection of METH on
a fixed ratio (FR) 20 schedule of reinforcement. For all four subjects, WAY pretreatments decreased response
rates during the SA sessions in a dose-dependent manner. In addition, METH SA was fully blocked in all
subjects following pretreatment with 1.0mg/kg WAY. These data further inform previous studies by
demonstrating that 5-HT2C receptor activation can suppress self-administration of METH as well as cocaine,
suggesting effectiveness across psychostimulants in general. The ability of WAY to fully suppress SA may be
due to its highly selective agonist activity at the 5-HT2C receptor compared to compounds that have been
evaluated previously. The results of this study may inform our search for novel, effective pharmaceutical
treatments for drug addiction.
Riley Perszyk, MSP
Poster #36
A NOVEL CLASS OF POSITIVE ALLOSTERIC NMDA RECEPTOR MODULATORS
Riley E. Perszyk1, Brooke M. Katzman2, Dennis C. Liotta2, Stephen F. Traynelis1
1
Department of Pharmacology, 2Department of Chemistry
N-methyl-D-aspartate (NMDA) receptors are ionotropic ligand-gated ion channels that are activated by
glutamate and glycine and mediate a slow component of excitatory synaptic currents. These receptors play
important roles in synaptic plasticity, neuronal development, and a number of neurological disorders. The
enhancement of NMDA receptor-mediated currents has been hypothesized to confer cognitive enhancement
and could be potentially beneficial in schizophrenia. Testing these hypotheses has been limited due to a lack
of potent and effective pharmacological compounds. During the development of a negative allosteric
modulator series, identified by screening efforts, we discovered a series of substitutions on the molecule that
converted these negative modulators into positive modulators. Using two-electrode voltage clamp recordings
of NMDA receptors expressed in Xenopus oocytes, we found that potentiators in this class can enhance
responses 150-250% with EC50 values ranging from 2 to 10ОјM. Co-application of positive and negative
modulators suggests that the positive modulators can compete with the negative modulators. This raises the
possibility that these modulators act at a single site. This class of compounds could be utilized to assess the
validity of enhancing NMDA receptor activity as a potentially beneficial strategy in neuropsychiatric
disorders and could lead to novel therapeutic strategies.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [45]В Katherine Reding, NS
Poster #37
CHRONIC PSYCHOSOCIAL STRESS AND ESTRADIOL ALTER INTRINSIC FUNCTIONAL
CONNECTIVITY IN RHESUS MACAQUES.
Katherine Reding1, David S. Grayson2, Oscar Miranda-Dominguez3, Siddharth Ray3, Martin Styner4, Mark E.
Wilson1, Donna Toufexis5, & Damien A. Fair3, Mar Sanchez1,6
1
Division of Developmental and Cognitive Neuroscience, Yerkes National Primate Research Center, Emory
University, 2Center for Neuroscience, University of California - Davis, 3 Departments of Behavioral
Neuroscience, Psychiatry, and Advanced Imaging Research Center, Oregon Health and Science University,
4
Department of Psychiatry, University of North Carolina, Chapel Hill, 5Department of Psychology, University
of Vermont, 6Department of Psychiatry & Behavioral Sciences, Emory University
The ovarian hormone estradiol (E2) has activational effects on female rhesus macaque prosociaI,
motivational, and sexual behavior that are attenuated during exposure to chronic psychosocial stress.
However, the mechanisms by which this attenuation occurs are unknown. In humans, analysis of neural
activation at rest using functional magnetic resonance imaging (fMRI) suggests that acute stress can increase
intrinsic functional connectivity (iFC) between the amygdala and midline cortical structures, including the
medial prefrontal cortex (mPFC). Studies on adult male rhesus macaques suggest that social subordination
can lead to changes in iFC between the superior temporal sulcus (STS) and the PFC. Furthermore, increased
iFC within amygdalar-cortical networks and between hemispheres is seen following exogenous E2
replacement therapy. Exposure to E2 in combination with chronic stress may exacerbate stress-related
increases in functional connectivity. To investigate this hypothesis, we studied ovariectomized adult female
rhesus macaques exposed to high levels of chronic social subordination stress, both with and without E2
replacement. Data show greater STS-mPFC iFC in Subordinate compared to Alpha females, which result
from chronic exposure to psychosocial stress. Furthermore, greater STS-mPFC iFC in Subordinate females
after E2 treatment suggests that E2 exacerbates the effects of subordination stress on iFC.
Jennifer Rha, BCDB
Poster #38
MUTATION OF A ZINC FINGER POLYADENOSINE RNA BINDING PROTEIN, ZC3H14, CAUSES
AUTOSOMAL RECESSIVE INTELLECTUAL DISABILITY
Jennifer Rha, Sara Leung, Gary Bassell, Anita Corbett.
Emory University School of Medicine, Atlanta, GA.
Patients with intellectual disability (ID) suffer from significantly subaverage intellectual function (IQ≤70),
which impinges on quality of life. We have recently identified the first gene encoding a polyadenosine RNA
binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), which is mutated in
inherited nonsyndromic autosomal recessive intellectual disability (NS-ARID). This finding uncovers the
molecular basis for disease in these patients and provides strong evidence that ZC3H14 is essential for proper
brain function. Studies of ZC3H14 orthologs in budding yeast and Drosophila provide insight into the role of
this protein in post-transcriptional regulation of gene expression and key evidence for its critical role in
neurons. Despite these studies, functional characterization of ZC3H14 in vertebrates is crucial for
understanding both normal brain function and the molecular mechanism underlying ID in these patients. We
have developed a conditional ZC3H14 knockout mouse utilizing the Cre/loxP system to extend our studies to
vertebrate ZC3H14 and address our hypothesis that ZC3H14 is required for proper expression of target
mRNAs that are critical for neuronal function. These ZC3H14 knockout mice provide us with an optimal
model to study the requirement for ZC3H14 in higher order brain function.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [46]В Samuel Rose, NS
Poster #39
NEUROCHEMICAL AND BEHAVIORAL DYSFUNCTION IN A MOUSE MODEL OF DOPARESPONSIVE DYSTONIA
Samuel J. Rose1,2, Xin Y. Yu2, H. A. Jinnah MD/PhD3,4,5, Ellen J. Hess PhD2,3
1
Neuroscience Graduate Program, Departments of 2 Pharmacology, 3 Neurology, 4 Human Genetics, and 5
Pediatrics
Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting
movements and postures. Although the pathogenesis of dystonia is not understood, reduced dopamine
neurotransmission is consistently observed across many different forms of dystonia, including doparesponsive dystonia (DRD). DRD is cause by mutations in genes integral to dopamine synthesis, including
tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. Therefore, to better understand
the role of dopamine in dystonia, we have created a knockin mouse bearing the DRD-causing TH mutation
1160C>A; Q381K (DRD knockin mice). TH activity is reduced by ~90% in vivo in these mice, causing a
dramatic reduction in dopamine concentration. Behaviorally, DRD knockin mice exhibit abnormal
movements reminiscent of human dystonia. These movements are exacerbated by a D1-like dopamine
receptor antagonist, suggestive of a mechanism for dystonia involving the D1 dopamine receptor. DRD
knockin mice also exhibit abnormal circadian regulation of locomotor activity, where DRD knockin mice are
more active than controls at the start of the dark cycle. However, by the start of the light cycle, DRD knockin
mice exhibit catalepsy. These studies establish DRD knockin mice as a useful model for examining
dopaminergic mechanisms underlying dystonia. Supported by PHS NS059645.
Chelsea Ruppersburg, BCDB
Poster #40
A NOVEL ROLE FOR THE CHLORIDE CHANNEL ANO1 (TMEM16A) IN CILIOGENESIS: THE
ANO1 NIMBUS
Chelsey Chandler Ruppersburg & H. Criss Hartzell.
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322 USA.
Many cells possess a single, non-motile, primary cilium that is thought to be a sensory transducer akin to a
cellular antenna. Although sensory transduction requires ion channels, relatively little is known about ion
channels in the primary cilium. We show here for the first time the presence of a Cl- channel, the Ca2+activated Cl- channel ANO1, in the primary cilium. Prior to cilia extension, ANO1 is organized into a torusshaped structure along with other ciliary proteins including the small GTPases CDC42 and Arl13b, the
exocyst complex component Sec6, and acetylated α-tubulin and γ-tubulin. This structure we call the “nimbus”
forms an interface between the microtubule cytoskeleton of the nascent cilium and the surrounding cortical
actin cytoskeleton. During ciliogenesis, the nimbus disassembles and ciliary components, including ANO1,
move into the cilium. Blocking ANO1 currents pharmacologically or knockdown of the ANO1 channel
disrupts ciliogenesis. Our data support a model where the nimbus provides a scaffold for staging of ciliary
components and cilia assembly and that Cl- transport by ANO1 is required for the genesis or maintenance of
cilia. Our data here may provide a mechanistic foundation for interpreting the roles of ANO1 in development
and cancer.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [47]В Emily Rye, BCDB
Poster #41
A NOVEL MECHANISM FOR THE GLUCOCORTICOID RECEPTOR IN INFLAMMATORY
GENE REGULATION
Emily Rye1, Will Hudson1, Kendall Nettles2, Eric Ortlund1
1
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA
303222Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458
Inflammation is a multifaceted process required to heal injuries and fight infections. However, chronic
inflammation underlies various debilitating diseases including asthma, rheumatoid arthritis, and heart disease.
Glucocorticoids (GC) are the most widely prescribed anti-inflammatory drug. GCs bind and activate the
glucocorticoid receptor (GR), inducing its translocation to the nucleus where it can stimulate or inhibit gene
expression. To activate transcription, GR binds to glucocorticoid response elements to drive the transcription
of metabolic and stress response genes. Conversely, GR can repress transcription by two distinct mechanisms,
either DNA-dependent or independent. The first mechanism involves direct binding of GR to negative
glucocorticoid response elements. The DNA-independent mechanism occurs when GR represses other proinflammatory transcription factors, most notably NF-ОєB and AP-1, through protein-protein interactions only.
This mechanism, known as “tethering”, is currently the only accepted model for GR-mediated repression of
inflammation. However, recent discoveries regarding DNA-dependent GR-mediated transrepression reveal
that the mutations used to develop the tethering hypothesis are inadequate. In addition, fluorescence
polarization binding assays and x-ray crystallographic structures show GR bound to inflammatory gene
promoters at AP-1 binding sites, suggesting that GR-mediated repression of inflammatory genes could occur
through an alternative mechanism, involving direct DNA binding.
Alessandra Salgueiro, CB
Poster #42
MECHANISTIC STUDIES OF VIMENTIN INTERMEDIATE FILAMENTS USING AN
INDUCIBLE TRANSFECTION SYSTEM
Alessandra M. Salgueiro1, Alessandra M. Salgueiro, Lauren S. Havel1, Adam I. Marcus1.
1
Winship Cancer Institute, Department of Hematology and Oncology, Emory University, Atlanta, GA 30322
Vimentin, an intermediate filament, is a marker of epithelial to mesenchymal transition (EMT), a mechanism
in cancer metastasis. Gain of vimentin expression correlates with EMT, but little is known about the
mechanisms by which vimentin function and dynamics contribute to EMT. We demonstrated that vimentin
regulates focal adhesion kinase (FAK) to stabilize focal adhesions essential for motility. Other groups have
shown that vimentin dynamics are mediated by serine phosphorylation sites in the head and tail domains. We
created an inducible GFP:vimentin system to recapitulate gain of vimentin expression observed during EMT
and use this model to determine how vimentin serine phosphorylation mediates invasion, and focal adhesion
stability. HEK293 cells (vim +/+) were used to optimize the transfection system. 16HBECs (human bronchial
epithelial cells) (vim null) were used for mechanistic studies of vimentin regulation of FAK and analysis of
serine phosphorylation sites. The four serine sites studied were mutated to either unphosphorylatable alanine
or phosphomimetic aspartic acid. Our studies in HEK293s demonstrate that wild type GFP:vimentin mimics
the morphology of endogenous vimentin. Similar experiments in 16HBECs show different morphologies
among GFP:vimentin phospho-mutants. These data demonstrate that mechanistic studies utilizing the
phospho-mutants will provide better understanding of vimentin dynamics focal adhesion regulation.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [48]В Eduardo Sanabria-Figueroa, MSP
Poster #43
TARGETING IGF-1R AND HER2 IN HER2-OVEREXPRESSING BREAST CANCER CELLS:
ROLE IN CELL VIABILITY, GROWTH, INVASION, MORPHOLOGY, AND ELUCIDATION OF
CROSSTALK MECHANISM
Eduardo Sanabria-Figueroa1, Siobhan M. Donnelly2, Elisavet Paplomata3, and Rita Nahta1,2,3,4.
1
Molecular & Systems Pharmacology Program, Graduate Division of Biological and Biomedical Sciences;
2
Department of Pharmacology, School of Medicine; 3Hematology & Medical Oncology, School of Medicine;
4
Winship Cancer Institute; Emory University, Atlanta, GA, 30322.
HER2 is overexpressed in ~25% of metastatic breast cancers in association with poor prognosis.
Trastuzumab, a monoclonal antibody that targets the extracellular domain of HER2, is the leading therapy for
this group of breast cancer patients. However, trastuzumab resistance often emerges, sometimes within a year
of treatment, allowing disease progression. Previous studies have indicated that IGF-1R signaling contributes
to trastuzumab resistance by crosstalk to HER2. Consistent with this idea, inhibition of IGF-1R restored
trastuzumab sensitivity in resistant cells. Here, we show that IGF-1R co-localized with HER2 in HER2positive cell lines. Inhibition of IGF-1R or inhibition of the intracellular kinase SRC reduced IGF-I-induced
phosphorylation of HER2. We also found that combinatorial pharmacological inhibition of IGF-1R and HER2
decreased cell viability, anchorage-independent growth, and invasion of trastuzumab-resistant HER2-positive
breast cancer cells. Our data also suggest that IGF-1R and HER2 co-inhibition might induce mesenchymal to
epithelial transition. These results support the presence of IGF-1R/HER2 crosstalk and suggest a potential
role for the intracellular kinase SRC in this crosstalk. Further studies will be performed to determine if cotargeting IGF-1R and HER2 will rescue the epithelial-mesenchymal transition and invasiveness of
trastuzumab-resistant HER2-positive breast cancers.
Karl Schmidt, NS
Poster #44
LOCUS COERULEUS NORADRENERGIC ACTIVATION REINFORCES OPERANT BEHAVIOR
Karl T. Schmidt1, 2, Chelsea A. Koller1, Elena M. Vazey3, Caroline E. Bass4, Ilana B. Witten5, Karl
Deisseroth6, Gary Aston- Jones3, David Weinshenker1, 2
1
Emory University, Department of Human Genetics 2 Emory University, Neuroscience Graduate Program
3
Medical University of South Carolina, Department of Neurosciences, Department of Psychiatry and
Behavioral Sciences 4SUNY Buffalo, Department of Pharmacology and Toxicology 5Princeton University,
Department of Psychology 6Stanford University
In order to probe the brain’s reward circuitry, early studies used electrodes to stimulate various regions
contingent upon an operant response (e.g. lever press), a technique called intracranial self-stimulation (ICSS).
In the 1970s, some studies found that electrical stimulation of the noradrenergic locus coeruleus (LC) and its
axons comprising the dorsal noradrenergic bundle maintained operant behaviors, while others obtained the
opposite result. Interpretation of the initial ICSS findings must be made cautiously as electrical stimulation is
limited by significant caveats such as electrode placement in the small nucleus and the activation of nonnoradrenergic sources including fibers of passage, innervating terminals from other brain regions, and nearby
cells with various chemical phenotypes. To overcome these technical obstacles and conclusively determine
whether noradrenergic activity can function as a reinforcer, we took an optogenetic approach. Using 2
different expression systems (a Cre-dependent AAV injected into tyrosine hydroxylase::Cre transgenic rats
and a Phox2b-dependent PRSx8 lentivirus), we have expressed channelrhodopsin selectively in LC
noradrenergic neurons. Response contingent activation of these neurons with light maintains behavior
comparable to identical stimulation parameters of optogenetic activation of VTA dopamine neurons. These
data provide new, convincing evidence implicating this noradrenergic nucleus as a component of the brain’s
reward system.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [49]В Marc Schureck, BCDB
Poster #45
MECHANISM OF HIGB-MEDIATED RIBOSOME-DEPENDENT MRNA DEGRADATION
Marc A. Schureck12, Tatsuya Maehigashi1, Jack Dunkle1, Stacey Miles1, Jhomar Marquez1, Ajchareeya
Ruangprasert1, and Christine M. Dunham1
1
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322
2
Biochemistry, Cell and Developmental Biology Graduate Program, Emory University, Atlanta, GA 30322
Proteus vulgaris (P. vulgaris) is a major cause of urinary tract bacterial infections. P. vulgaris can grow on
medical equipment, such as catheters, in an antibiotic-resistant state known as biofilms. In biofilms, bacteria
are highly adhesive and are protected from antibiotics by a self-produced extracellular matrix of nucleic acid,
protein and polysaccharides. My goal is to elucidate the molecular mechanism of the ribosome-dependent
RNase family of toxins, which play an important, but unknown, role in biofilm formation. P. vulgaris Host
inhibition of growth B (HigB) is a member of this family and cleaves ribosome-bound messenger RNA
(mRNA). HigB is unique because it cleaves adenosine-rich mRNA sequences rather than a three-base mRNA
codon, the unit by which translation factors and other better studied toxins, read mRNA. Towards the
elucidation of this novel mRNA decoding mechanism, we solved the X-ray crystal structure of HigB bound to
mRNA on the ribosome. This structure reveals that HigB binds mRNA in the A site of ribosome and highly
conserved HigB amino acids and ribosomal RNA appear important for recognition of mRNA. Our
biochemical studies of various adenosine-rich sequences cleaved by HigB have lead to our current model of
how HigB reads mRNA codon-independently.
Jill Seladi-Schulman, MMG
Poster #46
SPHERICAL INFLUENZA VIRUSES HAVE A FITNESS ADVANTAGE IN EMBRYONATED
EGGS, WHILE FILAMENT-PRODUCING STRAINS ARE SELECTED IN VIVO
Jill Seladi-Schulman, John Steel, and Anice C. Lowen
Emory University, Atlanta, GA, USA
Influenza viruses assume two morphologies: filamentous and spherical. Filaments are observed in lowpassage isolates while spheres are seen in strains grown extensively in laboratory substrates. Previous studies
have shown that filamentous morphology is lost upon passage in eggs in favor of spherical morphology. The
fact that filaments are maintained in nature but not in the laboratory raises the question of whether they
provide an advantage within the infected host. We examined the effect of serial adaptation to eggs, MDCK
cells, and guinea pigs on morphology. Two filamentous strains, A/Netherlands/602/2009(H1N1) and
A/Georgia/M5081/2012(H1N1), were passaged ten times in eggs and MDCK cells, substrates believed to
select for spheres. The spherical strain A/Puerto Rico/8/1934(H1N1) was passaged twelve times in guinea
pigs, aiming to select for filamentous virions. We found that an exclusively spherical morphology is not
required for improved growth in laboratory substrates. We did, however, identify two point mutations in the
matrix protein of egg passage 10 isolates that confer a spherical morphology and increased growth in eggs,
indicating that spherical viruses have a fitness advantage in eggs. Passage in guinea pigs resulted in the
selection of filament-forming viruses. These findings suggest a functional role for filaments in the infected
host.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [50]В Shardule Shah, IMP
Poster #47
p38 IS A NEGATIVE REGULATOR OF THE BORTEZOMIB-INDUCED HEAT SHOCK
RESPONSE IN MULTIPLE MYELOMA
Shardule P. Shah1,2, Vikas A. Gupta2, MD, PhD, Shannon M. Matulis2, PhD, Ajay K. Nooka2, MD, MPH,
Sagar Lonial2, MD, and Lawrence H. Boise1,2, PhD
1
Laney Graduate School, Emory University, Atlanta, GA, 2Hematology and Medical Oncology and Winship
Cancer Institute, Emory University, Atlanta, GA
Studies characterizing HSP regulation upon proteasome inhibition (PI) in multiple myeloma are limited. The
work displayed here sheds light on the regulation mechanism.
We show that HSF1 silencing and the proteasome inhibitor, bortezomib (VelcadeTM), have an additive death
effect in two myeloma cell lines, MM1.s and KMS18. HSF1 phosphorylation indicates activity and here we
demonstrate PI-induced phosphorylation in those lines and two patient samples.
There have been no published studies regarding kinases that phosphorylate HSF1 upon PI. To begin to
identify them, we treated cells with JNK, MEK, and p38 inhibitors to detect changes in bortezomib-induced
HSF1 phosphorylation. JNK and MEK inhibition yielded little change, but p38 inhibition ablated HSF1
constitutive phosphorylation and resulted in an increase in MM1.s and KMS18 inducible phosphorylation.
HSF1 activation leads to increased expression of downstream HSP genes via binding of HSF1 to heat shock
elements. We show that expression of HSP genes previously shown to be both induced upon PI and HSF1regulated, were increased when bortezomib is combined with p38 inhibition. Therefore, p38 may be a direct
negative regulator of the bortezomib-induced heat shock response.
Ultimately, the results shown here demonstrate a regulatory mechanism of the bortezomib-induced heat shock
response in multiple myeloma.
Jonathon Sia, IMP
Poster #48
MYCOBACTERIUM TUBERCULOSIS IMPAIRS DENDRITIC CELL FUNCTIONS THROUGH THE
SERINE HYDROLASE HIP1
Ranjna Madan-Lala1, Jonathan Kevin Sia1,2, Rebecca King1, Toidi Adekambi1, Bali Pulendran1,3, and
Jyothi Rengarajan1,4
1
Emory Vaccine Center, Emory University, Atlanta, GA, USA2Immunology and Molecular Pathogenesis
Graduate Program, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta,
GA, USA3Division of Pathology, Emory University School of Medicine, Atlanta, GA, USA4Division of
Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
Mycobacterium tuberculosis (Mtb) is a highly successful human pathogen that primarily resides in host
phagocytes and interferes with their functions. While multiple strategies used by Mtb to modulate
macrophage responses have been discovered, interactions between Mtb and DCs are less well understood.
DCs are the primary antigen presenting cells (APCs) of the immune system and play a central role in linking
innate and adaptive immune responses against microbial pathogens. We show that Mtb impairs DC cytokine
secretion and maturation through the cell envelope-associated serine hydrolase Hip1 via MyD88- and
TLR2/9-dependent pathways. These impaired processes led to suboptimal T-cell responses, as Mtb infected
DCs induced weaker Th1 and Th17 responses from antigen specific CD4 T-cells. Furthermore, we show that
priming of antigen specific T-cells in-vivo is affected during the early stages of the immune response in a low
dose aerosol mouse model of TB. Overall, these data show that Mtb impairs DC functions and modulates the
nature of antigen-specific T cell responses, with important implications for vaccination strategies.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [51]В Kristen Stout, MSP
Poster #49
THE SYNAPTIC VESICLE GLYCOPROTEIN 2C (SV2C) MEDIATES DOPAMINE VESICULAR
STORAGE AND RELEASE: IMPLICATIONS FOR PARKINSON’S DISEASE AND ADDICTION
Kristen A. Stout1, Amy R. Dunn1, Kelly M. Lohr1, Yingjie Li1, Thomas S. Guillot1, and Gary W. Miller1,2
1
Department of Environmental Health, 2Center for Neurodegenerative Disease, Emory University, Atlanta,
GA
Synaptic vesicle glycoproteins localize to synaptic vesicles and play a role in neurotransmission. Within this
family, the synaptic vesicle glycoprotein 2C (SV2C) localizes to regions associated with dopamine signaling,
such as the basal ganglia. Additionally, recent epidemiological data identified SV2C as mediating the
neuroprotective effects of nicotine in Parkinson’s disease, with minor SV2C allele frequencies reversing
nicotine neuroprotection. Thus, we hypothesize that SV2C contributes to dopamine storage and release. To
test this hypothesis, we generated SV2C knockout (SV2C-KO) mice. These mice show decreased dopamine
release in the dorsal striatum in comparison with WT littermate controls. Release within the nucleus
accumbens was also measured. Vesicular storage capacity in SV2C-KO mice is also reduced compared with
WT mice. These data suggest that SV2C plays an important role in the synaptic cycling of dopamine.
Further investigation is necessary to determine the contribution of SV2C to both neurodegenerative disease
and addiction.
E. Shannon Torres, GMB
Poster #50
UNDERSTANDING THE TRANSCRIPTIONAL REGULATORY MECHANISMS OF THE
HISTONE VARIANT, H2A.Z, THROUGH ITS INTERACTIONS WITH CHROMATIN
REMODELERS
E. Shannon Torres1 and Roger B. Deal2
1
Genetics and Molecular Biology Program in the Graduate Division of Biological and Biomedical Sciences,
Emory University, Atlanta, GA, 2Department of Biology, Emory University, Atlanta, GA
Differentiating cells acquire a stable transcriptional program necessary for proper development and
homeostasis, which is mediated through epigenetic processes, such as incorporation of histone variants into
nucleosomes. The histone H2A variant, H2A.Z, is involved in many genomic processes, including
transcriptional regulation. However, the mechanism through which it regulates transcription is currently
unclear. H2A.Z is necessary for transcription of the FLOWERING LOCUS C (FLC) gene in Arabidopsis, but
in the absence of transcriptional repressors, such as Brahma, H2A.Z is no longer required for transcription of
the gene. This project focuses on elucidating the mechanism by which Brahma antagonizes H2A.Z function to
infer the role of H2A.Z in transcriptional regulation. We will determine whether Brahma antagonizes H2A.Zmediated activation at FLC and similarly regulated loci through affecting nucleosome positioning or
occupancy and whether H2A.Z localization across the gene is impacted without Brahma. Additionally, we are
conducting a forward genetic suppressor screen to identify mutants that alleviate the need for H2A.Z
incorporation into nucleosomes for FLC activation. Currently, three candidate lines have been identified as
suppressor mutants. Future work to identify and characterize these genes will provide a deeper understanding
of H2A.Z function and broaden our understanding of transcriptional regulation through chromatin
remodeling.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [52]В MaKendra Umstead, CB
Poster #51
CHARACTERIZING A NOVEL AURORA KINASE A PROTEIN-PROTEIN INTERACTION IN
CANCER
MaKendra Umstead1,2, Cau Pham2, Zenggang Li2, Andrei Ivanov2, Yuhong Du2, Haian Fu2,3
1
Cancer Biology Graduate Program, 2Department of Pharmacology, 3Winship Cancer Institute
Cancer development is driven by genomic changes that affect normal protein function and induce a malignant
phenotype. Genomic data from The Cancer Genome Atlas (TCGA) provide opportunities to identifying the
mechanism by which aberrantly regulated proteins, often clustered in protein signaling pathways, contribute
to cancer. Therapeutic strategies can then be devised to target key protein hubs within the signaling network
and perturb oncogenic signaling while preserving tumor suppressive functions.
Proteins involved in many protein-protein interactions (PPIs) serve as signaling hubs. To identify these hubs,
we conducted high-throughput TR-FRET and PCA screening using a library of genes identified by TCGA to
be altered in cancer. One PPI hub revealed by the screen was Aurora Kinase A (AURKA). AURKA normally
facilitates mitosis; however, its overexpression in cancer suggests additional functions that remain to be
elucidated. For example, one novel binding partner of AURKA discovered by our screen was Ras. To
investigate the AURKA/Ras interaction, validation was completed using pull-downs, the structural properties
assessed by gene deletion analysis, and the impact of the interaction on cell signaling analyzed by western
blotting for downstream signaling. Ultimately, understanding the role of AURKA in cellular signaling will
provide new opportunities to develop targeted therapies for cancer.
Virginia Vachon, MMG
Poster #52
OPTIMAL ACTIVATION OF HUMAN 2’-5’ OLIGOADENYLATE SYNTHETASE-1 BY NONCODING VIRAL AND CELLULAR RNAS IS DEPENDENT UPON 3’ POLY-URIDYLATION
Virginia K. Vachon1,2, Brenda Calderon1 and Graeme L. Conn1
1
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta GA
30322, USA. 2Microbiology and Molecular Genetics (MMG) Program, Graduate Division of Biological and
Biomedical Sciences, Emory University.
The human 2’-5’ oligoadenylate synthetase-1 (hOAS1) is an important part of innate cellular immunity. Upon
binding viral dsRNA, hOAS1 synthesizes 2’-5’ linked oligoadenylates that activate RNAseL, thereby halting
replication through cleavage of viral mRNA or RNA genome. Both Adenovirus and Epstein Barr virus
produce non-coding RNAs (ncRNA), VA RNAI and EBER-1respectively, that inhibit the anti-viral dsRNA
binding protein PKR, ensuring continued translation of viral proteins. These Polymerase III (Pol III) RNA
transcripts are produced at high levels in later stages of infection, and are critical for replication of these
viruses. While both of these non-coding RNAs inhibit PKR’s anti-viral activity, they each activate rather than
inhibit hOAS1. In addition to these viral RNAs, a cellular Pol III transcript, non-coding RNA 886 (nc886),
has also been shown to inhibit PKR. Curiously, this cellular RNA is also a potent activator of hOAS1. We
found that presence of a 3’ poly U tail in each of these contexts is critical for optimal activity against hOAS1.
Activity of a model duplex RNA is also augmented by addition of a 3’ poly U tail, however a single U is
sufficient for maximal enhancement. Work is currently underway to determine the structural basis for these
observations.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [53]В Kellie Vinal, MMG
Poster #53
PATHOGEN-DERIVED 16S RRNA METHYLTRANSFERASE NPMA: MECHANISMS OF TARGET
RECOGNITION AND ENZYMATIC ACTIVITY
Kellie Vinal1,2, Pooja Desai1, Natalia Zelinskaya1, John Wang1 and Graeme L. Conn1
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta GA
30322, USA. 2Microbiology and Molecular Genetics (MMG) Program, Graduate Division of Biological and
Biomedical Sciences, Emory University.
1
Antibiotic resistance remains a pervasive problem in the treatment of bacterial infections due to the continual
evolution of resistance mechanisms in bacteria, as well as horizontal gene transfer between bacterial species.
The 16S rRNA methyltransferase NpmA specifically methylates nucleoside adenosine 1408 (A1408) in the
aminoglycoside binding site of the bacterial ribosome, conferring resistance to clinically relevant
aminoglycosides such as tobramycin and gentamicin. As the first A1408 16S rRNA methyltransferase
isolated from a human pathogen, it is critical to characterize the mechanism by which NpmA recognizes and
methylates its substrate as a platform for inhibitor development to combat this resistance determinant. Based
on data from rRNA probing experiments, residues predicted to interact with the 30S subunit were identified
and mutated. Kanamycin minimum inhibitory concentration (MIC) and 30S binding assays with these
mutants were used to pinpoint critical residues and characterize their role in conferring resistance.
Additionally, the crystal structure of the 30S subunit-NpmA complex was recently solved by our
collaborators. This new structural insight, in conjunction with functional data from the mutagenesis studies,
identified key residues for 30S binding and A1408 target base flipping, and allowed us to develop a molecular
model for the mechanism NpmA uses to methylate its substrate.
Jadiel Wasson, BCDB
Poster #54
THE FUNCTION OF LSD1 IN EPIGENETIC REPROGRAMMING AT FERTILIZATION
Jadiel A. Wasson1, Ashley L. King1, and David J. Katz1
Department of Cell Biology, Emory University
1
Epigenetic modifications are implicated in the maintenance and regulation of transcriptional programs. One
modification, H3K4me2 (dimethylation of lysine 4 on histone 3) is suggested to function as a type of
transcriptional memory by marking genes that were previously transcribed, establishing the cell’s
transcriptional program, and facilitating the transmission of this transcriptional program through cell division.
However, during cell fate change, H3K4me2 must be removed. At fertilization, the highly differentiated
sperm and egg, which have specific transcriptional programs, fuse and subsequently form the totipotent
zygote. This massive change in cell fate at fertilization implies there is a need to erase the gametic
transcriptional program in order to re-establish totipotency. The histone demethylase LSD1 specifically
erases H3K4me2 and is maternally deposited into the zygote at fertilization. We hypothesized that LSD1
plays a maternal role in epigenetic reprogramming at fertilization. To test our hypothesis, we have generated
two conditional deletion mouse models to specifically delete LSD1 in mouse oocytes. We find that maternal
deletion of LSD1 leads to a 1-2 cell arrest of progeny and a mis-expression of the oocyte transcriptional
program. In addition, by using a hypomorphic Cre allele, we find maternal loss of LSD1 leads to long range
epigenetic defects in progeny.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [54]В David Weir, CB
Poster #55
PROCASPASE-3 REGULATES FIBRONECTIN SECRETION AND INFLUENCES ADHESION,
MIGRATION AND SURVIVAL INDEPENDENT OF CATALYTIC FUNCTION
Matthew Brentnall1,2, David B. Weir1, Anthony Rongvaux3, Adam I. Marcus1, and Lawrence H. Boise1
1
Departments of Hematology and Medical Oncology and Cell Biology, Winship Cancer Institute of Emory
University, Atlanta, GA, 2Sheila and David Fuente Graduate Program in Cancer Biology, University of
Miami Miller School of Medicine, Miami, FL, 3Department of Immunobiology, Yale School of Medicine,
New Haven, CT
Apoptosis has profound effects on development and homeostasis. Inactivation of apoptosis can lead to
developmental disorders, carcinogenesis, autoimmunity, neurodegenerative disorders and defects in wound
healing. During apoptosis, caspase-3 (the main effector caspase) is activated downstream of Bax activation,
mitochondrial outer membrane permeablization and cytochrome c release. Previous work has shown that
caspase-3-deficient mouse embryonic fibroblasts are resistant to mitochondria-mediated cell death and exhibit
delayed Bax activation, mitochondrial outer membrane permeablization and cytochrome c release. There are
two ways to interpret this finding: caspase-3 is part of a feedback mechanism on mitochondria or,
alternatively, that caspase-3 has an independent non-apoptotic function that has an influence upstream of
mitochondrial events in apoptosis. Our laboratory has recently demonstrated that procaspase-3 functions as a
regulator of cell adhesion, migration and survival. This function is distinct and independent from the function
of activated caspase-3 in apoptosis. We plan to exploit mouse embryonic fibroblast cell lines to test our
overall hypothesis that fibronectin secretion is required for procaspase-3 regulation of cell adhesion,
migration and survival. Our long-term goal is to develop a comprehensive understanding of the molecular
mechanisms regulating cell adhesion, migration and survival, which will yield novel insight into the processes
of carcinogenesis and metastasis.
Amanda Wendt, NHS
Poster #56
UNDERSTANDING HEALTHCARE WORKERS’ PERCEPTIONS OF IRON AND FOLIC ACID
SUPPLEMENTATION IN BIHAR, INDIA
Amanda Wendt1, Melissa Young2, Amy Webb Girard1,2, Reynaldo Martorell1,2
1
Nutrition and Health Sciences, Laney Graduate School, Emory University, GA 30322
2
Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, GA 30322
High anemia prevalence and low iron and folic acid (IFA) consumption among pregnant women in Bihar is
an important public health issue. Surveys find 30% of Bihari women indicate receiving IFA and 23% report
IFA counseling during their last pregnancy. To address this, we conducted twenty in-depth interviews and
focus group discussions with three key types of frontline healthcare workers (FLWs) to explore their
knowledge of anemia and IFA counseling.
FLWs attributed anemia to factors including poor diet, close child spacing, high parity, and lack of rest, which
they perceived to stem from poverty, lack of awareness, low priority of women’s status, and family tension.
Many felt anemia was not a significant issue, estimating its prevalence as 0-20% (compared to survey
estimates of 60%). FLWs tended to restrict IFA consumption to the 4th-8th month. In conclusion, FLWs had
high knowledge of anemia causes but did not view it as a common issue. Further, their views of proper
consumption windows may prevent added health benefits of IFA consumption during the 9th month and
lactation. Efforts to allow IFA intake over a longer timeframe may increase opportunities for women to
receive IFA and be a key step to increasing IFA consumption.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [55]В Scott Wilkinson, CB
Poster #57
LKB1 REGULATES CELL POLARITY AND MOTILITY IN A FARNESYLATION-DEPENDENT
MANNER
Scott Wilkinson1, Adam I. Marcus2
1
Graduate Program in Cancer Biology, Emory University, Atlanta, GA, 2Hematology and Medical Oncology,
Emory University, Atlanta, GA
The tumor suppressor LKB1 is a serine/threonine kinase that serves as a master regulator of cell polarity, and
is mutated in 30% of non-small cell lung cancer tumors. LKB1 contains a C-terminal (CTD) farnesylation
motif to allow for plasma membrane insertion and interaction with other membrane-bound proteins. Using
confocal microscopy, we show that the LKB1 CTD regulates cell polarity and LKB1:actin association at the
leading edge in a farnesylation-dependent manner. We first created GFP constructs of both LKB1 wildtype
and CTD, as well as C430S farnesylation motif mutants of each. These constructs were transfected into
LKB1-null cells. Upon confluency, a scratch-wound assay was performed and 6 hours later cells were fixed
and permeabilized. We probed with a GM130 antibody (Golgi marker, used to identify polarization) and
stained with phalloidin to visualize actin filaments. Using confocal microscopy, we identified LKB1
farnesylation as critical for regulating cell polarity and leading edge actin association. Importantly, this is the
first report of kinase farnesylation in maintaining cell polarity. These data show that LKB1 farnesylation is
critical for regulating cell polarity and motility through its actin association, potentially leading to novel
insights into mechanisms of LKB1 function in regulating cell polarity and motility.
Kathryn Williams, BCDB
Poster #58
REPRESSION OF GAP-43 EXPRESSION BY THE MRNA BINDING PROTEIN HNRNP-Q1:
INVESTIGATING A LOCAL MECHANISM IN NEURONS
K.R. Williams1, L. Xing1, G.J. Bassell1
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA
1
mRNA binding proteins function to post-transcriptionally regulate gene expression, a key process that is
required for proper neuronal development. The mRNA binding protein hnRNP-Q1 is an ideal candidate
regulator of mRNA transport and/or local translation given that it is a component of transport mRNP granules
and regulates mRNA translation. We have identified GAP-43 mRNA as a new target of hnRNP-Q1. GAP-43
protein is highly enriched in axonal growth cones where it functions to regulate actin dynamics and ultimately
axonal growth and/or guidance. Additionally, GAP-43 mRNA is localized to developing axons and growth
cones. Therefore, we hypothesize that hnRNP-Q1 regulates the axonal localization and/or local translation of
GAP-43 mRNA as a mechanism to enrich GAP-43 protein and affect axon growth. Using RNAi methods to
knockdown hnRNP-Q1 in Neuro2a cells, our results suggest that hnRNP-Q1 suppresses the expression of
GAP-43 protein but does not affect steady state GAP-43 mRNA expression. Work in progress is to
knockdown hnRNP-Q1 in primary cortical neurons to assess possible impairments in GAP-43 mRNA
localization and local translation as well as axonal phenotypes that may result from impaired localization of
GAP-43 mRNA. hnRNP-Q1-mediated regulation of GAP-43 may be a key mechanism for regulating
neuronal development.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [56]В Myra Woodworth-Hobbs, NHS
DOCOSAHEXAENOIC ACID
SIGNALING IN MYOTUBES
Poster #59
COUNTERACTS
PALMITATE-INDUCED
PROTEOLYTIC
Myra E. Woodworth-Hobbs1,2Вё Matthew B. Hudson2, Jill A. Rahnert2, Bin Zheng2, Harold Franch2, S. Russ
Price2,3.
1
Nutrition and Health Sciences, Graduate Division of Biological and Biomedical Sciences,
2
Medicine/Nephrology, Emory University, Atlanta, GA; 3Atlanta VAMC, Decatur, GA
Dyslipidemia is a comorbidity common to illnesses like diabetes which contributes to debilitating muscle
atrophy. The saturated fatty acid palmitate (PA) induces insulin resistance and disrupts protein metabolism in
cultured myotubes, whereas the omega-3 fatty acid docosahexaenoic acid (DHA) has beneficial metabolic
effects. We evaluated the effects of PA and DHA on atrophy-related signaling in skeletal muscle, with the
hypothesis that DHA prevents PA-induced myotube atrophy by counteracting the stimulatory effects of PA on
protein degradation. C2C12 myotubes were treated with 500uM PA and/or 100uM DHA for up to 28h. PA
increased the rate of protein degradation, while co-treatment with DHA completely prevented the response.
Akt inhibits the FoxO3 transcription factors which regulate “atrogene” expression. PA reduced the activation
state of Akt as well as increased the level of nuclear FoxO3 protein while decreasing cytosolic FoxO3. PA
also increased the mRNA levels of two FoxO3 atrogene targets, the E3 ubiquitin ligase atrogin-1/MAFbx and
the autophagy mediator Bnip3. DHA reversed the effects of PA on Akt, FoxO3, and both atrogenes. These
data indicate that PA induces myotube atrophy by inducing components of the ubiquitin-proteasome and
autophagic proteolytic systems and that DHA counters the catabolic effects of PA by improving Akt
signaling.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [57]В ACKNOWLEDGEMENTS
The GDBBS Division Student Advisory Council (DSAC) would first like to express our
sincerest gratitude to all of the participants-students, faculty, and post-docs- who made this 11th
Annual DSAC Student Research Symposium possible.
Our deepest gratitude goes to the Graduate Student Council (GSC) for funding the event. In
addition we want to thank Monica Taylor for her continued efforts to help make the symposium a
success. We would also like to thank the GDBBS office and program staff for their assistance. Lastly
we thank the GDBBS Director, Keith Wilkinson for continuing to support our efforts to do more than
sponsor mixers.
Again, thank you, and we look forward to seeing you next year as we convene again to make
the symposium a signature event for all of us in GDBBS.
TheВ EleventhВ AnnualВ DSACВ StudentВ ResearchВ SymposiumВ [58]В 

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