2015 graduate student symposium

Transcription

GRADUATE
STUDENT
SYMPOSIUM
The Graduate School of Biomedical Sciences
Baylor College of Medicine
Thursday, October 29, 2015
THE 27th ANNUAL GRADUATE STUDENT
RESEARCH SYMPOSIUM
The Graduate School of Biomedical Sciences
Baylor College of Medicine
Houston, Texas
October 29, 2015
Program
Awards Ceremony
Welcome
Graduate Student Council
Message from Dr. Kuspa
The Joseph L. Melnick Lecturer – Dr. Christopher Mason
Student Speaker Abstracts
Poster Presenters
Awards 2014-15
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Cover Legend:
“Immunofluorescent staining of mouse female reproductive organs showing distribution of
cilia through the lumen of the oviduct. Beta tubulin (green) labeling cilia microtubules, DAPI
(blue) labeling the nuclei, phalloidin (red) labeling actin in smooth muscle cells. Image
nd
submitted by Jason Burton, a 2 year IMBS graduate student in the lab of Irina Larina, PhD.
A publication of The Graduate School of Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030
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Oral Presentations – Cullen
Poster Presentations – Rayzor Lounge
9:15 – 9:30
9:30 – 9:35
9:35 – 9:40
9:40 – 9:45
Breakfast (Coffee & snacks provided by the Graduate Student Council)
Introductions
Welcome
Timothy Dosey, Graduate Student Council President
Alicia Monroe, M.D., Provost & Sr. VP of Academic
& Faculty Affairs
Gayle Slaughter, Ph.D. Sr. Associate Dean of
Graduate Education & Diversity
Announce Poster
Finalist
9:45 – 10:00
10:00 – 10:15
10:15 – 10:30
10:30 – 10:45
10:45 – 11:00
Biochemistry & Molecular Biology (pg 7)
Pharmacology (pg 8)
Translational Biology & Mol. Medicine (pg 9)
Molecular Physiology (pg 10)
Monica GalazMontoya
Xiang Feng
Simran Madan
John Leach
Break (Coffee & snacks provided by the Graduate Student Council)
The Joseph L. Melnick Distinguished Guest Speaker
11:00 – 12:00
12:00 – 1:15
1:15 – 1:30
1:30 – 1:45
1:45 – 2:00
2:00 – 2:15
2:15 – 2:30
2:30 – 2:45
2:45 – 3:00
3:00 – 3:15
3:15 – 3:30
3:30 – 3:45
Christopher Mason, Ph.D.
“Single-cell, city-scale, and inter-planetary genomics”
Finalist Poster Session
3:45 – 4:00
Leah Gates
Molecular & Cellular Biology (pg 11)
Vitor Onuchic
Structural & Computational Biology (pg 12)
Ran You
Immunology (pg 14)
Daniel Mendoza
Clinical Scientist Training Program (pg 14)
Wenyi Zhu
Integrative Molecular & Biomedical Sciences (pg 15)
Alexander
Developmental Biology (pg 16)
Herman
Neuroscience (pg 17)
Molecular & Human Genetics (pg 18)
Lucy Liu
Chih-Chun Lin
Molecular Virology & Microbiology (pg 19)
Jason Kaelber
4:00 – 5:00
Awards Ceremony
Adam Kuspa, Ph.D., Sr. VP & Dean of Research and
Interim Dean of the Graduate School
Reception (Coffee & snacks provided by the Graduate Student Council)
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Awards Ceremony & Reception
Thursday, October 29, 2014
4:00 – 5:00 PM
8-Stranded Beta-Barrel Jelly Roll Awards
Professor John J. Trentin Scholarship Awards
Deborah K. Martin Achievement Award in Biomedical Sciences
Marc Dresden Excellence in Graduate Education Award
Poster Awards
Student Speaker Awards
Abstract Book can be located at:
https://www.bcm.edu/education/schools/graduate-school-of-biomedicalsciences/current-students/graduate-student-council/activities-and-opportunities/gsbssymposium
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Welcome to Baylor College of Medicine’s 27th Annual Graduate Student
Symposium! Thank you for joining us as we honor all BCM graduate students and
showcase some of their exceptional biomedical research.
This year’s symposium features student poster presentations and platform
talks delivered by student representatives from each department and graduate
program. In addition to the outstanding student work, we are pleased to have
Associate Professor Christopher Mason PhD from the Weill Cornell Medical College
of Cornell University deliver the Joseph L. Melnick Distinguished Lecture.
The events of today would not have been possible without the help of many
dedicated students and faculty. Members of the Graduate Student Council have
graciously volunteered their time and efforts all year. For their hard work and their
indispensable help with the symposium, we extend our sincerest thank you.
Special thanks are also owed to Melissa Houghton, Dr. Gayle Slaughter, and
Dr. John Rodgers for their leadership roles in planning and organizing all aspects of
today’s symposium. We also thank Dr. Carolyn Smith for her work in organizing the
symposium and continued mentorship of the Graduate Student Council.
It has been our pleasure serving you over the last year and we hope you enjoy
today’s symposium.
Tim Dosey, GSC President
Cameron Landers, GSC Vice President
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2015 GRADUATE STUDENT COUNCIL MEMBERS
President: Tim Dosey, Integrative Molecular and Biomedical Sciences
Vice-President: Cameron Landers, Translational Biology & Molecular Medicine
Treasurer: Shrenik Mehta, Pharmacology
Secretary: Marissa Scavuzzo, Developmental Biology
Representatives:
Jessica Scott, Integrative Molecular and Biomedical Sciences
Jordan Kho, Developmental Biology
Ninad Oak, Molecular and Human Genetics
Chinh Nguyen, Molecular Virology and Microbiology
Jenny Sun, Neuroscience
Marissa Scavuzzo, Developmental Biology
James Arnold, Biochemistry and Molecular Biology
Michelle Rubin, Integrative Molecular and Biomedical Sciences
Sungwoo Choi, Developmental Biology
Amanda Rodriguez, Molecular and Cellular Biology
Amanda Webb, Molecular and Human Genetics
Ann Quick, Molecular Physiology and Biophysics
Anisha Misra, Molecular Virology and Microbiology
Asante Hatcher, Neuroscience
Papiya Sinha, Pathology and Immunology
Cameron Brown, Structural and Computational and Molecular Biophysics
James Campbell, Structural and Computational and Molecular Biophysics
Valencia Potter, Medical Scientist Training Program
Katharina Schulze, Molecular and Human Genetics
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“Baylor College of Medicine is a health sciences university that creates knowledge
and applies science and discoveries to further education, healthcare and community service
locally and globally”. This mission statement is meant to convey our institutional character that all that we do as a health sciences university is grounded in research. Research at Baylor
College of Medicine, guided by our faculty, is accomplished in large part through the
ingenuity and dedicated effort of our trainees, especially the graduate students whose work
we celebrate today. It is awe inspiring to see all of the great science presented at this
symposium every year and to learn how our students are pushing the boundaries of basic,
translational, and clinical sciences. I want to congratulate each of you for your research
accomplishments over the past year. I look forward to another exciting program filled with
remarkable discoveries.
Adam Kuspa, Ph.D.
Senior Vice President and Dean of Research
Interim Dean of the Graduate School
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THE JOSEPH L. MELNICK DISTINGUISHED
GUEST LECTURER
Christopher Mason Ph.D.
Associate Professor in the Department of Physiology and
Biophysics, Weill Cornell Medical College
Associate Professor in the Institute for Computational
Biomedicine, Weill Cornell Medical College
Christopher E. Mason completed his dual B.S. in Genetics and Biochemistry from University
of Wisconsin-Madison in 2001, his Ph.D. in Genetics from Yale University in 2006, and his postdoctoral training at Yale Medical School, while also holding a fellowship at Yale Law School. He is
currently an associate professor at Weill Cornell Medical College.
Dr. Mason founded his laboratory at Weill Cornell Medical College, in the Department
of Physiology and Biophysics and at the Institute for Computational Biomedicine, as well as
appointments at the Tri-Institutional Program on Computational Biology and Medicine between
Cornell, Memorial Sloan-Kettering Cancer Center and Rockefeller University, the Weill Cornell
Cancer Center, and the Feil Family Brain and Mind Research Institute.
Since 2010, he has won NIH’s Transformative R01 Award, the Hirschl-Weill-Caulier Career
Scientist Award, the Vallee Foundation Young Investigator Award, the CDC Honor Award for
Standardization of Clinical Testing, the WorldQuant Foundation Research Scholar Award, and he
was named as one of the “Brilliant Ten” Scientists in the world by Popular Science magazine and
spoken for TEDMED. His over 100 papers and briefs on genomics and metagenomics have been
featured on the covers of Science, Nature Biotechnology, Nature Collections, Cell Systems, Neuron,
Genome Biology and Evolution, on the cover of the Wall Street Journal and The New York Times,
featured on TV (CNN, PBS, ABC, FoxNews), cited by the U.S. District Court and U.S. Supreme
Court, and covered by 300 other media outlets around the world
The Graduate Student Research Symposium’s Joseph L. Melnick Guest Lectureship is made
possible through a generous endowment from the late Joseph L. Melnick, Ph.D., the first Dean of
the Graduate School of Baylor College of Medicine. Dr. Melnick served in that capacity from
1968-1991.
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A cAMP INDEPENDENT PATHWAY FOR CALCIUM MOBILIZATION BY BETA-2
ADRENERGIC RECEPTOR
Monica Laura Galaz-Montoya
Department of Biochemistry & Molecular Biology
Advisor:
Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
Beta adrenergic receptors (β2-AR) are important for vascular regulation in the heart
and lung and for physiological responses to the hormones/neurotransmitters adrenaline and
noradrenaline. They are found in the nervous system and throughout the body and are the
targets of numerous widely used drugs. Their canonical signaling pathway involves
activation of adenylyl cyclase (AC) and a rise in cyclic AMP (cAMP) levels, which leads to
activation of cAMP-dependent protein kinase (PKA). Beta adrenergic receptors have long
been thought to activate distinct signaling pathways from those downstream of G-protein
coupled receptors which activate phospholipase C (PLC) and elevate intracellular Ca2+. By
monitoring intracellular Ca2+ levels in real time using a fluorescent indicator dye we found
that an endogenous receptor in HEK-293 cells responds to the adrenergic agonist
norepinephrine by a delayed rise in intracellular [Ca2+]. The response is blocked by ICI
118,551, a selective antagonist for β2-AR, and the relative potency of agonists is
isoproterenol > epinephrine > norepinephrine, consistent with the pharmacological profile of
β2-AR. Treatment with thapsigargin (an inhibitor of the SERCA Ca2+ pump of the
endoplasmic reticulum) and chelation of extracellular Ca2+ revealed that the Ca2+ is
released from intracellular stores. The release is sensitive to inhibition of PLC with U73122
and of InsP3 receptors with 2-APB. Treatment with cholera toxin, a drug that activates Gαs,
indicated that direct activation of this G protein is not sufficient for Ca2+ release, although it
increases intracellular cAMP. Additionally, treatment with adenylyl cyclase inhibitors SQ
22536 and dideoxyadenosine did not inhibit isoproterenol-induced Ca2+ responses whereas
both inhibitors were able to suppress cAMP production by AC. Additionally, an increase in
intracellular cyclic AMP levels by treatment with the phosphodiesterase inhibitors IBMX does
not potentiate the response of adrenergic agonists. Furthermore, treatment with PKA
inhibitors H-89 and KT5720 had no effect on the Ca2+ signal, and treatment with the cAMP
analogue 8-bromo-cAMP, which selectively activates PKA, did not trigger a Ca2+ response
even though it was effective at triggering PKA induced CREB phosphorylation. These
results strongly support the conclusion that AC, cAMP and the cAMP effector PKA are not
involved in this signaling pathway. Together these findings indicate that activation of β2-AR
leads to an increase in cytoplasmic [Ca2+] by a previously unrecognized signaling pathway.
A novel mechanism for Ca2+ mobilization by β2AR has broad implications for adrenergic
signaling, and in particular for the effects of βAR-directed drugs.
Contributors: Galaz-Montoya, Monica; Rodriguez, Gustavo; Lichtarge, Olivier and Wensel,
Theodore
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HIGH-RESOLUTION PREDICTION AND DESIGN OF STRUCTURALLYUNCHARACTERIZED G PROTEIN-COUPLED RECEPTOR WITH NOVEL
LIGAND BINDING SELECTIVITES
Xiang Feng
Department of Pharmacology
Advisor:
Patrick Barth, Ph.D.-Department of Pharmacology
Ligand docking is a key component of computer-aided rational drug discovery
methods, which play a crucial role in modern drug development. It has been shown
that ligand docking can effectively decrease the number of compounds necessary to
screen while retaining the same level of lead compound discovery.
It has long been known that a simplistic rigid ‘lock-and-key’ model of ligandreceptor interaction is inadequate and incorporation of conformational
rearrangement that accounts for ’induced fit’ or ‘conformational selection’ model of
ligand-receptor interaction is required for accurately predicting ligand-receptor
binding. However, receptor flexibility remains a challenge for ligand docking
applications because of the large number of conformational degrees of freedom that
have to be considered in calculation. Besides such algorithm limitation, another
factor that limits the application of ligand docking to membrane proteins and GProtein Coupled Receptors (GPCR) in particular is the difficulty in obtaining accurate
structural information. Although GPCRs represent the targets of around 40%
approved drugs, less than 5% have solved crystal structures. Comparative modeling
of GPCR structures can greatly increase the number of potential drug targets that
can be used in ligand docking, but currently suffers from inaccurate ligand binding
pocket modeling.
To address these two challenges simultaneously, we developed a novel
protocol in the software Rosetta to build accurate models of ligand docked GPCR
complexes for a wide range of structurally uncharacterized receptors. The new
protocol not only models explicitly receptor flexibility but also improves the accuracy
of the ligand binding site by integrating loop rebuilding and ligand docking steps. Our
technique outperforms existing methods in its ability to recover native ligand binding
conformation and ligand-protein interactions in blind prediction and benchmark tests.
We also show that our approach can be used to predict the effects of receptor
sequence changes on ligand binding affinities and specificities. We also applied our
technique to design structurally uncharacterized dopamine D2 receptor variants with
novel ligand binding selectivities.
Our method should prove useful in the drug discovery process of structurally
uncharacterized GPCRs and sets the stage for engineering receptors with novel
pharmacological properties.
Contributors: Feng, Xiang; Chen, Kuang-Yui Michael; Garrido, Joaquin Ambia; Barth,
Patrick
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HEPATIC GLYCOGEN ACCUMULATION AND LIVER DYSFUNCTION IN ASL
DEFICIENCY
Simran Arun Madan
Program in Translational Biology & Molecular Medicine
Advisor:
Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Brett Graham, M.D./Ph.D.-Department of Molecular & Human Genetics
Argininosuccinate lyase (ASL) is a urea cycle enzyme responsible for conversion of
argininosuccinate to arginine and fumarate. ASL deficiency (ASLD) is an autosomal
recessive disorder. Neonatal symptoms of ASLD include hyperammonemia, vomiting,
lethargy and seizures. Despite early intervention, some patients develop long-term
complications including liver dysfunction, neurocognitive deficits, developmental delays and
hypertension. Liver pathology in patients with ASLD may include swollen hepatocytes,
glycogen accumulation, fibrosis and cirrhosis, and liver transplantation may eventually be
required in some patients. The ASLneo/neo hypomorphic mouse serves as a good model
for evaluating liver dysfunction in ASLD because we have discovered that these mice, like
the human patients, have hepatomegaly, elevated liver enzymes, and hepatic glycogen
accumulation. To further evaluate this glycogen accumulation, we evaluated the activity of
hepatic enzymes involved in glycogen metabolism and discovered that ASL deficient mice
have decreased activity of hepatic glycogen phosphorylase, an enzyme responsible for
breakdown of glycogen. Thus, we hypothesize that this decreased activity of glycogen
phosphorylase may explain the hepatic glycogen accumulation and may contribute to liver
dysfunction observed in mice and humans with ASLD. Preliminary studies investigating the
cause for decreased hepatic glycogen phosphorylase activity revealed similar hepatic RNA
expression levels of glycogen phosphorylase between ASL deficient and wild type mice but
reduced protein levels in ASL deficient mice. We are now investigating post-translational
modifications on glycogen phosphorylase. In particular, we have shown that ASL is required
for nitric oxide production and that loss of ASL leads to a global decrease in protein
nitrosylation in several tissues, including the liver.
Interestingly, hepatic glycogen
phosphorylase is one protein that is differentially nitrosylated in the liver of ASL deficient vs.
wild type mice. To address whether loss of nitrosylation of glycogen phosphorylase
contributes to the hepatic glycogen accumulation in ASLD, we are testing if supplementation
with a nitric oxide donor increases the activity of glycogen phosphorylase in vitro and in vivo
and whether this results in improvement in the hepatic glycogen accumulation or liver
dysfunction phenotype in ASL deficient mice. Studies evaluating the mechanism underlying
hepatic glycogen accumulation and liver dysfunction in this mouse model of ASLD may
potentially lead to the discovery of new therapeutic strategies for liver dysfunction in human
patients with ASLD.
Contributors: Madan, Simran*; Burrage, Lindsay*; Paschalis, Doulias; Cela, Racel; Jiang,
Ming Ming; Chen, Yuqing; Bertin, Terry; Ischiropoulos, Harry; Finegold, Milton; Lee,
Brendan.
*Contributed equally to this work
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HIPPO SIGNALING DELETION REVERSES SYSTOLIC HEART FAILURE
John Preston Leach
Department of Molecular Physiology & Biophysics
Advisor:
James Martin, M.D./Ph.D.-Department of Molecular Physiology &
Biophysics
Mammalian organs vary widely in regenerative capacity. All organs are prone
to failure, but poorly regenerative organs like the heart are particularly vulnerable to
tissue loss and organ failure. However, newborn mice possess an astonishing ability
to regenerate heart tissue. This ability is limited and is completely lost by seven days
after birth, thus, developmental pathways could be the key to unlocking regenerative
potential.
The organ-intrinsic, cell-cycle regulating Hippo-pathway activates during this
seven day window after birth. Therefore we postulate that Hippo signaling inhibits
regeneration in the adult heart. Indeed, we have previously shown that the Hippo
pathway controls cardiomyocyte proliferation during development to restrain heart
size, and knockout of Hippo pathway proteins Salvador and Lats1/2 leads to an
increase in cardiomyocyte renewal and is cardioprotective against ischemic damage
in the adult heart.
Furthermore, heart failure is the result of a vicious cycle deriving from the
physiologic response to low oxygen perfusion; this places stress on residual
cardiomyocytes, leading to the further loss of cardiomyocytes and decreased cardiac
function. Therefore, we investigated whether Hippo pathway deletion in failing
hearts, provided therapeutic benefits.
Hippo pathway inactivation resulted in functional recovery with extensive
repopulation of the left ventricle with contractile muscle. Sequential labeling with
nucleoside analogs revealed rare stem cell–like growth in Hippo-deficient
cardiomyocytes. An increase in capillary density provides evidence for improved
vascularization at the interface between muscle and scar tissue. Finally, RNA-seq
reveals involvement of circadian rhythm in improved cardiac function. Thus far we
have determined Hippo pathway deletion in the failing heart results in functional
recovery and has the potential for therapeutic approaches to treat systolic heart
failure.
Contributors: Leach, John P.; Heallen, Todd; Zhang, Min; Hill, Matthew C.; Rahmani,
Mahdis; Willerson, James T.; Martin, James F.
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THE ROLE OF H3K4ME3-PROMOTED HISTONE ACETYLATION IN
TRANSCRIPTION
Leah Ashley Gates
Department of Molecular & Cellular Biology
Advisor:
Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Epigenetic modifications such as histone marks are critical regulators of gene
expression and chromatin structure. Determining the molecular mechanisms of how
these modifications impact transcription is therefore necessary to understand cell
physiology and pathologies resulting from altered gene expression. These histone
marks serve to promote the recruitment or dismissal of coregulators that can
influence the transcriptional outcome through the direct binding of effector proteins,
or “readers”. Trimethylation on histone H3 at lysine 4 (H3K4me3) stimulates both
transcription and subsequent histone acetylation selectively on histone H3 at lysine
9 (H3K9Ac). We therefore hypothesized that H3K9Ac recruits specific reader
proteins for progression through the transcription cycle. By performing biotinylated
histone peptide pulldowns with HeLa nuclear extract followed by mass spectrometry
and immunoblotting, we identified the super elongation complex (SEC) as being
recruited to H3K9Ac through the reader protein AF9. Importantly, reduction of
H3K4me3 results in the global decrease of H3K9Ac and histone H4 acetyl marks.
Additionally, loss of H3K4me3 and H3K9Ac results in reduced AF9 occupancy on
select genes, as well as reduced gene expression. However, RNA polymerase II
occupancy increases at select gene promoters with the loss of these histone marks,
indicating a defect in promoter clearance and transcription elongation. Based on this
data, we propose a model in which H3K4me3 is required for transcription initiation
and promotes acetylation of H3K9 through the recruitment of select histone
acetyltransferases. This acetylation can then recruit transcription elongation
machinery via AF9. Next, we will determine the direct functional relevance of
H3K9Ac in transcription using biochemical functional assays. Importantly, these
studies will advance our understanding of how epigenetic marks can regulate
transcription at the level of RNA polymerase II elongation.
Contributors: Gates, Leah A.; Feng, Qin; Rohira, Aarti D.; Bedford, Mark T.; Sagum, Cari A.;
Jung, Sung Yun; Qin, Jun; Tsai, Sophia; Tsai, Ming Jer; Foulds, Charles E.; O’Malley, Bert
W.
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EPIGENOMIC DECONVOLUTION YIELDS INSIGHTS INTO THE BIOLOGY OF
BREAST TUMOR CELLS WITHIN THEIR NATIVE MICROENVIRONMENT
Vitor Onuchic
Program in Structural and Computational Biology and Molecular Biophysics
Advisor:
Aleksandar Milosavljevic, Ph.D.-Department of Molecular & Human
Genetics
Tumor phenotypes result from interactions of a diversity of cell types and yet it is
currently not possible to measure epigenomic and transcriptomic states of constituent cell
types without perturbing their interactions by physically isolating them from their
microenvironment. To gain insights into cancer-related processes within epigenetically
defined subpopulations of breast tumor cells within their native microenvironment, we
developed Epigenomic Deconvolution (EDec), a two-stage procedure for estimating
methylation and gene expression profiles of constituent cell types, as well as proportions of
constituent cell types in each input sample. We validate high accuracy of EDec using both
experimental and simulated mixtures of purified cell types in known proportions. When
applied to 1184 breast tumor methylation profiles from the TCGA collection the method
infers methylation profiles of constituent cell types that closely match the reference
methylation profiles of cell types known to constitute breast tumors. The inferred cell type
proportions are highly concordant with pathologist’s estimates based on H&E staining. By
comparing the cell type specific gene expression profiles, we detected gene expression
changes that are highly consistent with known hallmarks of cancer. We confirmed
significantly longer survival for triple negative breast cancer patients with high immune cell
infiltration within their tumors. We detected down-regulation of cellular respiration pathways
in triple negative tumor associated stroma in conjunction with an up-regulation of those
same pathways in the cancerous epithelial cells, a metabolic shift consistent with the
previously proposed Reverse Warburg Effect. Lastly, by analyzing gene expression changes
that are specific to epithelial cells of triple negative tumors, we identified gene expression
changes in a large number of SP1 regulated genes, including CDH1. Such changes are
consistent with the down-regulation of SP1 that is also identified specifically in epithelial
cells of basal-like breast cancers. The down-regulation of SP1 is consistent with a single
copy deletion affecting the SP1 gene that is present in nearly 60% of basal-like breast
cancers, but is rarely detected in other subtypes of breast cancer. Despite not being
previously reported, the down-regulation of SP1 and deregulation of its targets specifically in
basal-like breast cancers is highly consistent with the more aggressive and EMT-like
phenotype of that subtype of breast cancers. We show that these cancer cell perturbations
could not be detected without EDec because of averaging across diverse cell types within
complex tumor tissue. We therefore conclude that EDec in conjunction with newly available
reference epigenomes provides unique new insights into the biology of tumor cells within
their native microenvironment. Contributors: Onuchic, Vitor; Hartmaier, Ryan; Boone, David;
Oesterreich, Steffi; Samuels, Michael L.; Roth, Matt E.; Lee, Adrian V.; Milosavljevic,
Aleksandar
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CARBON BLACK-INDUCED INFLAMMATION IN EMPHYSEMA AND LUNG
CANCER
Ran You
Department of Pathology & Immunology
Advisor:
Farrah Kheradmand, M.D.-Department of Medicine
Environmental pollutants and cigarette smoke are major causative factors for lung
diseases, such as COPD (chronic obstructive pulmonary diseases) and lung cancer, which
are respectively the 3rd leading cause of death and the 1st cancer-related death, however
the shared pathogenic substance by all risk factors for these diseases is still unknown. In
our study, carbon black (CB), as the component of coal mine dust, cigarette smoke and
airborne particles, is found to deposit in the lung antigen-presenting cells (APCs) of
emphysema/COPD patients and mice exposed to chronic cigarette smoke. Therefore, we
hypothesized that CB-induced activation of APCs and lung inflammation promote
progression of emphysema and lung cancer.
By using mouse model with the intranasal challenge of CB, we found that CBchallenged mice developed similar immunopathological changes in emphysema patients
and mice exposed to chronic smoke, including enlarged lung volume, infiltration of immune
cells into the lungs, upregulated disease-related gene and strong Th17 responses.
Moreover, CB directly activate APCs by the upregulation of pro-Th17 cytokines, IL-6 and IL1β. Furthermore, by comparing the soluble nanoparticle polyethylene glycol (PEG)-CB
(hydrophilic) and elemental CB (hydrophobic), we found that hydrophobic CB induced more
severe inflammation and emphysema, indicating that surface features of CB contribute to its
pathogenesis.
More interestingly, CB-induced inflammation also decreases lung tumor latency. In
airway specific Pten/Smad4 deficient mice, CB promotes early lung cancer progression
concomitant with early stomach metastasis. Further on, CB directly increases the invasion
of human bronchoepithelial cells with Pten/Smad4 knockdown.
To figure out the underlying mechanism of how hydrophobic CB induces emphysema
and promotes cancer progression, we did RPPA analysis of CB-treated cells and found that
CB directly induced DNA double strand break and Erk signaling, which can stimulate
genomic instability, inflammation and cell invasion. APCs treated with the inhibitors to either
protein kinases sensing DNA damage or MAPK kinase were less sensitive to CB treatment
indicated by less IL-6 production.
In all, CB, as the shared pathogenic substance in environmental pollutants and
cigarette smoke, induces severe inflammation and promotes emphysema and early lung
cancer progression by direct induction of DNA damage. Potential mechanisms of CBinduced APC activation revealed in our study provide new insights into the
immunopathogenesis of lung diseases caused by environmental risk factors.
Contributors: You, Ran; Shan, Ming; Lu, Wen; Cho, Sungnam; Seryshev, Alexander;
Marcano, Daniela; Song, Lizhen; Yuan, Xiaoyi; Demayo, Francesco; Tour, James; Corry,
David; Kheradmand, Farrah
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DENDRITIC CELL RESPONSES TO PNEUMOCOCCAL VACCINES ARE
IMPAIRED IN HIV-INFECTED SUBJECTS ON ANTIRETROVIRAL THERAPY
Daniel Mendoza
Clinical Scientist Training Program
Advisor:
David Corry, M.D.-Department of Medicine
Background. HIV+ subjects have high burden of invasive pneumococcal disease
(IPD). The pneumococcal vaccines, the 23-valent polysaccharide vaccine (PPV) and the 13valent polysaccharide conjugate vaccine (PCV), have reduced efficacy in HIV+ patients.
PPV contains pure pneumococcal capsular polysaccharides (PS), and PCV has PS
conjugated to the protein carrier CRM197 to enhance immunogenicity. Dendritic cells (DC)
play essential roles in vaccine responses by producing cytokines. We hypothesize that DC
produce cytokines in response to pneumococcal vaccines and that HIV impairs this DC
function. Methods. We performed a clustering analysis of 41 cytokines produced in the
supernatant of peripheral blood mononuclear cells (PBMC) exposed to pneumococcal
vaccines for 6 h ex vivo using a Luminex machine. PBMC were from healthy unvaccinated
adults. We exposed PBMC to PCV and PPV for 6 h to measure production of the identified
cytokines in DC using flow cytometry. We used PBMC of HIV+ patients off antiretroviral
therapy (ART), HIV+ patients on >6 months of ART and healthy controls. We evaluated
whether pre-vaccine DC production of cytokines ex vivo predicted post-vaccine serum
opsonophagocytic killing activity (OPA), which is an in vitro assay that correlates with
protection against IPD. Results. PPV and PCV induced similar cytokine profiles consistent
with an antibacterial innate response. Among the cytokines produced were the chemokine
IL-8, and IL-6, which induces inflammation and B cell differentiation. DC produced IL-8 in
response to pneumococcal vaccines (median percent of DC producing IL-8: media: 5.1%,
PPV 10%, PCV 23%, p<0.001). These DC responses targeted the PS component in both
vaccines and not the protein carrier: median percent of DC producing IL-8: media: 11.1%,
CRM197: 9.3%, PS: 21.2%, p=0.01; and of DC producing IL-6: media: 1.6%, CRM197:
2.2%, PS: 5.4%, p=0.02. DC production of cytokines against PCV was reduced in HIV+
subjects (percent of DC producing IL-6: HIV+ subjects off ART: 0%, HIV+ subjects on ART:
0.7%; uninfected subjects: 17.5%, p<0.0001; percent of DC producing IL-8: HIV+ subjects
off ART: 6%, HIV+ subjects on ART: 10%, uninfected subjects: 43%, p<0.0001. Pre-vaccine
DC production of IL-8 in response to PCV predicted OPA against PS (serotype 6B)
measured 1 month post-vaccination (r=0.48, p=0.001). Conclusion. DC production of
cytokines against pneumococcal vaccines is impaired in HIV+ patients and this defect is not
reversed by ART. This could explain why HIV+ patients have reduced vaccine responses.
DC production of cytokines in response to pneumococcal vaccines predicted post-vaccine
OPA, highlighting the presence of a mechanism that could be exploited to improve vaccine
efficacy and reduce IPD among HIV+ subjects.
Contributors: Mendoza, Daniel; De La Rosa, Indhira; Xu, Yi; Rodriguez-Barradas, Maria;
Lewis, Dorothy; Keitel, Wendy; Chen, Min; Corry, David.
xvi
DAAM2 FUNCTIONS AS A NOVEL CONVERGENCE POINT OF SIGNALING
PATHWAYS IN GLIOBLASTOMA
Wenyi Zhu
Integrative Program in Molecular and Biomedical Sciences
Advisor:
Benjamin Deneen, Ph.D.-Department of Neuroscience
Glioblastoma multiforme (GBM) is one of the most aggressively malignant gliomas in
humans. Genes involved in glia fate development have been linked to GBM tumorigenesis.
Considering tumorigenesis is viewed as a convergence of genetic mutation and
developmental context, we want to understand the mechanisms which govern glia
development contribute to GBM formation. Our lab had identified Daam2 inhibits
oligodendrocyte differentiation through Wnt pathway. The Wnt signaling pathway also has
well defined functions in several cancers except GBM, which accentuates the importance of
our study. Moreover Daam2 was reported also by our lab to function through a direct
interaction with PIP5K during development, which raises the possibility that Daam2 interacts
with genes unrelated to Wnt pathway in GBM.
We first characterized that Daam2 is highly expressed in both human GBM xenograft
and tissue array samples. To further analyze the function of Daam2 in GBM, we took
advantages of both human GBM xenograft cell line model and mouse GBM brain tissue
generated from In Utero Electroporation (IUE) model. The mouse model that uses
PiggyBac(PB) and In Utero Electroporation (IUE) targets astro-glial lineages with
oncogenes, which can efficiently generate GBM in three weeks. We found that Daam2
potentiates tumor proliferation. In detail, in human GBM model, the result from the growth
curve and agar assay indicated that Daam2 over-expression increases proliferation.
Coherent results were observed from mouse IUE model coinjected with oncogene and
Daam2. In these tissues, we observed more tumor growth and proliferation as indicated by
in vivo luciferase imaging and staining of BrdU/pH3. These results were further supported by
the complementary study performing IUE on Daam2 knockout mice. Next, we delineate how
Daam2 is participated in GBM formation/progression. We first verified that Daam2 functions
through Wnt pathway. TOP reporter signal in GBM cell lines was dramatically increased with
the over-expression of Daam2. To fully explore the mechanism, MS and RPPA were applied
on mouse brain tissues. It shows that Daam2 binds to PI3K and also negative correlates
with VHL expression, both of which have also been verified in mouse model.
In sum, we demonstrated that Daam2 plays critical roles in GBM tumorigensis, which
potentiates proliferation and tumor growth. Its roles were found not only in Wnt signaling
pathway, but also involved in PI3K/AKT and VHL, which made Daam2 as a convergence
point of multiple pathways in GBM.Contributors: Zhu, Wenyi; Lee, Hyun-Kyoung; Loturco,
Joseph; Mohila, Carrie; Deneen, Benjamin
xvii
A CHOLINERGIC BASAL FOREBRAIN FEEDING CIRCUIT MODULATES
APPETITE SUPPRESSION
Alexander Michael Herman
Program in Developmental Biology
Advisor:
Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Irregular food intake, either consuming too much or too little, is a primary
cause of obesity and other eating and metabolic disorders. Highly coordinated by
the brain, food intake must be balanced with energy expenditure in order to maintain
proper body weight homeostasis. To date, insight into the neural control of feeding
has largely focused on signaling mechanisms classically associated with the
hypothalamus, the major center in the brain which regulates body weight
homeostasis.
However, the role of non-canonical signaling mechanisms in
regulating feeding behavior has been largely uncharacterized. Acetylcholine has
long been proposed to influence feeding behavior, but this idea has been largely
based on anecdotal evidence due to functional similarity between acetylcholine and
nicotine, the addictive component in tobacco that acts as an appetite suppressant.
Although this connection has not been formally validated, it is noteworthy that
nicotine is an exogenous agonist for acetylcholine receptors, suggesting that
endogenous cholinergic signaling may play a role in normal physiological regulation
of body weight homeostasis. However, it remains unclear if cholinergic neurons in
the brain regulate food intake, and how their circuits function to modulate feeding.
Here, we report that cholinergic neurons of the basal forebrain potently influence
food intake and body weight. While impairment of cholinergic signaling increases
food intake and results in severe obesity, enhanced cholinergic signaling decreases
food consumption. Accordingly, we found that this cholinergic population regulates
appetite-suppressing POMC neurons of the hypothalamus. Together, our data
pinpoint the cholinergic basal forebrain as a major modulatory center underlying
feeding behavior, highlighting a new role of acetylcholine in modulating food intake,
which may provide insight towards the development of effective treatments to
manage eating disorders.
Contributors: Herman, Alexander M.; Ortiz-Guzman, Joshua; Kochukov, Mikhail; Garcia,
Isabella; Quast Katie; Carlson, Jeffrey C.; Selever, Jennifer; Tong, Qingchun; Arenkiel,
Benjamin R.
xviii
GLIAL LIPID DROPLETS AND ROS INDUCED BY MITOCHONDRIAL DEFECTS
PROMOTE NEURODEGENERATION
Lucy Liu
Department of Neuroscience
Advisor:
Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Reactive oxygen species (ROS) and mitochondrial defects are implicated in
many neurodegenerative diseases. My colleagues and I found that a key
consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of
lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK)
and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons, leading
to LD accumulation in glia prior to or at the onset of neurodegeneration. The
accumulated lipids are peroxidated in the presence of ROS. Reducing LD
accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or
lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a
similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal
mitochondrial defects, suggesting that LD accumulation following mitochondrial
dysfunction is an evolutionarily conserved phenomenon, and represents an early,
transient indicator and promoter of neurodegenerative disease. In these mutant
animals, lipid synthesis originates in the neurons and is transferred to the glia where
LDs are formed. Lipid transfer between neuron and glia has not been previously
documented and the mechanism of lipid transport is ill-defined. I found that
monocarboxylate (lactate) transporters are critical for the transport of lipids from the
neuron to the glia irrespective of ROS levels. The pharmacological or genetic
perturbation of monocarboxylate transporters and/or lactate levels reduces cell
death in vitro and ameliorates neurodegeneration in mutant animals with ROS
induced LD accumulation.
Contributors: Liu L, Zhang K, Sandoval H, Yamamoto S, Jaiswal M, Sanz E, Li Z, Hui J,
Graham BH, Quintana A, Bellen HJ
xix
MICROBE-HOST CHEMICAL COMMUNICATION REGULATES HOST
METABOLIC ADAPTATION TO ENVIRONMENTAL VARIATIONS
Chih-chun Janet Lin
Department of Molecular & Human Genetics
Advisor:
Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Gut microbiota exists virtually in all coelomate animals. Besides, microbederived metabolites can directly influence specific cellular pathways in the host.
These microbe-host metabolic axes play pivotal roles in maintaining the
homeostasis of host physiology, and in modulating host susceptibility to diseases.
Although the composition of the gut microbiota is essentially stable throughout
adulthood, microbial metabolism is highly dynamic and flexible in response to
environmental variations. Therefore, environmental factors not only directly exert
their effects on host physiology but also indirectly through tuning microbe-host
metabolic communication. However, the molecular interactions between
environment, microbe and host remain largely unknown. In this project, we showed
that environmental methionine tunes bacterial methyl metabolism, which in turn
modulates mitochondrial dynamics and lipid metabolism in Caenorhabditis
elegans through NR5A nuclear hormone receptor NHR-25. We discovered that
methionine deficiency decreases the production of bacterial metabolites that are
essential for phosphatidylcholine synthesis in C. elegans. Reduction of diundecanoyl
and dilauroyl phosphatidylcholines suppressed the activation of their receptor NHR25, and led to increased lipid accumulation by promoting mitochondrial
fragmentation. Together, our work reveals a molecular mechanism that connects
host-microbe interactions with environmental variations. The components in this
pathway have well-conserved homologous counterparts in human. Therefore, these
results could advance our knowledge on the evolutionarily conserved chemical
dialogues between microbiota and human under environmental variations.
Contributors: Wang, Meng
xx
THE SIMPLEST NATURAL REOVIRUS AND ITS EVOLUTIONARY TRAJECTORY
Jason T Kaelber
Department of Molecular Virology & Microbiology
Advisor:
Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Fako virus is a novel mosquito-specific dsRNA reovirus with 9 genomic segments, the
fewest of any reovirus. While most reoviruses have two or three capsid layers, Fako virus is
single-shelled. The 4.5Å structure of this virus reveals a streamlined capsid organization with
fewer peptides per asymmetric unit than any other reovirus. The innermost shell of FAKV
displays a “pseudo T=2” symmetry with 120 subunits arranged with one dimer per
asymmetric unit. The fold of the core protein is so conserved as to be recognizable in all
reoviruses and two other families. In the turreted reovirus subfamily (Spinareovirinae), a
clamp protein decorates the exterior of the major capsid protein. Unique among the
Spinareovirinae, the Fako virus clamp does not obey “pseudo T=2” symmetry and is present
in only 60 copies per virion. Fako virus’s “clamp” protein has no statistically significant
sequence homology to any other protein, yet structural analysis reveals 10 out of 13
secondary-structural elements are shared with cytoplasmic polyhedrosis virus. Future work
will reveal the mechanism by which this clamp selects only one of two similar binding sites
on the surface of the major capsid protein dimer.
Ancestral trait reconstruction was performed to infer the character states of nodes within
the subfamily’s phylogeny, allowing me to propose a model for the natural history for singleshelled reoviruses. The common ancestor of all turreted reoviruses had a second shell and
120 clamp proteins per virion. Fako virus emerged by serial loss-of-function events. The
second shell became dispensable in a 10-segmented insect-infecting common ancestor of
three genera: Fako virus experienced a deletion of the gene, while in cytoplasmic
polyhedrosis virus, the outer shell gene was exapted to form intracellular occlusions, and in
Rice ragged stunt virus, 70% of the shell quasi-equivalent sites are unoccupied. This
highlights the role of reductive evolution among reoviruses.
Additionally, we determined the atomic structure of human picobirnavirus by
cryoelectron microscopy, revealing similarities in capsid configuration between these two
dsRNA virus families. Despite the lack of sequence homology, underlying structural
similarities between the various dsRNA virus families point to a common origin of the extant
dsRNA viruses.
Contributors: Kaelber, Jason T; Auguste, A Jonathan; Hryc, Corey F; Collier, Aaron M; Tao,
Yizhi J; Weaver, Scott C; Chiu, Wah
xxi
NAME
Cheng, Jinxuan
Haley, Ryan
Kaushik, Akash
Khatiwada, Sanjeev
Mitra, Sayantan
Ren, Zhenning
Stojanoski, Vlatko
Xu, Xiaowei
Xue, Zenghui
Hollier, John
Lindsay, Holly
Premkumar, Muralidhar
Stevens, Alexandra
Alcott, Callison
Birol, Onur
Brown, Rogers
Chaboub, Lesley
Chen, Kuchuan
Choi, Sungwoo
Cullen, Sean
David, Gabriela
De Maio, Antonia
Haelterman, Nele & Tan,
Kai Li
Huang, Yung-Hsin
Jen, Hsin-I
Kho, Jordan
Laug, Dylan
Li, Tongchao
Lu, Hsiang-Chih
Ozseker, Ayse
Scavuzzo, Marissa
PROGRAM
Biochemistry & Molecular
Biology
Biology
Biology
Biology
Biology
Biology
Biology
Biology
Biology
Clinical Scientist Training
Program
Program
Program
Program
Developmental Biology
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Semerci, Fatih
Senturk, Mumine
Tepe, Burak
Tsai, Chang-Ru
Ugur, Berrak
Ung, Kevin
Vue, Zer
Yeh, Szu-Ying
Yen, Shuo-Ting
Zeng, Huan-Chang
Bertolet, Grant
Gu, Bon-Hee
Gwalani, Lavesh
Hsu, Hsiang Ting
Lee, Thomas
Lesteberg, Kelsey
Liang, Dan
Mata, Melinda
Sinha, Papiya
Tung, Hui-Ying
You, Ran
Barrasso, Anthony
Burton, Jason
Chung, Hsiang-Ching
Clark, Justin
Dawson, Emily
Dosey, Timothy
Fujiwara, Kenichiro
Graves, Joshua
Hilton, Tyler
Kim, Ik Sun
Kodali, Srikanth
Lee, Yi-Chien
Litvinchuk, Alexandra
Lo, Hin Ching Flora
Immunology
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Integrative Mol & Biomedical
Sciences
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Lu, Hengyu
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Mak, Keng Hou
McCue, Tyler
Morra, Christina
Muscarella, Aaron
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Tu, Yen-Kuei
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Zou, Winnie
Bader, David
Brinegar, Amy
Call, Steven
Foley, Christopher
Freire, Pablo
Haller, Meade
Hamilton, Mark
Hein, Sarah
Kettner, Nicole
Lewis, Kyle
McNamara, Catherine
Monkkonen, Teresa
O'Neill, Marisol
Pankowicz, Francis
Pew, Braden
Rajasekharan, Vivek
Roberts, Justin
Rodriguez, Amanda
San Martin, Rebeca
Sciences
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Molecular & Cellular Biology
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Saylor, Stephen
Spike, Aaron
Sreekumar, Amulya
Szwarc, Maria
Zhelyazkova, Boryana
Barreto, Brittany
Bondar, Vitaliy
Chen, Chun-An
Haines, Katherine
Han, Seung Yeop
Jones, Evan
Li, Cheng-Lin
Lin, Chih-Chun
Lopez, Angel
Nair, Amritha
Neve, Isaiah
Oak, Ninad
Siehr, Meagan
Wang, Li
Webb, Amanda
Yin, Jiani
Zhai, Yijie
Zhang, Xiaotian
Abo-Zahrah, Reem
August, Brandon
Cao, Zhijuan
Huq, Redwan
Jordan, Valerie
Leach, John
Li, Lele
Loehr, James
Monroe, Tanner
Piazza, Victor
Quick, Ann
Tajhya, Rajeev
Yosef, Nejla
Barker, Anna
Hall, Anne
Hornstein, Benjamin
Minor, Marissa
Misra, Anisha
Molecular & Human Genetics
Molecular Physiology
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Microbiology
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Tsai, Wei-Chih
Chen, Chien-Ju
Crommett, Lexi
Huang, Yu-Mei
Johnson, Jennifer
Lakshminarasimhan,
Janakiraman
Lien, Steven
Meng, Xiangling
Patterson, Jaclyn
Shen, Shan
Stay, Trace
Sun, Jenny
van der Heijden, Meike
White, Joshua
Wu, Chun-Ting
Brown, Cameron
Haines, Emily
Jiang, Xiqian
Mehta, Shrenik
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Campbell, James
Koire, Amanda
Laitman, Andrew
Onuchic, Vitor
Raman, Ayush
Taylor, Aaron
Wang, Mengyu
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Microbiology
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Fofanova, Tatiana
Gastelum, Grady
Ghosh-Choudhury,
Triparna
Griffin, Deric
Hurwitz, Amy
Kruse, Robert
Ku, Amy
Lee, Yu-Ju
Lewis, Phoebe
Lim, Phaik Har Karen
Martini-Stoica, Heidi
Nguyen, Tuan
Patel, Maha
Roy, Ethan
Stone , Adrianne
Sukumaran, Sujita
Tanner, Mark
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Williams, Laterrica
Yong, Lin-Kin
Medicine
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2014 SYMPOSIUM AWARD WINNERS
BECKMAN 1st PLACE PLATFORM SPEAKER WINNER:
Andrew Folick, Developmental Biology
ALUMNI 2ND AND 3RD PLACE PLATFORM SPEAKER AWARDS:
Redwan Huq, Molecular Physiology (2nd place)
Corey Hryc, Structural & Computational Biology & Molecular Biophysics (3rd place)
BECKMAN POSTER WINNER:
Amy Ku, Translational Biology & Molecular Medicine
POSTER PRESENTATION WINNERS:
1st Place
Viktor Feketa – Molecular Physiology
Amy Ku – Translational Biology & Molecular Medicine
John Leach– Molecular Physiology
Meagan Pitcher– Translational Biology & Molecular Medicine
2nd Place
Meghan Buckley – Integrative Molecular & Biomedical Sciences
Charlene Emerson – Molecular & Human Genetics
Jennifer Johnson – Neuroscience
3rd Place
Alexander Herman – Developmental Biology
Jana Knezevic– Molecular & Cellular Biology
Phoebe Lewis – Translational Biology & Molecular Medicine
Keng Hou Mak – Integrative Molecular & Biomedical Sciences
Austen Terwilliger – Integrative Molecular & Biomedical Sciences
Ran You – Immunology
Honorable Mention
Lisa Atkins – Molecular Virology & Microbiology
Zachary Conley – Biochemistry and Molecular Biology
Aaron Kelly – Struct & Computational Biology & Molecular Biophysics
Xiangling Meng – Neuroscience
Ayse Ozseker – Developmental Biology
Yen-Kuei Tu – Integrative Molecular & Biomedical Sciences
Laterrica Williams – Translational Biology & Molecular Medicine
Liuliu Zheng – Biochemistry & Molecular Biology
xxviii
2014 AWARD WINNERS
MARC DRESDEN EXCELLENCE IN GRADUATE EDUCATION AWARD:
Benjamin Arenkiel, Ph.D., Molecular & Human Genetics
DEBORAH K. MARTIN ACHIEVEMENT AWARD IN BIOMEDICAL SCIENCES:
Ian Campbell, Molecular & Human Genetics
PROFESSOR JOHN J. TRENTIN SCHOALRSHIP AWARDS:
Matthew Hill -- Developmental Biology
Tuan Nguyen -- Translational Biology & Molecular Medicine
Yoon Joo Rah -- Neuroscience
Derrick Chu -- Translational Biology & Molecular Medicine
8-STRANDED BETA-BARREL JELLY ROLL AWARDS:
BEST OVERALL COURSE
Neuroscience
BEST TEACHING
Dr. Thomas Cooper, Pathology
Dr. Hamed Jafar-Nejad, Molecular & Human Genetics
Dr. Jon Levitt, Immunology
BEST TEACHING ASSISTANT
Kathleen Seger Manning, Integrative Molecular & Biomedical Sciences
xxix
Table of Contents
Abo-Zahrah, Reem Sharon ................................................................................. 1
Advisor: George Rodney, Ph.D. - Department of Molecular Physiology & Biophysics
Acevedo-Rodriguez, Alexandra ......................................................................... 2
Advisor: Shailaja Mani, Ph.D. - Department of Molecular & Cellular Biology
Adams, Joshua Matthew ..................................................................................... 3
Advisor: Benjamin Deneen, Ph.D. - Department of Neuroscience
Adams, Nyssa .................................................................................................... 4
Advisor: Francesco Demayo, Ph.D. - Department of Molecular & Cellular Biology
Chandrasekhar Yallampalli, Ph.D.-Department of Obstetrics & Gynecology
Alcott, Callison Edward ...................................................................................... 5
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Alexander, Stefanie Louise ................................................................................. 6
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Amin, Samirkumar B .......................................................................................... 7
Advisor: Roeland Verhaak, Ph.D. - Bioinformatics & Computational Biology
Amin, Viren R ..................................................................................................... 8
Advisor: Aleksandar Milosavljevic, Ph.D. - Department of Molecular & Human Genetics
Anderson, Andrew Peter Porten ......................................................................... 9
Advisor: Y. Yin, D.Phil - Chemistry & Biochemistry
Araya, Mussie .................................................................................................. 10
Advisor: William Brownell, Ph.D. - Department of Otolaryngology-Head and Neck Surgery
Arnold, James Michael ..................................................................................... 11
Advisor: Arun Sreekumar, Ph.D. - Department of Molecular & Cellular Biology
Atkins, Lisa Michelle ........................................................................................ 12
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Atri, Benu ......................................................................................................... 13
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
August, Brandon Christopher ........................................................................... 14
Advisor: Ergun Sahin, M.D. - Department of Molecular Physiology & Biophysics
Aviles-Padilla, Kevin ....................................................................................... 15
xxx
Advisor: Silke Paust, Ph.D. - Department of Pediatrics
Bader, David A ................................................................................................. 16
Advisor: Sean Mcguire, M.D./Ph.D. - Department of Molecular & Cellular Biology
Bajaj, Lakshya .................................................................................................. 17
Advisor: Marco Sardiello, Ph.D. - Department of Molecular & Human Genetics
Barker, Anna Kathryn ....................................................................................... 18
Advisor: David Bates, Ph.D. - Department of Molecular & Human Genetics
Barrasso, Anthony Patrick ................................................................................ 19
Advisor: Ross Poche, Ph.D. - Department of Molecular Physiology & Biophysics
Barreto, Brittany Marie ..................................................................................... 20
Advisor: Susan Rosenberg, Ph.D. - Department of Molecular & Human Genetics
Barry, Meagan Amelia ...................................................................................... 21
Advisor: Peter Hotez, M.D./Ph.D. - Department of Pediatrics
Laila Woc-Colburn, M.D.-Department of Medicine
Bartanus, Justin Ray.......................................................................................... 22
Advisor: Fuli Yu, Ph.D. - Department of Molecular & Human Genetics
Benton, Susan Michelle .................................................................................... 23
Advisor: Jason Heaney, Ph.D. - Department of Molecular & Human Genetics
Bertolet, Grant Daniel ....................................................................................... 24
Advisor: Dongfang Liu, M.D./Ph.D. - Department of Pediatrics
Biggerstaff, Kristin Schmid .............................................................................. 25
Advisor: Donna White, Ph.D./M.P.H. - Department of Medicine
Jennifer Kramer, Ph.D./M.P.H.-Department of Medicine
Bimler, Lynn .................................................................................................... 26
Birol, Onur ....................................................................................................... 27
Advisor: Andrew Groves, Ph.D. - Department of Neuroscience
Bodine, Truston J .............................................................................................. 28
Advisor: E. Zechiedrich, Ph.D. - Department of Molecular Virology & Microbiology
Richard Hamill, M.D.-Department of Medicine
Bondar, Vitaliy V .............................................................................................. 29
xxxi
Brinegar, Amy Elizabeth .................................................................................. 30
Advisor: Thomas Cooper, M.D. - Department of Pathology & Immunology
Brown, Cameron August .................................................................................. 31
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Brown, Rogers, II Milton .................................................................................. 32
Burton, Jason Christopher................................................................................. 33
Advisor: Irina Larina, Ph.D. - Department of Molecular Physiology & Biophysics
Byrd, Tiara T ..................................................................................................... 34
Advisor: Nabil Ahmed, M.D. - Department of Pediatrics
Robert Grossman, M.D.-Department of Neurosurgery
Call, Lee Thomas .............................................................................................. 35
Advisor: Douglas Burrin, Ph.D. - Department of Pediatrics
Robert Britton, Ph.D.-Department of Molecular Virology & Microbiology
Call, Steven Gregory......................................................................................... 36
Advisor: Orla Conneely, Ph.D. - Department of Molecular & Cellular Biology
Campbell, James Christopher ........................................................................... 37
Advisor: Choel Kim, Ph.D. - Department of Pharmacology
Cao, Felicia ...................................................................................................... 38
Advisor: Stephen Gottschalk, M.D. - Department of Pediatrics
Lisa Wang, M.D.-Department of Pediatrics
Cao, Zhijuan ..................................................................................................... 39
Program in Cardiovascular Sciences
Advisor: Sean Marrelli, Ph.D. - Department of Anesthesiology
Carlson, Jeffrey Clifford ................................................................................... 40
Carpenter, Jennifer Leigh.................................................................................. 41
Advisor: Mary Brandt, M.D. - Department of Surgery
Carrero, Ivenise ................................................................................................ 42
Carter, Angela .................................................................................................. 43
Advisor: Anne Anderson, M.D. - Department of Pediatrics
Chaboub, Lesley S. ........................................................................................... 44
xxxii
Cham, Henry Xin .............................................................................................. 45
Advisor: J. Dickman, Ph.D. - Department of Neuroscience
Chang-Graham, Alexandra L. ........................................................................... 46
Advisor: Joseph Hyser, Ph.D. - Department of Molecular Virology & Microbiology
Chapple, Richard H ........................................................................................... 47
Advisor: Daisuke Nakada, B.A.Sc. - Department of Molecular & Human Genetics
Chen, Bo .......................................................................................................... 48
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Chen, Chien-Ju ................................................................................................. 49
Advisor: Mauro Costa-Mattioli, Ph.D. - Department of Neuroscience
Chen, Chun-an .................................................................................................. 50
Advisor: Christian Schaaf, M.D./Ph.D. - Department of Molecular & Human Genetics
Chen, Kuchuan ................................................................................................. 51
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Chen, Min-Shan ............................................................................................... 52
Advisor: Noah Shroyer, Ph.D. - Department of Pediatrics
Chen, Muyuan .................................................................................................. 53
Advisor: Steven Ludtke, Ph.D. - Department of Biochemistry & Molecular Biology
Cheng, Jinxuan ................................................................................................. 54
Advisor: Kimberley Tolias, Ph.D. - Department of Neuroscience
Chiang, Angie Chi An ...................................................................................... 55
Advisor: Joanna Jankowsky, Ph.D. - Department of Neuroscience
Choi, Sungwoo ................................................................................................. 56
Advisor: David Moore, Ph.D. - Department of Molecular & Cellular Biology
Chung, Hsiang-Ching ...................................................................................... 57
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Ciupek, Andrew Mark Robinson ...................................................................... 58
Advisor: Suzanne Fuqua, Ph.D. - Department of Medicine
Mothaffar Rimawi, M.D.-Department of Medicine
Clark, Justin Ryan ............................................................................................. 59
Advisor: Anthony Maresso, Ph.D. - Department of Molecular Virology & Microbiology
xxxiii
Collinson-Pautz, Matthew R ............................................................................. 60
Advisor: David Spencer, Ph.D. - Department of Pathology & Immunology
Kevin Slawin, M.D.-Department of Urology
Conley, Zachary Christopher ............................................................................ 61
Courtland, Michael .......................................................................................... 62
Advisor: Qinghua Wang, Ph.D. - Department of Biochemistry & Molecular Biology
Crommett, Lexi E.............................................................................................. 63
Advisor: Jeffrey Yau, Ph.D. - Department of Neuroscience
Cullen, Sean Michael ........................................................................................ 64
Advisor: Margaret Goodell, Ph.D. - Department of Pediatrics
Dai, Hang ......................................................................................................... 65
Advisor: Suzanne Leal, Ph.D. - Department of Molecular & Human Genetics
David-Morrison, Gabriela Riva ........................................................................ 66
Davis, Shaun Michael ....................................................................................... 67
Advisor: Herman Dierick, M.D. - Department of Molecular & Human Genetics
Dawson, Emily Packard .................................................................................... 68
Advisor: Jason Heaney, Ph.D. - Department of Molecular & Human Genetics
De Maio, Antonia ............................................................................................. 69
Denfield, George Hilton.................................................................................... 70
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Derrick, Chu Michael........................................................................................ 71
Advisor: Kjersti Aagaard, M.D./Ph.D. - Department of Obstetrics & Gynecology
Stephen Welty, M.D.-Department of Pediatrics
Dharmat, Rachayata ......................................................................................... 72
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
DiNardo, Andrew R .......................................................................................... 73
Advisor: Anna Mandalakas, M.D. - Department of Pediatrics
Dogruluk, Turgut ............................................................................................. 74
Advisor: Kenneth Scott, Ph.D. - Department of Molecular & Human Genetics
xxxiv
Dominguez Vidana, Rocio ............................................................................... 75
Dosey, Timothy L ............................................................................................. 76
Advisor: Theodore Wensel, Ph.D. - Department of Biochemistry & Molecular Biology
Egunsola, Adetutu ............................................................................................ 77
Emerson, Charlene Hannah .............................................................................. 78
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Fachini, Joseph Mario ....................................................................................... 79
Advisor: Joel Neilson, Ph.D. - Department of Molecular Physiology & Biophysics
Farinholt, Timothy ........................................................................................... 80
Advisor: Adam Kuspa, Ph.D. - Department of Biochemistry & Molecular Biology
Feng, Xiang ...................................................................................................... 81
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Fleet, Tiffany Chantal ....................................................................................... 82
Advisor: Bert O'Malley, M.D. - Department of Molecular & Cellular Biology
Clifford Dacso, M.D./M.P.H.-Department of Molecular & Cellular Biology
Fofanova, Tatiana Y.......................................................................................... 83
Richard Kellermayer, M.D./Ph.D.-Department of Pediatrics
Foley, Christopher James .................................................................................. 84
Advisor: Nicholas Mitsiades, M.D./Ph.D. - Department of Medicine
Foster, Jennifer Haunani ................................................................................... 85
Advisor: Terzah Horton, M.D./Ph.D. - Department of Pediatrics
Fountain, Michael David .................................................................................. 86
Daryl Scott, M.D./Ph.D.-Department of Molecular & Human Genetics
Fousek, Kristen Nicole...................................................................................... 87
Advisor: Nabil Ahmed, M.D. - Department of Pediatrics
Helen Heslop, M.D.-Department of Pediatrics
Freire, Pablo Riera ............................................................................................ 88
Fuja, Daniel G ................................................................................................... 89
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Advisor: Jason Yustein, M.D./Ph.D. - Department of Pediatrics
Fujiwara, Kenichiro ......................................................................................... 90
Advisor: Lawrence Donehower, Ph.D. - Department of Molecular Virology & Microbiology
Fullerton, Jr., Paul Thomas ............................................................................... 91
Advisor: Martin Matzuk, M.D./Ph.D. - Department of Pathology & Immunology
Fultz, Robert Steven.......................................................................................... 92
Advisor: James Versalovic, M.D./Ph.D. - Department of Pathology & Immunology
Gahremanpour, Amir Ali .................................................................................. 93
Advisor: Emerson Perin, M.D. - Department of Medicine
Gala, Upasana .................................................................................................. 94
Galaz-Montoya, Monica Laura ......................................................................... 95
Gallion, Jonathan ............................................................................................. 96
Garcia, Courtney Dawn .................................................................................... 97
Advisor: Dora Angelaki, Ph.D. - Department of Neuroscience
Gastelum, Grady Manuel .................................................................................. 98
Advisor: Yi Li, Ph.D. - Department of Molecular & Cellular Biology
Polly Niravath, M.D.-Department of Medicine
Gates, Leah Ashley ........................................................................................... 99
Ghosh-Choudhury, Triparna .......................................................................... 100
Advisor: Matthew Anderson, M.D./Ph.D. - Department of Obstetrics & Gynecology
Creighton Edwards, M.D.-Department of Obstetrics & Gynecology
Gillentine, Madelyn Arlene ............................................................................ 101
Girard, Mary Elizabeth ................................................................................... 102
Advisor: Christophe Herman, Ph.D. - Department of Molecular & Human Genetics
Godoy, Guilherme .......................................................................................... 103
Advisor: Seth Lerner, M.D. - Department of Urology
Gokul, Nisha Thara ......................................................................................... 104
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Advisor: Stephanie Pangas, Ph.D. - Department of Pathology & Immunology
Graves, Joshua Daniel ..................................................................................... 105
Advisor: Weei-Chin Lin, M.D./Ph.D. - Department of Medicine
Griffin, Deric Maurice .................................................................................... 106
Advisor: Robia Pautler, Ph.D. - Department of Molecular Physiology & Biophysics
Rajesh Krishnamurthy, M.B.,B.S.-Department of Radiology
Grzeskowiak, Caitlin L. .................................................................................. 107
Gu, Bon-Hee .................................................................................................. 108
Advisor: Farrah Kheradmand, M.D. - Department of Medicine
Gu, Franklin ................................................................................................... 109
Gundry, Michael ............................................................................................ 110
Gunesch, Justin Tyme ..................................................................................... 111
Advisor: Jordan Orange, M.D. - Department of Pediatrics
Gwalani, Lavesh Amar ................................................................................... 112
Haelterman, Nele A ........................................................................................ 113
Haines, Emily Grace ....................................................................................... 114
Haines, Katherine ........................................................................................... 115
Haley, Ryan Christopher ................................................................................. 116
Advisor: Zheng Zhou, Ph.D. - Department of Biochemistry & Molecular Biology
Hall, Anne Elizabeth ....................................................................................... 117
Haller, Meade Elspeth ..................................................................................... 118
Advisor: Dolores Lamb, Ph.D. - Department of Urology
Hamilton, Mark Patrick .................................................................................. 119
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Advisor: Sean Mcguire, M.D./Ph.D. - Department of Molecular & Cellular Biology
Han, Seung Yeop ........................................................................................... 120
Advisor: Hamed Jafar-Nejad, - Molecular Medicine- UT Houston
Harrigal, Lindsay Jeanette .............................................................................. 121
Advisor: Janet Butel, Ph.D. - Department of Molecular Virology & Microbiology
Hatcher, Asante .............................................................................................. 122
Advisor: Jeffrey Noebels, M.D./Ph.D. - Department of Neurology
Hause, Anne M ............................................................................................... 123
Advisor: Pedro Piedra, M.D. - Department of Molecular Virology & Microbiology
Robert Atmar, M.D.-Department of Medicine
He, Zongxiao .................................................................................................. 124
Hecksel, Corey W ........................................................................................... 125
Hein, Sarah ..................................................................................................... 126
Herman, Alexander Michael ........................................................................... 127
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Hill, Matthew Craig ........................................................................................ 128
Advisor: James Martin, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
Hilton, Tyler John ........................................................................................... 129
Hinton, Jr., Antentor Othrell ........................................................................... 130
Advisor: Yong Xu, Ph.D. - Department of Pediatrics
Holdman, Xue Bin .......................................................................................... 131
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Hollier, John M ............................................................................................... 132
Advisor: Hashem El-Serag, M.D./M.P.H. - Department of Medicine
Holt, Matthew Valle........................................................................................ 133
Advisor: Jun Qin, Ph.D. - Department of Biochemistry & Molecular Biology
Christopher Peters, Ph.D.-Department of Biochemistry & Molecular Biology
Hooker, Stanley Eugene.................................................................................. 134
xxxviii
Hornstein, Benjamin Daniel............................................................................ 135
Horstman, Molly Jane ..................................................................................... 136
Advisor: Barbara Trautner, M.D./Ph.D. - Department of Medicine
Hryc, Corey F.................................................................................................. 137
Hsieh, Yi-Chen .............................................................................................. 138
Advisor: Joshua Shulman, M.D./Ph.D. - Department of Neurology
Hsu, Hsiang-Ting ........................................................................................... 139
Hsu, Teng-Kuei .............................................................................................. 140
Huang, Yu-Mei .............................................................................................. 141
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Huang, Yung-Hsin ......................................................................................... 142
Huq, Redwan .................................................................................................. 143
Advisor: Christine Beeton, Ph.D. - Department of Molecular Physiology & Biophysics
Hurwitz, Amy Marie ....................................................................................... 144
Ihekweazu, Faith D ......................................................................................... 145
Advisor: Richard Kellermayer, M.D./Ph.D. - Department of Pediatrics
James, Regis Aaron ......................................................................................... 146
Advisor: Chad Shaw, Ph.D. - Department of Molecular & Human Genetics
Jarrett, Kelsey Elizabeth ................................................................................. 147
Advisor: William Lagor, Ph.D. - Department of Molecular Physiology & Biophysics
Jen, Hsin-I ...................................................................................................... 148
Jiang, Xiqian .................................................................................................. 149
Advisor: Jin Wang, Ph.D. - Department of Pharmacology
xxxix
Jiang, Yajian .................................................................................................. 150
Johnson, Jennifer Leigh .................................................................................. 151
Johnston, Alyssa N.......................................................................................... 152
Jones, Evan Michael ....................................................................................... 153
Jordan, Valerie K. ........................................................................................... 154
Advisor: Daryl Scott, M.D./Ph.D. - Department of Molecular & Human Genetics
Kaelber, Jason T.............................................................................................. 155
Kaushik, Akash Kumar ................................................................................... 156
Kee, Sara Elizabeth ......................................................................................... 157
Kelly, Aaron Josef .......................................................................................... 158
Advisor: Tsz-Kwong Man, Ph.D. - Department of Pediatrics
Kettner, Nicole Michelle ................................................................................. 159
Advisor: Loning Fu, Ph.D. - Department of Pediatrics
Khatiwada, Sanjeev ........................................................................................ 160
Kho, Jordan .................................................................................................... 161
Kim, Cynthia J ................................................................................................ 162
Kim, Ik Sun ................................................................................................... 163
Advisor: Xiang Zhang, Ph.D. - Department of Molecular & Cellular Biology
Kodali, Srikanth ............................................................................................. 164
Advisor: Jin Wang, Ph.D./D.V.M. - Department of Pathology & Immunology
Koire, Amanda Michele .................................................................................. 165
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Kruse, Robert Layne ....................................................................................... 166
Advisor: Karl-Dimiter Bissig, M.D./Ph.D. - Department of Molecular & Cellular Biology
Fasiha Kanwal, M.D.-Department of Medicine
Ku, Amy Tsu ................................................................................................... 167
Advisor: Hoang Nguyen, Ph.D. - Department of Molecular & Cellular Biology
Abdul Diwan, Ph.D.-Department of Pathology & Immunology
Kurbanov, Suhrab .......................................................................................... 168
Advisor: Leonid Metelitsa, M.D./Ph.D. - Department of Pediatrics
Kyle, Stephanie Marie .................................................................................... 169
Advisor: Monica Justice, Ph.D. - Department of Molecular & Human Genetics
David Nelson, Ph.D.-Department of Molecular & Human Genetics
Laitman, Andrew Marc ................................................................................... 170
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D. - Department of Pediatrics
Lakshminarasimhan, Janakiraman Kaushik ................................................... 171
Landers, Cameron Taylor ............................................................................... 172
Advisor: David Corry, M.D. - Department of Medicine
Adrian Casillas, M.D.-Department of Medicine
Laug, Dylan James .......................................................................................... 173
Le, Henry H .................................................................................................... 174
Advisor: Mary Dickinson, Ph.D. - Department of Molecular Physiology & Biophysics
Leach, John Preston ........................................................................................ 175
Lee, Thomas ................................................................................................... 176
Advisor: Matthew Bettini, Ph.D. - Department of Pediatrics
Lee, Yi-Chien ................................................................................................. 177
Lee, Yu-Ju ...................................................................................................... 178
Advisor: Weei-Chin Lin, M.D./Ph.D. - Department of Medicine
Lemon, Laramie Denise .................................................................................. 179
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Lesteberg, Kelsey Elizabeth............................................................................ 180
Advisor: George Makedonas, Ph.D. - Department of Pediatrics
Levine, Amber T ............................................................................................. 181
Lewis, Andrew H ............................................................................................ 182
Advisor: H Lacorazza, Ph.D. - Department of Pathology & Immunology
Lewis, Kyle Joseph Edward ............................................................................ 183
Advisor: Nikolaj Timchenko, Ph.D. - Department of Pathology & Immunology
Richard Sifers, Ph.D.-Department of Pathology & Immunology
Lewis, Phoebe Elnora ..................................................................................... 184
Advisor: Qizhi Yao, M.D./Ph.D. - Department of Surgery
Shital Patel, M.D.-Department of Medicine
Li, Cheng-Lin ................................................................................................. 185
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Li, Jiani .......................................................................................................... 186
Advisor: Fuli Yu, Ph.D. - Department of Molecular & Human Genetics
Li, Lele ........................................................................................................... 187
Li, Tongchao .................................................................................................. 188
Andrew Groves, Ph.D.-Department of Neuroscience
Liang, Dan ...................................................................................................... 189
Advisor: William Decker, Ph.D. - Department of Pathology & Immunology
Lien, Steven Donald........................................................................................ 190
Advisor: J. Dickman, Ph.D. - Department of Neuroscience
Lim, Karen Phaik Har ................................................................................... 191
Advisor: Carl Allen, M.D./Ph.D. - Department of Pediatrics
Donald Parsons, M.D./Ph.D.-Department of Pediatrics
Lin, Angelique ............................................................................................... 192
Lin, Chih-chun Janet ....................................................................................... 193
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
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Lindsay, Holly Brianne ................................................................................... 194
Advisor: Xiaonan Li, M.D./Ph.D. - Department of Pediatrics
Litvinchuk, Alexandra ................................................................................... 195
Advisor: Hui Zheng, Ph.D. - Department of Molecular & Human Genetics
Liu, Lucy ........................................................................................................ 196
Lo, Hin Ching ................................................................................................ 197
Lo, Yuan-Hung .............................................................................................. 198
Advisor: Noah Shroyer, Ph.D. - Department of Pediatrics
Loehr, James Anthony .................................................................................... 199
Advisor: George Rodney, Ph.D. - Department of Molecular Physiology & Biophysics
Lopez, Angel Y ............................................................................................... 200
Advisor: Edward Cooper, M.D./Ph.D. - Department of Neurology
Lu, Hengyu .................................................................................................... 201
Lu, Hsiang-Chih ............................................................................................. 202
Lu, Yang (Daisy) ........................................................................................... 203
Luk, Berkley .................................................................................................. 204
Ma, Boxue ...................................................................................................... 205
Madan, Simran Arun ....................................................................................... 206
Madison, Matthew Cameron........................................................................... 207
Anita Sabichi, M.D.-Department of Medicine
Mahapatra, Sanjana ........................................................................................ 208
xliii
Mak, Keng Hou .............................................................................................. 209
Advisor: Jue Wang, Ph.D. - Department of Molecular & Human Genetics
Manning, Kathleen Seger................................................................................ 210
Advisor: Thomas Cooper, M.D. - Department of Pathology & Immunology
Mao, Dongxue ................................................................................................ 211
Marin, Miguel Alejandro ................................................................................ 212
Martinez, Jarrod Don ...................................................................................... 213
Advisor: Jianming Xu, Ph.D. - Department of Molecular & Cellular Biology
Martini-Stoica, Heidi ..................................................................................... 214
Joshua Shulman, M.D./Ph.D.-Department of Neurology
Mata, Melinda ................................................................................................ 215
Matinyan, Nick .............................................................................................. 216
Advisor: Koen Venken, Ph.D. - Department of Biochemistry & Molecular Biology
Mayle, Ryan ................................................................................................... 217
Advisor: Grzegorz Ira, Ph.D. - Department of Molecular & Human Genetics
Maynard, Janielle P ......................................................................................... 218
Advisor: Sundararajah Thevananther, Ph.D. - Department of Pediatrics
John Goss, M.D.-Department of Surgery
McCue, Tyler Jordan ...................................................................................... 219
McNamara, Catherine Teresa ......................................................................... 220
Mehta, Shrenik Chetan.................................................................................... 221
Mendoza, Daniel ............................................................................................ 222
Meng, Xiangling ............................................................................................ 223
xliv
Minnick, Phillip Jay ........................................................................................ 224
Minor, Marissa Mie-Kehaulani ....................................................................... 225
Advisor: Betty Slagle, Ph.D. - Department of Molecular Virology & Microbiology
Misra, Anisha ................................................................................................. 226
Advisor: Jason Kimata, Ph.D. - Department of Molecular Virology & Microbiology
Mitchell, Patrick Gerald .................................................................................. 227
Mitra, Sayantan .............................................................................................. 228
Advisor: B Prasad, Ph.D. - Department of Biochemistry & Molecular Biology
Monkkonen, Teresa ........................................................................................ 229
Advisor: Michael Lewis, Ph.D. - Department of Molecular & Cellular Biology
Monroe, Tanner Oliver ................................................................................... 230
George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Morra, Christina Narie .................................................................................... 231
Murry, Jaclyn Bravo ....................................................................................... 232
Advisor: Ignatia Van Den Veyver, M.D. - Department of Obstetrics & Gynecology
Muscarella, Aaron Matthew............................................................................ 233
Mysore, Krupa Ramaprasad............................................................................ 234
Advisor: Robert Shulman, M.D. - Department of Pediatrics
Xian Li, M.D./Ph.D.-
Nair, Amritha ................................................................................................. 235
Namwanje, Maria ........................................................................................... 236
Advisor: Chester Brown, M.D./Ph.D. - Department of Molecular & Human Genetics
Nash, Andrea Kathleen ................................................................................... 237
Neill, Nicholas J .............................................................................................. 238
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Nelson, James Willard .................................................................................... 239
Advisor: Robert Bryan, Ph.D. - Department of Anesthesiology
Neve, Isaiah Adam Alexander ........................................................................ 240
Nguyen, Chinh Thi Quynh.............................................................................. 241
Nguyen, Lena ................................................................................................. 242
Nguyen, Tuan Minh ........................................................................................ 243
Novikov, Ilya B ............................................................................................... 244
O’Neil, Derek Steven ...................................................................................... 245
William Gibbons, M.D.-Department of Obstetrics & Gynecology
O’Neill, Marisol Ann ...................................................................................... 246
Oak, Ninad R .................................................................................................. 247
Advisor: Sharon Plon, M.D./Ph.D. - Department of Pediatrics
Oakes, Joshua ................................................................................................. 248
Advisor: Susan Hamilton, Ph.D. - Department of Molecular Physiology & Biophysics
Olarte, Liset C ................................................................................................. 249
Advisor: Sheldon Kaplan, M.D. - Department of Pediatrics
Onuchic, Vitor ................................................................................................ 250
Ortiz, Joshua .................................................................................................. 251
Ozseker, Ayse Sena ....................................................................................... 252
Paine, Ingrid Sophie ........................................................................................ 253
Advisor: Michael Lewis, Ph.D. - Department of Molecular & Cellular Biology
xlvi
Pandit, Lavannya M. ....................................................................................... 254
Advisor: Robert Bryan, Ph.D. - Department of Anesthesiology
Pankowicz, Francis Peter ................................................................................ 255
Advisor: Karl-Dimiter Bissig, M.D./Ph.D. - Department of Molecular & Cellular Biology
Park, Ji Young ................................................................................................ 256
Advisor: Stelios Smirnakis, M.D./Ph.D. - Department of Neurology
Parulekar, Amit Dilip ...................................................................................... 257
Advisor: Nicola Hanania, M.B.,B.S./M.S. - Department of Medicine
Patel, Maha Praful ........................................................................................... 258
Patterson, Jaclyn Sky ...................................................................................... 259
Pejerrey, Sasha M ........................................................................................... 260
Peterson, Kassandra Leigh .............................................................................. 261
Advisor: Rebecca Rico, M.P.H. - Department of Molecular Virology & Microbiology
Pew, Braden Kyle ........................................................................................... 262
Dennis Bier, M.D.-Department of Pediatrics
Piazza, Victor George ..................................................................................... 263
Pohodich, Amy E ............................................................................................ 264
Pradhan, Geetali ............................................................................................. 265
Advisor: Yuxiang Sun, M.D./Ph.D. - Department of Pediatrics
Susan Samson, M.D.-Department of Medicine
Premkumar, Muralidhar H .............................................................................. 266
Pribis, John P .................................................................................................. 267
Qin, Liying ..................................................................................................... 268
xlvii
Advisor: Choel Kim, Ph.D. - Department of Pharmacology
Quick, Ann Pepper .......................................................................................... 269
Advisor: Xander Wehrens, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
Quiros, Joel Patrick ......................................................................................... 270
Rajasekharan, Vivek ...................................................................................... 271
Advisor: Frederick Pereira, Ph.D. - Department of Molecular & Cellular Biology
Ramachandran, Prasanna V ............................................................................ 272
Raman, Ayush ................................................................................................ 273
Advisor: Zhandong Liu, Ph.D. - Department of Pediatrics
Rau, Rachel .................................................................................................... 274
Regenbogen, Sam Julian ................................................................................. 275
Ren, Zhenning ................................................................................................ 276
Advisor: Ming Zhou, Ph.D. - Department of Biochemistry & Molecular Biology
Renwick, Alexander ....................................................................................... 277
Reyes, Natalie Michelle .................................................................................. 278
Roberts, Justin Michael ................................................................................... 279
Advisor: Nancy Weigel, Ph.D. - Department of Molecular & Cellular Biology
Rodriguez, Amanda ....................................................................................... 280
Advisor: Stephanie Pangas, Ph.D. - Department of Pathology & Immunology
Rodriguez, Perla Janet ................................................................................... 281
Advisor: Henry Pownall, Ph.D. - Department of Medicine
Roy, Ethan ...................................................................................................... 282
Claudia Robertson, M.D.-Department of Neurosurgery
Rubin, Michelle Jenny .................................................................................... 283
xlviii
Sabharwal, Jasdeep Singh ............................................................................... 284
Advisor: Samuel Wu, Ph.D. - Department of Ophthalmology
Salvo, Jason Scott ........................................................................................... 285
San Martin, Rebeca ........................................................................................ 286
Advisor: David Rowley, Ph.D. - Department of Molecular & Cellular Biology
Satterfield, Laura ............................................................................................ 287
Advisor: Jason Yustein, M.D./Ph.D. - Department of Pediatrics
Saylor, Stephen Michael ................................................................................. 288
Scavuzzo, Marissa Ann................................................................................... 289
Advisor: Malgorzata Borowiak, Ph.D. - Department of Molecular & Cellular Biology
Scott, Jessica Diane......................................................................................... 290
Seilheimer, Robert Lionel ............................................................................... 291
Advisor: Samuel Wu, Ph.D. - Department of Ophthalmology
Semerci, Fatih ................................................................................................ 292
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D. - Department of Pediatrics
Senturk, Mumine ............................................................................................ 293
Seth, Abhishek ............................................................................................... 294
Seymour, Michelle L. ..................................................................................... 295
Advisor: Frederick Pereira, Ph.D. - Department of Molecular & Cellular Biology
Shah, Rajesh Rasik.......................................................................................... 296
Shaw, Perris S. ................................................................................................ 297
Advisor: Richard Hurwitz, M.D. - Department of Pediatrics
Patricia Chevez-Barrios, M.D.-Department of Ophthalmology
Shelly, Katharine Elizabeth ............................................................................ 298
Advisor: David Nelson, Ph.D. - Department of Molecular & Human Genetics
Shen, Shan ...................................................................................................... 299
xlix
Shen, Ye ......................................................................................................... 300
Advisor: H Lacorazza, Ph.D. - Department of Pathology & Immunology
Sheng, Kuanwei ............................................................................................. 301
Advisor: Chenghang Zong, Ph.D. - Department of Molecular & Human Genetics
Shivanna, Binoy ............................................................................................. 302
Advisor: Bhagavatula Moorthy, Ph.D. - Department of Pediatrics
Shum, Thomas ............................................................................................... 303
Advisor: Cliona Rooney, Ph.D. - Department of Pediatrics
Stephen Gottschalk, M.D.-Department of Pediatrics
Siehr, Meagan ................................................................................................ 304
Advisor: Jeffrey Noebels, M.D./Ph.D. - Department of Neurology
Simon, Lukas Mikolaj ..................................................................................... 305
Sinha, Papiya .................................................................................................. 306
Sivaramakrishnan, Priya ................................................................................ 307
Advisor: Christophe Herman, Ph.D. - Department of Molecular & Human Genetics
Soens, Zachry Tore ......................................................................................... 308
Sowa, Jessica N............................................................................................... 309
Spike, Aaron Jonathon .................................................................................... 310
Sprouse, Maran Lee ........................................................................................ 311
Advisor: Maria Bettini, Ph.D. - Department of Pediatrics
Sreekumar, Amulya ....................................................................................... 312
Stay, Trace L ................................................................................................... 313
Roy Sillitoe, Ph.D. - Department of Pathology & Immunology
Stevens, Alexandra McLean ........................................................................... 314
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Advisor: Michele Redell, M.D./Ph.D. - Department of Pediatrics
Stinnett, Gary R. ............................................................................................. 315
Advisor: Robia Pautler, Ph.D. - Department of Molecular Physiology & Biophysics
Stojanoski, Vlatko .......................................................................................... 316
Stone, Adrianne Elayne .................................................................................. 317
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Straker, Lauren Elizabeth................................................................................ 318
Stuhlsatz, Danielle Dee ................................................................................... 319
Advisor: B. Pettitt, Ph.D. - Biochemistry
Su , Qingtai ................................................................................................... 320
Advisor: Li Xin, Ph.D. - Department of Molecular & Cellular Biology
Sukumar, Ann Tabitha .................................................................................... 321
Sukumaran, Sujita .......................................................................................... 322
Advisor: Ann Leen, Ph.D. - Department of Pediatrics
William Fisher, M.D.-Department of Surgery
Sun, Jenny J. ................................................................................................... 323
Advisor: Russell Ray, Ph.D. - Department of Neuroscience
Sun, Jiayi Monika ........................................................................................... 324
Advisor: Ching Lau, M.D./Ph.D. - Department of Pediatrics
Sun, Yu .......................................................................................................... 325
Advisor: Weiwei Dang, Ph.D. - Department of Molecular & Human Genetics
Szwarc, Maria Magdalena .............................................................................. 326
Tajhya, Rajeev Babu ....................................................................................... 327
Tan, Kai Li ..................................................................................................... 328
Tanner, Mark R ............................................................................................... 329
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Taylor, Aaron Michael .................................................................................... 330
Advisor: Ching Lau, M.D./Ph.D. - Department of Pediatrics
Tepe, Burak .................................................................................................... 331
Terrón-Díaz, María Elisa ................................................................................ 332
Terwilliger, Austen Lee .................................................................................. 333
Tian, Gengwen ............................................................................................... 334
Advisor: Leonid Metelitsa, M.D./Ph.D. - Department of Pediatrics
Tian, Lin ......................................................................................................... 335
Tran, Baouyen ................................................................................................ 336
Advisor: Edward Cooper, M.D./Ph.D. - Department of Neurology
Tran, Linda Lien ............................................................................................. 337
Advisor: David Rowley, Ph.D. - Department of Molecular & Cellular Biology
Tsai, Chang-Ru .............................................................................................. 338
Advisor: Michael Galko, Ph.D. - Biochemistry and Molecular Biology
Tsai, Wei-Chih ............................................................................................... 339
Advisor: Richard Lloyd, Ph.D. - Department of Molecular Virology & Microbiology
Tu, Yen-Kuei ................................................................................................. 340
Advisor: Kimberley Tolias, Ph.D. - Department of Neuroscience
Tung, Hui-Ying .............................................................................................. 341
Ugur, Berrak .................................................................................................. 342
Ung, Kevin ..................................................................................................... 343
Van der Heijden, Meike Esther ....................................................................... 344
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Vandeventer, Paul Jerrod ................................................................................ 345
Advisor: Koen Venken, Ph.D. - Department of Biochemistry & Molecular Biology
Veith, Alex Christopher .................................................................................. 346
Advisor: Bhagavatula Moorthy, Ph.D. - Department of Pediatrics
Visser, Bryan Joseph....................................................................................... 347
Advisor: David Bates, Ph.D. - Department of Molecular & Human Genetics
Vogt, Megan Brittany ..................................................................................... 348
Advisor: Rebecca Rico, M.P.H. - Department of Molecular Virology & Microbiology
Vue, Zer ......................................................................................................... 349
Advisor: Richard Behringer, M.D. Anderson
Walker, Edgar Yasuhiro.................................................................................. 350
Wang, Chih-Chuan ........................................................................................ 351
Wang, Hui ...................................................................................................... 352
Advisor: Susan Hamilton, Ph.D. - Department of Molecular Physiology & Biophysics
Lawrence Chan, D.Sc.-Department of Medicine
Wang, Li ........................................................................................................ 353
Wang, Mengyu ............................................................................................... 354
Advisor: Ido Golding, Ph.D. - Department of Biochemistry & Molecular Biology
Wang, Yumeng .............................................................................................. 355
Advisor: Han Liang, Ph.D. - Bio informatics and Computational Biology
Webb, Amanda Nicole .................................................................................... 356
Wei, Xing ....................................................................................................... 357
Advisor: Li Xin, Ph.D. - Department of Molecular & Cellular Biology
Wetendorf, Margeaux .................................................................................... 358
Advisor: Francesco Demayo, Ph.D. - Department of Molecular & Cellular Biology
Ming-Jer Tsai, Ph.D.-Department of Molecular & Cellular Biology
White, Janson James ....................................................................................... 359
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Advisor: James Lupski, M.D./Ph.D. - Department of Molecular & Human Genetics
White, Joshua J ............................................................................................... 360
Advisor: Roy Sillitoe, Ph.D. - Department of Pathology & Immunology
Whittle, Sarah Brackley .................................................................................. 361
Advisor: Peter Zage, M.D./Ph.D. - Department of Pediatrics
Williams, LaTerrica Chemise ......................................................................... 362
Xiao-Tong Song, Ph.D.-Department of Pathology & Immunology
Wilson, Stephen Joseph .................................................................................. 363
Wu, Chun-Ting .............................................................................................. 364
Advisor: Daoyun Ji, Ph.D. - Department of Molecular & Cellular Biology
Xia, Jun .......................................................................................................... 365
Xie, Wei ......................................................................................................... 366
Xu, Mingchu .................................................................................................. 367
Xu, Xiaowei ................................................................................................... 368
Advisor: Rachel Schiff, Ph.D. - Department of Medicine
Xue, Zenghui .................................................................................................. 369
Advisor: Anna Sokac, Ph.D. - Department of Biochemistry & Molecular Biology
Yang, Diane ................................................................................................... 370
Advisor: Malgorzata Borowiak, Ph.D. - Department of Molecular & Cellular Biology
Ye, Hui ........................................................................................................... 371
Advisor: Joshua Shulman, M.D./Ph.D. - Department of Neurology
Yeh, Szu-Ying ................................................................................................ 372
Yen, Shuo-Ting .............................................................................................. 373
Advisor: Richard Behringer, - Molecular Genetics - M.D. Anderson
Yin, Jiani ........................................................................................................ 374
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Yong, Lin-Kin ................................................................................................ 375
Advisor: Qizhi Yao, M.D./Ph.D. - Department of Surgery
Yosef, Nejla ................................................................................................... 376
You, Ran ........................................................................................................ 377
Young, Melvin Chen....................................................................................... 378
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Zeng, Huan-Chang ......................................................................................... 379
Zhai, Yijie ...................................................................................................... 380
Advisor: John Belmont, M.D./Ph.D. - Department of Molecular & Human Genetics
Zhang, Hanzhi ................................................................................................ 381
Advisor: Ming Zhou, Ph.D. - Department of Biochemistry & Molecular Biology
Zhang, Jing ..................................................................................................... 382
Advisor: Ido Golding, Ph.D. - Department of Biochemistry & Molecular Biology
Zhang, Xiaotian .............................................................................................. 383
Zhao, Li .......................................................................................................... 384
Zhao, Linhai ................................................................................................... 385
Zhao, Mingkun ............................................................................................... 386
Advisor: Jin Wang, Ph.D. - Department of Pharmacology
Zhelyazkova, Boryana Hristova...................................................................... 387
Zhou, Quan .................................................................................................... 388
Advisor: Yongtao Guan, Ph.D. - Department of Pediatrics
Zhou, Ying ..................................................................................................... 389
Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
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Zhu, Lin L ....................................................................................................... 390
Advisor: Michael Beauchamp, Ph.D. - Department of Neurosurgery
Zhu, Wenyi .................................................................................................... 391
Zhu, Ying ....................................................................................................... 392
Advisor: Fabrizio Gabbiani, Ph.D. - Department of Neuroscience
Zou, Winnie Y ................................................................................................ 393
Advisor: Mary Estes, Ph.D. - Department of Molecular Virology & Microbiology
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2015 GRADUATE STUDENT SYMPOSIUM
TARGETING SRC KINASE AND THE MICROTUBULE NETWORK IN THE
TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY
Reem Sharon Abo-Zahrah
Advisor: George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused
by mutations in the gene encoding dystrophin. Dystrophin is an important scaffolding
protein in the dystroglycan complex, as it connects the cytoskeleton with the
extracellular matrix. Disruption of this complex reduces the structural and mechanical
integrity of the muscle fiber, resulting in progressive skeletal muscle degeneration,
weakness, and death. Although viral gene therapy and exon skipping strategies have
been used in attempts to correct the genetic defect at the molecular level, these
attempts have not been successful. Consequently, identifying downstream cellular
pathways affected by dystrophin deficiency is a critical strategy to develop therapies to
slow down or halt disease progression. While there are several factors that contribute to
the pathologies seen in DMD, oxidative stress has emerged as a key facilitator, with
NADPH oxidase (Nox) being the main source of reactive oxygen species production.
We have identified a novel Nox2/Src pathway that leads to increased oxidative stress
and impairment in autophagy in mdx mice (a model of DMD). Pharmacological inhibition
of Nox2/Src or deletion of p47phox in-vivo decreased oxidative stress and improved
autophagic flux in mdx muscle. Additionally, studies have suggested that the observed
increase in Nox2 ROS production is partially regulated by the stiffening of the
microtubule network. Furthermore, it has been established that microtubule density and
dynamics are critically involved in regulating several autophagy steps. Therefore, we
posit that repurposing Dasatinib, an oral and potent inhibitor of Src Kinase, or KX-01, a
dual inhibitor of Src Kinase and tubulin polymerization, will rescue mdx skeletal muscle.
Preliminary data from mdx mice treated with Dasatinib (15 mg/kg daily) for 14 days
resulted in a decrease in the phosphorylation level of Src, decreased ROS production,
and improved autophagy. In-vitro treatment of dystrophic muscle with KX-01 led to
decreased microtubule density. Taken together, our data strongly support our
hypothesis that targeting Src Kinase and the microtubule network will improve the
pathophysiology of dystrophic skeletal muscle.
Contributors: Abo-Zahrah, Reem; Pal, Rituraj; Rodney, George
1
BAYLOR COLLEGE OF MEDICINE
DETERMINING THE ROLE OF OXYTOCIN IN ESTROGEN RECEPTOR BETA’S
MODULATION OF STRESS
Alexandra Acevedo-Rodriguez
Advisor: Shailaja Mani, Ph.D.-Department of Molecular & Cellular Biology
Anxiety and mood disorders affect 20% of the population and confer large
decrements to the patient’s quality of life. These disorders are frequently associated
with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis which is a system
activated in response to stressors. The sex steroids can modulate HPA axis activity
which may contribute to gender disparities observed in the prevalences of these
disorders. In particular, estrogen receptor β (ERβ) activity has been found to confer
anxiolytic effects and to reduce HPA axis reactivity. This receptor is expressed in the
paraventricular nucleus (PVN) of the hypothalamus which is a key component of the
HPA axis. Within the PVN, ERβ is highly expressed in oxytocin neurons, and in vitro
studies find that ERβ interacts with the oxytocin promoter to regulate oxytocin
transcription. Oxytocin is being researched as a potential therapeutic for numerous
psychiatric disorders, and peripheral oxytocin administration has been found to
decrease HPA axis reactivity and anxiety related behaviors. With ERβ’s expression on
oxytocin neurons and ERβ and oxytocin’s shared anxiolytic behavior, this suggests that
ERβ’s anxiolytic effect may be due to modulating oxytocin signaling. To determine if
oxytocin modulates ERβ mediated anxiolysis, oxytocin null and wild-type animals will
perform a behavioral battery to measure anxiety related behaviors and social aversion.
To determine the locus of oxytocin signaling necessary for ERβ mediated anxiolysis,
oxytocin receptors will be ablated from the PVN, central amygdala, and the median
raphe, regions important in stress circuitry. This will be done by utilizing stereotaxic
injection of an adeno-associated viral (AAV) vector associated with Cre-recombinase,
which will knockdown oxytocin receptor expression in infected cells of OxtrloxP/loxP
animals. In order to determine if ERβ binds to the oxytocin promoter in vivo and if stress
alters the level of this interaction, wild-type animals will be injected with ERβ agonist or
vehicle and then undergo restraint stress or be sacrificed from their home cage. The
PVN will be microdissected out and in vivo chromatin immunoprecipitation will be
performed on this tissue to determine if ERβ interacts with the oxytocin promoter. For
all experiments, male and female animals will be gonadectomized, to prevent
endogenous sex steroid interactions with ERβ, and treated with R-DPN or vehicle.
These experiments will illustrate the interaction between ERβ and oxytocin and how this
interaction relates to physiological stress and anxiety related behaviors.
Contributors: Acevedo-Rodriguez, Alexandra; Oyola, Mario; Malysz, Anna; Kudwa, Andrea;
Carbone, David; Handa, Robert; Mani, Shaila
2
ROLE OF ASTROCYTE HETEROGENEITY IN NEURAL DEVELOPMENT
Joshua Matthew Adams
Program in Developmental Biology/M.D.-Ph.D. Program
Advisor: Benjamin Deneen, Ph.D.-Department of Neuroscience
Astrocytes comprise 50% of the cellular constituency of the central nervous
system (CNS). They diversely function in synaptogenesis, neurotransmitter
homeostasis and blood-brain barrier formation/maintenance. Astrocyte function has
been linked to both developmental disorders and repair. Our understanding of
astrocytes has been limited by a lack of molecular markers and tools to study their
diverse functions. Ramon y Cajal recognized as early as the late nineteenth century
astrocytes’ diverse morphologies. While we currently know much on where astrocytes
function, we still know surprising little on how diverse astrocytes function in the brain.
The astrocyte development and physiology field has been limited by a lack of a
tractable entry point into astrocyte heterogeneity.
Our laboratory has recently utilized Fluorescence-activated cell sorting (FACS) to
isolate and identify five distinct astrocyte subpopulations from the mouse olfactory bulb
(OB) and across the mouse brain. Our previous gene expression analysis led us to
identify a subpopulation highly enriched for genes associated with neuronal chemotaxis
and synapse development/pruning.
We are utilizing FACS to determine the heterogeneous astrocyte expression of
genes of interest in the OB using In Situ Hybridization (ISH) and Immunohistochemistry.
We are coculturing this astrocyte subpopulation with neurons to evaluate the number,
nature and physiology of synapses. In addition we are performing neuronal stem cell
migration assays to determine the neuronal attractive/repulsive potential of the
subpopulation. Finally, we are determining the synaptic and migratory effects of five
select genes of interest, CCL2, CCL3, CCL4, Grn and SLAMF8 using gain-of-function
assays in OB astrocytes utilizing AAV vectors in in vivo mouse olfactory bulbs utilizing
the olfactory bulb’s feature of ongoing adult neurogenesis.
Our aim is to determine the role of astrocyte heterogeneity in synaptogenesis
and neural migration and our findings will help shed light on how different astrocyte
subpopulations help govern synapse formation and maintenance in the brain.
Contributors: Adams, Joshua; Lin, John; Deneen, Benjamin
3
THE ROLE OF WNK1 IN THE ABILITY OF THE UTERUS TO SUPPORT
DECIDUALIZATION AND MAINTENANCE OF PREGNANCY
Nyssa Adams
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Francesco Demayo, Ph.D.-Department of Molecular & Cellular Biology
Chandrasekhar Yallampalli, Ph.D.-Department of Obstetrics & Gynecology
The differentiation of endometrial stromal cells into decidual cells is an integral
step in the establishment and maintenance of pregnancy. This process, termed
decidualization, is regulated by a complex interplay of signaling pathways. Previous
work by our lab showed that epidermal growth factor receptor (EGFR) signaling is
required for decidualization. We demonstrated that the protein, with no lysine kinase 1
(WNK1), is activated downstream of EGFR in decidualizing stromal cells. WNK1 has
not been described in the uterus, although it is known to activate mitogen-activated
protein kinase (MAPK) pathway proteins in other cell types. We hypothesize that WNK1
plays an integral role in the proliferation and differentiation of decidualizing endometrial
stromal cells by activating downstream MAPK proteins. To test this hypothesis, we
examined the effect of siRNA knockdown of WNK1 in primary human endometrial
stromal cells (HESCs). We show that WNK1 knockdown results in failure of HESCs to
decidualize and decreases the proliferation of HESCs. To examine the effects of WNK1
on gene expression during decidualization, we performed RNA-Seq following WNK1
knockdown. We show that WNK1 regulates the expression of 1,858 genes in
decidualizing HESCs. Pathway analysis of differentially expressed genes shows that
WNK1 regulates the expression of genes involved in cellular growth and proliferation,
cellular movement, and cell death and survival. We also show that the MAPK protein,
extracellular-signal-regulated kinase 5 (ERK5), is activated during decidualization
downstream of WNK1. Further analysis of protein phosphorylation by kinome array
shows that WNK1 regulates the activation of multiple signaling kinases in decidualizing
HESCs. Our data suggest that WNK1 regulates downstream kinases, including ERK5,
to drive proliferation of endometrial stromal cells during decidualization. This work was
supported by NIH Grants: R01HD042311, 5U54HD007495, and NURSA grant:
U19DK62434.
Contributors: Adams, Nyssa; Vasquez, Yasmin; Mo, Qianxing; DeMayo, Francesco J.
4
NUDT21-SPANNING CNVS LEAD TO NEUROPSYCHIATRIC DISEASE AND
ALTERED MECP2 ABUNDANCE VIA ALTERNATIVE POLYADENYLATION
Callison Edward Alcott
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
The brain is sensitive to the dose of MeCP2 such that small fluctuations in
protein quantity lead to neuropsychiatric disease. Despite the importance of MeCP2
levels to brain function, little is known about its regulation. In this study, we report
eleven individuals with neuropsychiatric disease and copy-number variations spanning
NUDT21, which encodes a subunit of pre-mRNA cleavage factor Im. Investigations of
MECP2 mRNA and protein abundance in patient-derived lymphoblastoid cells from one
NUDT21 deletion and three duplication cases show that NUDT21 regulates MeCP2
protein quantity. Elevated NUDT21 increases usage of the distal polyadenylation site in
the MECP2 3′ UTR, resulting in an enrichment of inefficiently translated long mRNA
isoforms. Furthermore, siRNA-mediated normalization of NUDT21 in duplication patient
lymphoblasts restores their MeCP2 level to that of healthy controls. Ultimately, we
identify NUDT21 as a novel candidate for intellectual disability and neuropsychiatric
disease, and elucidate a mechanism of pathogenesis by MeCP2 dysregulation via
altered alternative polyadenylation.
Contributors: Alcott, Callison; Gennarino, Vincenzo; Chen, Chun-An; Chaudhury, Arindam;
Gillentine, Madelyn; Li, Wei; Neilson, Joel; Schaaf, Christian; Zoghbi, Huda
5
THE ROLES OF TGFΒ SIGNALING AND SMALL LEUCINE-RICH
PROTEOGLYCANS IN OSTEOGENESIS IMPERFECTA
Stefanie Louise Alexander
Integrative Program in Molecular and Biomedical Sciences/M.D.-Ph.D. Program
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Osteogenesis imperfecta (OI) is a debilitating genetic osteodysplasia that results
in low bone mass, bone deformity, and bone fractures. Most cases of OI are caused by
mutations in the structural protein type I collagen (dominant OI) or in protein complexes
that post-translationally modify type I collagen (recessive OI); both types of mutations
can lead to biochemical overmodification. Type I collagen has structural roles and
regulates cell signaling in the matrix. Due to the phenotypic similarities between OI
mouse models and TGFβ overexpression models, we hypothesized that altered TGFβ
signaling could contribute to OI pathogenesis in both recessive (Crtap-/-) and dominant
(G610C OI) OI mouse models. In support of this, we found increased mRNA expression
of TGFβ target genes with quantitative PCR and an increased ratio of phosphorylated
Smad2 to total Smad2 with western blot in bone samples from both OI mouse models
suggesting increased TGFβ signaling. In a bone marrow stromal cell (BMSC) in vitro
system, which mimics osteoblast differentiation, we found that osteoblasts from both OI
mouse models demonstrated increased TGFβ signaling and increased free TGFβ in the
media, suggesting altered TGFβ bioavailability. Treatment of both OI mice with a TGFβ
antibody (1D11) significantly rescued the OI bone phenotype. Together, these
experiments demonstrate that increased TGFβ signaling contributes to the
pathogenesis of OI Small leucine-rich proteoglycans (SLRPs) are matrix components
that sequester TGFβ by binding to type I collagen, and changes in type I collagen in OI
may disturb SLRP binding, altering TGFβ availability in the matrix. Surface plasmon
resonance (SPR) demonstrated that the SLRP decorin (Dcn) binds Crtap-/- type I
collagen 45% less effectively than wildtype type I collagen. SLRP expression is
significantly altered in an in vitro bone marrow stromal cell (BMSC) system that models
osteoblast differentiation. To fully elucidate the role of SLRPs in bone, I have generated
two SLRP knockout mouse models for asporin (Aspn) and osteomodulin (Omd) and am
assessing for OI phenotype and TGFβ signaling. Together, these data suggest that
increased TGFβ signaling contributes to the pathogenesis of OI and that altered SLRP
biology may be important in OI. Further studies will be done to investigate the
relationship between the increased TGFβ signaling, increased SLRP expression, and
the binding of SLRPs to the ECM. Ultimately, establishing the molecular mechanisms
behind altered ECM signaling in OI will lead to more specific treatments for this disease.
Contributors: Alexander, Stefanie; Grafe, Ingo; Lee, Brendan
6
PANCANCER ANALYSIS OF LONG NONCODING RNA INTERACTIONS IN GENE
REGULATORY REGIONS AND EPIGENETIC REMODELING IN CANCER
Samirkumar B Amin
Advisor: Roeland Verhaak, Ph.D.-Bioinformatics & Computational Biology
The primary focus of this study is to uncover emerging functional role of long
non-coding RNA (lncRNA) in the development and progression of cancer. Recent
studies have consistently shown that lncRNAs play important role in gene regulation by
providing modular scaffold at promoter and distal regulatory sites. Although structural
alterations are found to be driver events in most of cancers, large scale studies like the
cancer genome atlas (TCGA) have repeatedly showed subgroups of cancer patients
with relatively fewer mutations and copy number alterations. However, in such
seemingly quiet genome, several studies, especially in pediatric gliomas have shown
dominant role of epigenetic remodeling in driving oncogenic transcriptional program. At
the transcription core, such remodeling can take place by differential interaction
patterns of transcription factors (TFs) with their co-regulators at target regulatory sites
by action of chromatin remodelers. However, it is unknown whether these TF-coregulator-DNA interactions at known cancer gene sites are coordinated by an
abundance of tethering elements. Based on evidence supporting lncRNAs acting as a
modular scaffold, we hypothesize that abundance of lncRNAs harboring sequencespecific motif or structural motif coordinate TF-DNA interactions at promoter regions of
known cancer genes and thus, aid in driving oncogenesis. Using pancancer matched
RNA-seq and WGS data from TCGA/ICGC (n=969), we observe tissue- and subtypespecific lncRNA expression with enrichment of several lncRNAs in subgroups of tumorspecific samples having fewer structural alterations, e.g., SPOP mutant prostate cancer
and IDH1 mutant glioblastoma. There is significant expression of lncRNAs which are
proximal (< 5kb) to coding genes as compare to intergenic lncRNAs, suggesting their
potential role at promoter regions of those coding genes. lncRNAs exonic regions have
been shown to be enriched in repeat elements which can server as sequence-specific
motifs. We find abundance of a few transposable elements from AluYc and LINE-1
families in coding region of these lncRNAs and promoter regions of expressed
consensus cancer genes. We are currently integrating TCGA matched RNA-seq and
WGS data with RNA-seq and ChIP-seq data from GTEx and ENCODE data,
respectively to find significant associations between lncRNAS and chromatin
remodelers mediated recruitment of TFs at target regulatory sites. Our analysis pipeline
will be scalable and extensible in understanding mechanistic and functional role of
lncRNA in other cancers. Further, such integrated analyses should better help us
characterize tumor subtypes based on downstream transcriptional program, and
potentially improve existing expression based prognostic prediction.
Contributors: Amin, Samir B. ; Verhaak RG.
7
EPIGENOMIC FOOTPRINTS ACROSS 111 REFERENCE EPIGENOMES REVEAL
TISSUE-SPECIFIC EPIGENETIC REGULATION OF LINCRNAS
Viren R Amin
Advisor: Aleksandar Milosavljevic, Ph.D.-Department of Molecular & Human Genetics
Tissue-specific expression of lincRNAs suggests developmental and cell-type
specific functions, yet tissue-specificity was established for only a small fraction of
them. By analyzing 111 reference epigenomes from the NIH Roadmap Epigenomics
project we determined tissue-specific epigenetic regulation for 3,753 (69% examined)
lincRNAs, with 54% active in one of the fourteen cell/tissue clusters and an additional
15% in two or three clusters. Larger fraction of lincRNA TSSs are marked in a tissuespecific manner by H3K4me1 than H3K4me3. The tissue-specific lincRNAs are strongly
linked to tissue-specific pathways and undergo distinct chromatin state transitions
during cellular differentiation. Polycomb-regulated lincRNAs reside in the bivalent state
in embryonic stem cells and many of them undergo H3K27me3-mediated silencing at
early stages of differentiation. The exquisitely tissue-specific epigenetic regulation of
lincRNAs and the assignment of a majority of them to specific tissue types will inform
future studies of this newly discovered class of genes.
Contributors: Amin, Viren; Harris, Alan R.; Onuchic, Vitor; Jackson, Andrew R.; Charnecki, Tim;
Paithankar, Sameer; Subramanian, Sai Lakshmi; Riehle, Kevin; Coarfa, Cristian; Milosavljevic,
Aleksandar
8
OXIDATION OF MITOCHONDRIAL SSB ALTERS THE SINGLE-STRANDED DNA
BINDING PROFILE, CHANGING THE PROTEINS REGULATORY EFFECT
Andrew Peter Porten Anderson
Advisor: Y. Yin, D.Phil-Chemistry & Biochemistry
The human mitochondrial genome contains the genes for 13 essential proteins,
2 rRNAs, and 22 vital tRNAs. Human mitochondrial Single Stranded DNA Binding
Protein (SSB) is responsible for the stimulation of multiple elements of the
mitochondrial replisome, including the DNA Polymerase (Pol γ) and inhibition of
Exonuclease activity. The mitochondrial matrix is host to significant quantities of
reactive oxygen species, which are generated continuously by the electron transport
chain even while operating normally. Oxidation of human mitochondrial single stranded
binding protein has a marked effect on the ssDNA binding affinity and binding modes
exhibited. This is due to disruption of the charge based, arginine-rich, protein-ligand
interface rather than a catastrophic disruption of protein structure. A loss of affinity, in
the oxidized form, is coupled with a shift in binding mode, and considering that SSB
binding is synergistic over large strands of ssDNA, this oxidation will cause a large shift
in binding, and therefore alter the biological effect of the mtSSB.
Contributors: Anderson, Andrew; Yin, Whitney
9
ION CHANNEL COOPERATIVITY THROUGH MEMBRANE ELECTROMECHANICS
Mussie Araya
Advisor: William Brownell, Ph.D.-Department of Otolaryngology-Head and Neck Surgery
The ability of neuronal populations to encode rapidly varying stimuli and respond
quickly is crucial for basic neuronal computations, such as coincidence detection,
grouping by synchrony, spike-timing-dependent plasticity and boosting the processing
speed of neuronal networks. Theoretical analyses have linked these abilities to the fastonset dynamics of action potentials (APs). While Hodgkin Huxley theory fails to explain
the speed of AP onset, a computational analysis invoking cooperative activation of Na+
ion channels at the axon initial segment (AIS) does. The near simultaneous gating of
ion channels results in a hyperpolarized shift in the population activation curve
producing a rapid AP initiation. The biophysical basis for intra-channel coupling is
unknown and Ca++ or GTP based signaling is too slow. Axons show dimensional
changes during the AP production and membrane tethers have been shown to
generate electromechanical force at frequencies up to 10 kHz. It is also known that
membrane mechanics modulate ion channel function. We propose an
electromechanical mechanism for cooperative gating of sodium channels at the AIS.
Specifically, the rapid modulation of membrane tension by membrane potential can
provide a fast and direct mechanism for inter-channel coupling.
We combined optical tweezers and voltage clamp apparatus to pull membrane
tethers from the cells in order to make precise measurement of membrane
electromechanical force generation. We pulled membrane tethers from the soma and
AIS of hippocampal pyramidal neurons to probe the membrane-cytoskeleton adhesion
strength. We find stronger strength of membrane-cytoskeleton adhesion in the AIS
suggesting ion channels are firmly anchored to the actin based cytoskeleton at AIS.
Cooperative activation is highly dependent on the density of Na+ channels. The
clustering of Na+ channels at the AIS by the actin cytoskeleton can set the stage for
cooperative gating between ion channels. Once a tether is formed, the
electromechanical force is measured by applying a sinusoidal voltage and measuring
the tether pulling force. Testing for concomitant variation between electromechanical
force and channel function to determine the membrane’s role in ion channel gating will
be discussed.
Contributors: Mussie K. Araya1 and William E. Brownell1,2
1Molecular Physiology and Biophysics Baylor College of Medicine, Houston, Tx, USA
2Otolaryngology – H&N Surgery Baylor College of Medicine, Houston, Tx, USA
10
FOXM1 AS A REGULATOR OF BREAST CANCER LIPID METABOLISM
James Michael Arnold
Advisor: Arun Sreekumar, Ph.D.-Department of Molecular & Cellular Biology
Breast cancer is a significant public health concern and there remain unmet
challenges in the diagnosis and treatment of triple negative breast cancer (TNBC).
Gene expression profiling has revealed that TNBC is composed of a diverse set of
disease states, which serve as an imperfect predictor of response to therapy, thus there
is a need for a more integrated approach to describe phenotypic subtypes within TNBC.
Metabolites are the final products of cell signaling pathways, and the relative levels of
metabolites can be considered a direct readout of the current phenotypic state of the
cell. In a recently published study, our lab profiled 536 metabolites across 67 matchedtumor-normal pairs of breast tissue, all containing matched gene expression profiles
and epidemiological data. Our results suggest altered levels of specific metabolites
within a subset of TNBC, predominantly of African American (AA) descent. These
include elevated levels of fatty acids as well as cholesterol metabolites. Interestingly,
this same group of TNBC tumors is highly enriched for the expression of FOXM1, an
oncogenic transcription factor associated with cell cycle progression known to be
associated with aggressive breast cancer subtypes. Within the TNBC cell line model
MDA-MB-231 we have found cholesterol accumulates following FOXM1 knockdown.
Datamining of public datasets and preliminary knockdown studies in vitro suggests
FOXM1 negatively regulates transcription of the cholesterol biosynthetic enzymes
HMG-CoA Synthase (HMGCS1) and HMG-CoA Reductase (HMGCR), suggesting a
novel regulatory network linking cholesterol production and FOXM1 activity. These
preliminary results suggest FOXM1 may play a significant role in the regulation and
maintenance of lipid metabolism in breast cancer.
Contributors: Konde, Sai A.; Gu, Franklin; Rasaily, Uttam; Rao, Arundhati; Ambs, Stefan
11
A GENOMICS-DRIVEN APPROACH TO UNDERSTANDING MECHANISMS OF
ATTENUATION AND VIRULENCE IN FRANCISELLA TULARENSIS
Lisa Michelle Atkins
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
Francisella tularensis (Ft) is a Gram-negative, intracellular coccobacillus and the
etiological agent of the zoonotic disease tularemia. Subspecies tularensis (type A) and
holarctica (type B) are responsible for all tularemia-associated fatalities in the U.S., with
type A infections resulting in significantly higher mortality compared to type B. Ft is
classified as a Tier One select agent due to its low infectious dose, high morbidity, and
lack of a licensed vaccine. An attenuated Live Vaccine Strain (LVS) was empirically
derived from repeated passage of a virulent type B strain. While it was later shown that
LVS is less effective against inhalational type A, it is to date the only vaccine for which
formal efficacy data in humans exists. However, LVS remains unlicensed and the exact
mechanism of attenuation is unknown.
We aligned the LVS genome to those of virulent type B strains and identified 17
genes that are disrupted in LVS, implicating these genes in LVS attenuation and Ft
virulence. We hypothesize that the disruption of a subset of candidate genes in Ft Type
B will resemble attenuating mutations in LVS, thus identifying genes required for an
effective vaccine against Ft Type A.
Few studies have characterized virulent type B strains in vitro or in vivo. We
report that WT type B replicates to higher numbers in both human and murine
macrophages compared to LVS, and that TH1 cytokine patterns of macrophages
infected with WT type B are more similar to negative controls, while those of
macrophages infected with LVS are similar to positive controls. Quantitative real-time
RT-PCR confirms higher 16S rRNA copies for WT type B compared to LVS, and
reveals differences in mRNA levels of acute-phase proteins at specific time points postinfection. Furthermore, LVS-infected macrophages, which are able to control LVS
replication, undergo cell death 10-24 hours earlier than those infected with WT type B,
which are permissive for growth of WT. To our knowledge, this is the first systematic
evaluation of innate immune response kinetics in murine and human macrophages
following infection of a virulent type B strain compared to its attenuated counterpart,
LVS.
Having established valid thresholds for attenuation and virulence, studies to
determine if disruptions for specific candidate genes attenuate type B in vitro are
currently underway. Ft virulence mutants will reveal further underlying mechanisms of
pathogenesis, and may also identify novel, conserved virulence factors for other
intracellular pathogens.
Contributors: Atkins, Lisa; Ayvaz, Tulin; Petrosino, Joseph
12
FUNCTIONAL EFFECTS OF AMINO ACID SUBSTITUTION IN E.COLI RECA
Benu Atri
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
The impact of amino acid substitutions may vary from no effect to a total loss of
protein function. It is critical to develop methods to estimate this impact robustly and on
a large scale in order to guide mutational studies of protein function, distinguish
disease–causing from harmless mutations in clinical settings, and interpret the
numerous polymorphisms revealed by exome sequencing. In order to achieve this, we
hypothesized that the impact of a substitution depends on two factors: the functional
importance of the residue and size of substitution. This hypothesis was found true
retrospectively, when we used a large amount of mutational data from prokaryotic,
eukaryotic and viral proteins. Prospectively, we applied it to generate mutations that
tune protein function to expected levels in E.coli RecA. This protein plays a central role
in homologous recombination, DNA damage repair, and is a key component of the
bacterial SOS response, where it controls the expression of many other DNA damage
repair genes. We experimentally measured the impact of RecA mutations by assaying
for DNA damage repair and for recombination. The results reveal a correlation between
the computed impact and the experimental measurements of the functions of RecA
mutants. Together these data shed new light on the relationship between genotype and
phenotype variations and suggest that the impact of mutations can be estimated by
computing patterns in the evolutionary history of proteins.
Contributors: Katsonis P, Adikesavan AK, Lichtarge, O
13
GENERATION OF NOVEL KNOCK-IN CELL LINES TO STUDY TELOMERASE
Brandon Christopher August
Advisor: Ergun Sahin, M.D.-Department of Molecular Physiology & Biophysics
Telomeres are the repetitive TTAGGG sequences at the ends of chromosomes,
which function as chromosomal caps that prevent DNA degradation, chromosomal
fusions, and activation of DNA damage response checkpoints. Telomeres are
synthesized by a specialized reverse transcriptase known as telomerase, which
consists of two essential components at its core – hTERT, the reverse transcriptase
subunit, and hTERC, the RNA template subunit. In humans, most somatic cells do not
express hTERT, which results in telomere shortening with each round of cell division
due to the lack of detectable telomerase activity to extend telomeres. However,
telomerase reactivation and telomere maintenance are required for the progression of
approximately 90% of human cancers, which utilize telomerase to obtain proliferative
immortality. The remaining 10% of human cancers utilize a mechanism known as
Alternative Lengthening of Telomeres (ALT), a recombination-based method of
telomere lengthening. To date, the development and mechanisms of ALT are poorly
understood, and no therapeutic interventions for ALT-positive cancers are available.
Despite the numerous studies of hTERT regulation, the mechanisms leading to
repression of hTERT in somatic cells and reactivation of hTERT in cancer cells are
unknown, suggesting that additional factors and mechanisms remain to be identified.
This gap in our understanding is in part due to the lack of a relevant human model
system in which endogenous hTERT regulation can be studied. The emergence of new
genome editing tools, including TALENS and CRISPR, allows for the manipulation of
the human genome at distinct locations, providing the opportunity to generate human
knock-in and knockout cell lines to study biological relevance. Here, we have utilized
these novel tools to generate conditional human knock-in cell lines that permit the
identification of novel regulators of telomerase expression and key activators of ALT.
We have used TALENs to knock-in a loxP-flanked fluorescent-hTERT fusion gene into
the ATG site of hTERT in 293T and HAP1 cells, the latter of which is a human haploid
leukemia cell line. The generation of such conditional knock-in cell lines will facilitate
robust genetic screens to identify key regulators of hTERT and key components of the
ALT pathway.
Contributors: August, Brandon; Sahin, Ergun
14
DEVELOPING A CURE TO CHRONIC HIV INFECTION WITH ADOPTIVE NATURAL
KILLER CELL IMMUNOTHERAPY
Kevin Aviles-Padilla
Advisor: Silke Paust, Ph.D.-Department of Pediatrics
Despite over 25 years of HIV active research, HIV infections is still a major
international health crisis, with an estimated 40 million people living with the disease1.
Combination antiretroviral therapy (cART) prolongs the life expectancy of individuals
infected with HIV-1 and improved quality of life, but fails to cure HIV infection,
presumably due to the inability of the host immune system to eradicate latently HIV
infected cells2, primarily CD4+ T cells and monocytes that reside in the gastrointestinal
mucosa, the central nervous system, and heart. Improved therapeutic approaches are
necessary to eradicate latently HIV infected cells to cure HIV3-6. Increasing
experimental evidence supports the important role of Natural Killer (NK) cell- mediated
effector function in HIV disease. Therefore, we hypothesize that adoptive NK cell based
immunotherapy can be used to attack the latent HIV reservoir and cure chronic HIV
infection. We propose to assess the effects of NK adoptive immunotherapy in a
humanized mouse model. Specifically, we aim to assess the optimal stimulus for NK
attack of HIV infected cells, the effect of NK adoptive immunotherapy on the frequency
of HIV infected cells in multiple organs, and to determine the optimal amount of HIV
reactivation needed for NK mediated attack of the latently infected cells. If successful,
our approach will open the door for novel strategies that seek to eradicate latent HIV
infection.
Özkaya Şahin G, Bowles EJ, Parker J, et al. PLoS Pathogens.
2010;6(9):e1001084.
Costagliola, D., et al. Journal of the International AIDS Society 17, 19481 (2014).
Kim, M., et al. Nature protocols 9, 2755-2770 (2014).
Kumar, A., Abbas, W. & Herbein, G. Viruses 6, 1837-1860 (2014).
Battistini, A. & Sgarbanti, M.Viruses 6, 1715-1758 (2014).
Contributors: Aviles-Padilla, Kevin; Hernandez-Sanabria, Mayra; Tri Le, Duy; Rooney, Cliona;
Angelo, Laura and Paust, Silke
15
FUNCTIONAL INVESTIGATION OF THE MITOCHONDRIAL PYRUVATE CARRIER IN
PROSTATE CANCER
David A Bader
Department of Molecular & Cellular Biology/M.D.-Ph.D. Program
Advisor: Sean Mcguire, M.D./Ph.D.-Department of Molecular & Cellular Biology
Androgen receptor (AR)-induced de-novo lipid synthesis allows the growth and
survival of hormone sensitive and castrate-resistant prostate cancer cells, but the
metabolic substrate trafficking alterations required to support lipogenesis in prostate
cancer are poorly understood, hindering the development of targeted therapeutic
strategies. Recent work in our lab has demonstrated a subunit of the mitochondrial
pyruvate carrier (MPC), MPC2, is a direct AR target gene, suggesting a link between
AR action and pyruvate trafficking. In addition, pharmacologic inhibition of the MPC in
AR positive prostate cancer cell lines (LNCaP, LNCaP C4-2, and LNCaP-ABL) has a
profound effect on in-vitro proliferation while AR negative cell lines (PC3 and DU145)
are virtually unaffected. These data suggest both castrate sensitive and resistant AR
positive models of prostate cancer rely on pyruvate flux through the MPC to sustain
proliferation. In contrast, AR negative models of prostate cancer may rely on the
classic, ‘Warburg’ model of cancer metabolism for growth. These observations begin to
explain why MPC inhibition fails to slow growth in AR negative cell lines and are in line
with recent reports positioning the MPC as a tumor suppressor in colon cancer. The
overall objective of the current project is to determine the mechanism by which AR
alters pyruvate trafficking to fuel lipogenesis in prostate cancer. Our central hypothesis
is that AR regulates the MPC to increase pyruvate flux into mitochondria to enable
oncogenic growth by increasing lipogenic capacity of tumor cells. The rationale of our
proposal is that characterization of the mechanism underlying increased pyruvate
trafficking in prostate cancer will likely enable the development of strategies to
therapeutically manipulate pyruvate flux. Such treatments could be applied to both
hormone sensitive and treatment refractory castrate-resistant prostate cancer.
Contributors: Bader, David A; Hartig, Sean M; Sreekumar, Arun; Coarfa, Cristian; O’Malley,
Bert W; McGuire, Sean E
16
A CLN6-CLN8 COMPLEX ESCORTS LYSOSOMAL ENZYMES FROM THE ER TO
THE GOLGI
Lakshya Bajaj
Advisor: Marco Sardiello, Ph.D.-Department of Molecular & Human Genetics
One-third of the proteome is synthesized in the endoplasmic reticulum (ER) and
then sorted across the secretory route. Upon proper folding, newly synthesized proteins
exit the ER and are translocated to the Golgi, where addition of complex sugars may
assist in their correct subsequent delivery to endosomes, lysosomes, plasma
membrane, or extra-cellular matrix. Together, this transportation system accounts for
the secretory pathway of the cell. The 50 lysosomal hydrolases are synthesized in the
ER and delivered to lysosomes through this route. Extensive studies have dissected the
Golgi-to-lysosome trafficking of lysosomal hydrolases, however, how these proteins exit
the ER has not been thoroughly examined under the guise that there is a bulk flow of
forward protein trafficking from the ER to the Golgi. Most recently, the concept of
proteins being sorted in the cell through specific cargo receptors is gaining strength.
CLN8 is one such cargo receptor, recently shown to assist in the ER exit of lysosomal
hydrolases. We show here that the Batten disease protein CLN6 complexes with CLN8,
and that the CLN6-CLN8 complex escorts the lysosomal hydrolases from the ER to the
Golgi. We demonstrate that CLN6 interacts with the homodimer of CLN8 and that
depletion of CLN6 leads to loss of interaction of CLN8 with lysosomal hydrolases,
indicating that CLN6 and CLN8 form a transport complex necessary for transportation
of hydrolases. Using a naturally occurring mouse line deficient for Cln6 (nclf Batten
mouse) we observe a general depletion of various lysosomal hydrolases from the
lysosomal compartment. Thus, we conclude that CLN6 plays an essential role in the
trafficking of lysosomal hydrolases from the ER to the Golgi and that, in its absence, the
hydrolases are missorted or degraded, resulting in a disease of lysosomal function. This
study identifies the function of CLN6 as essential to lysosomal biogenesis and function
and sheds light on the pathogenesis of a devastating childhood neurodegenerative
disease.
Contributors: Lakshya Bajaj, Alberto Di Ronza, Pal rituraj, Jai Prakash Sharma, Marco Sardiello
17
PROTEIN BARRIERS BLOCK REPLICATION FORKS BY CONSTRAINING DNA
TOPOLOGY
Anna Kathryn Barker
Advisor: David Bates, Ph.D.-Department of Molecular & Human Genetics
Replication forks in all cells are faced with a multitude of challenges as they
move along the chromosome, including topological fluctuations and DNA-bound
proteins. These challenges act as physical barriers to DNA replication by prohibiting
progression of the replisome, consequently resulting in chromosome breakage,
mutation, and cell cycle conflicts due to delayed replication. Replication fork barriers are
a source of genomic instability that fuels the evolution of antibiotic resistance in
pathogenic bacteria, as well as cancer and genetic diseases in higher organisms. It is
now apparent that replication conflict with barriers is the primary source of mutation and
chromosome rearrangements in non-stressed cells, and that this phenomenon
underpins the most basic root of reduced cellular fitness and human disease evolution.
However, the factors that serve as the “first line of defense” against genomic instability
by facilitating fork progression at replication barriers are unknown, and the frequency of
barrier-mediated replisome stalling and its consequences on DNA integrity are
undefined.
Using the Fluorescent Repressor-Operator System (FROS), which has been
previously established as a replication roadblock in E. coli, we are able to assay
genome-wide replication progression and arrest at a DNA-bound protein complex with
Next Generation Sequencing. Our preliminary data demonstrates that (1) replisomes
are pausing 0.5Mb upstream of a DNA-bound protein, suggesting that replisome arrest
is not dependent on direct interactions with the protein barrier. (2) Type II
topoisomerases, Topoisomerase IV and DNA Gyrase, promote fork progression during
replication roadblock by the repressor-operator complex, indicating a role for DNA
topology in replication arrest at protein barriers. (3) Our data also demonstrates
extensive DNA degradation at a paused fork, suggesting that fork regression and
degradation may underlie DNA and cell fates. Collectively, these data suggest the
formation of a multi-fork catastrophe caused by the topological stress induced by a
DNA-bound protein, and implicate a mechanism of how general DNA-bound proteins
cause genomic instability thereby increasing our understanding of DNA replication as
the most basic origin of disease evolution.
Contributors: Barker, Anna K.; Bates, David
18
CYCLIN D1 POTENTIALLY COUPLES RETINAL PROGENITOR CELL
PROLIFERATION AND MITOCHONDRIAL BIOGENESIS
Anthony Patrick Barrasso
Advisor: Ross Poche, Ph.D.-Department of Molecular Physiology & Biophysics
The retina is a thin, neuronal tissue layer of the eye that processes and transmits
visual stimuli to the brain via the optic nerve. Composed of 7 major cell types derived
from a common pool of multipotent retinal progenitor cells (RPCs), the mammalian
retina serves as a useful model to address questions regarding neural progenitor fate
determination and terminal differentiation. Retinogenesis requires a tightly regulated
balance between proliferation and differentiation. A critical point in balancing these
mechanisms occurs in G1 phase of the cell cycle. Early in G1 phase, it is determined
whether a cell exits the cell cycle and stops proliferating, or continues into S phase to
replicate DNA and prepare for G2 phase and division. Premature cell cycle exit of
retinal progenitor cells may cause developmental defects that result in abnormally small
eyes. In proliferating cells, Cyclin D1 normally promotes S phase progression, and
genetic ablation of Cyclin D1 in mice results in thinner retinae and degenerative lesions
in the photoreceptor cell layer. This phenotype is mirrored in mice with retinal-specific
ablation of Ronin, a transcription factor that regulates mitochondrial gene expression. In
addition, it has been found that mitochondrial activity promotes cell cycle progression.
Together, this suggests that Cyclin D1 and mitochondria work together to promote
proliferation during retinogenesis. However, current literature has conflicting reports on
the relationship between Cyclin D1 and mitochondrial activity. Some claim Cyclin D1
represses mitochondrial activity in liver and mammary tissue, but a study of Drosophila
indicates that Cyclin D promotes mitochondrial biogenesis and metabolic activity. Thus,
we investigated whether Cyclin D1 influences mitochondrial activity to promote cell
cycle progression in the developing retina. To test this hypothesis, we used
immunohistochemistry to show that Cyclin D1 ablation suppresses G1/S phase
progression in RPCs, which is consistent with previous conclusions about the role of
Cyclin D1 in RPCs. In addition, we found that Cyclin D1 ablation decreases electron
transport chain activity in RPCs, and Western blot analysis of mitochondrial lysates
isolated from wildtype P0 retinal tissue revealed that Cyclin D1 is present in
mitochondria. Together, these data suggest that Cyclin D1 may interact with
mitochondrial proteins to drive electron transport chain activity, a known promoter of cell
cycle progression. Finally, we have developed a technique to live image retinal slice
cultures, which allows us to track mitochondrial dynamics and cell cycle kinetics in
developing retinae. Future studies aim to further characterize a relationship between
Cyclin D1 and mitochondrial activity, and determine the effect it has on proliferation.
Contributors: Barrasso, Anthony P.; Rueda, Elda; Fox, Donald A.; Poché, Ross A.
19
ROLE OF SMALL RNA, GcvB, IN MUTAGENIC DNA BREAK REPAIR UNDER
STRESS
Brittany Marie Barreto
Advisor: Susan Rosenberg, Ph.D.-Department of Molecular & Human Genetics
In organisms from bacteria to human, stress responses upregulate mutagenesis
mechanisms, potentially accelerating adaptation. In Escherichia coli repair of DSBs by
homologous recombination becomes error-prone under the control of the
general/starvation (RpoS) and DNA-damage (SOS) stress responses. Mutagenic break
repair (MBR) also occurs in yeast and human cancers, though whether controlled by
stress responses is unknown. A network of 93 proteins promotes MBR in starvationstressed E. coli, most by sensing stress and signal transduction that activates the stress
responses. But the screen that identified these genes was biased against small genes
including small RNAs (sRNAs). E. coli has at least 80 sRNA genes. We tested 10
candidate sRNAs and found that one, GcvB, is required for MBR. Deletion of gcvB
reduces MBR 5 ± 0.03-fold in a standard assay. Using a variety of assays, we show
that GcvB does not promote formation of spontaneous DSBs required for MBR,
activation of the RpoE stress response, which promotes spontaneous DSBs in some
genomic regions, DSB repair itself, or activation of the SOS response. Rather, cells
lacking GcvB show reduced induction of the general (RpoS) stress response during
starvation, shown by flow cytometry with a fluorescent reporter gene and via a colony
assay. The general stress response is activated by RpoS protein, a bacterial sigma
factor—a transcriptional activator that plugs into the RNA polymerase directing it to
specific (RpoS-dependent) promoters.
However, western blots show no RpoS
decrease in ∆gcvB cells. Preliminary data support a model in which RpoS activity is
decreased in ∆gcvB cells by strong upregulation of the RpoE membrane-stress
response, causing decreased RpoS response by sigma-factor competition for RNA
polymerase. First, RpoS activity in the fluorescent-reporter and colony assays is fully
restored in ∆gcvB cells carrying rpoE::Tn, which ablates RpoE stress-response activity
but not its essential function. Second, DSB-dependent mutagenesis is also restored in
∆gcvB cells carrying rpoE::Tn in a MBR assay in which chromosomal DSBs are
supplied by I-SceI endonuclease, which makes MBR RpoE-independent. These data
imply that strong induction of the RpoE/membrane stress response in ∆gcvB cells
decreases ability to induce the RpoS/general stress response, which decreases MBR,
and suggest sigma-factor competition for RNA polymerase as the mechanism of this
shift.
Contributors: Barreto, Brittany; Rogers, Elisabeth; Frisch, Ryan; Rosenberg, Susan
20
NON-INVASIVE FUNCTIONAL CARDIAC MONITORING IN A MOUSE MODEL OF
CHAGASIC CARDIOMYOPATHY TO EVALUATE A NEW THERAPEUTIC VACCINE
Meagan Amelia Barry
Advisor: Peter Hotez, M.D./Ph.D.-Department of Pediatrics
Laila Woc-Colburn, M.D.-Department of Medicine
New estimates from the World Health Organization indicate that 1.17 million
people suffer from chagasic cardiomyopathy in Latin America. Initially, cardiac
dysfunction is characterized by conduction disorders, which can then progresses to
cardiomyopathy and even sudden cardiac death. We have utilized state-of-the-art noninvasive functional cardiac monitoring offered by a core facility at Baylor College of
Medicine as an innovative approach to evaluate new therapeutics in a mouse model of
chagasic cardiomyopathy. Current pharmacological treatments are plagued by
significant side effects and poor efficacy. There is an urgent need for new treatment
modalities. A therapeutic vaccine has potential advantages that include reduced
adverse effects, cost savings, and the potential to be used as a replacement for current
therapies or when paired with chemotherapy. Our laboratory has previously shown
promising cell-mediated protective immunity and therapeutic efficacy of a nanoparticle
vaccine in an acute mouse model of Chagas disease. In order to test new treatment
modalities in a model that mimics key aspects of human disease, we have optimized a
mouse model of chagasic cardiomyopathy. When female ICR mice (Taconic
Biosciences, Inc) are infected with 500 trypomastigotes of a H1 strain originally isolated
from a patient in Yucatán, Mexico, 70% of the mice survive through the acute phase of
the disease and enter into the chronic stage. Of these mice, 22% have evidence of
ECG abnormalities in the early chronic stage of disease including altered basal heart
rates, ectopic activity, and conduction blocks. As these mice progress further into
chronic infection 17% show severe conduction blocks by echocardiography. Currently,
we are evaluating our therapeutic vaccine in this mouse model of chagasic
cardiomyopathy. This work optimizes a mouse model of chagasic heart disease, and
utilizes non-invasive functional cardiac monitoring as a novel translational technique to
evaluate new therapeutic vaccines against Chagas disease.
Contributors: Barry, Meagan; Respress, Jonathan; Jones, Kathryn; Wang, Qian; Bottazzi, Maria
Elena; Heffernan, Michael; Hotez, Peter.
21
FUNCTIONAL PRIORITIZATION OF STRUCTURAL VARIANTS THROUGH A
COMBINATION OF SIGNALS FOR PURIFYING SELECTION
Justin Ray Bartanus
Advisor: Fuli Yu, Ph.D.-Department of Molecular & Human Genetics
As the cost of sequencing has fallen dramatically over the past decade, it has
become more feasible to sequence large cohorts of individuals from a diverse range of
populations. While many studies have focused on analyzing human variation, the vast
majority of the focus has been directed at single-nucleotide variants (SNVs) and short
indels even though copy number variants (CNVs) account for variants covering a larger
proportion of the human genome. Due to this fact, our understanding of CNVs and their
relative contributions to both common and rare disease is incomplete. Though more
recent tests for directional selection have begun incorporating functional properties to
traditional statistical analyses of regional variations, the overall picture of the
contribution of these regional variants to disease susceptibility remains unclear. It is
well known that mutations in functionally important regions undergo directional
selection. Through the application of population genetic principles on large cohorts such
as the 1000 Genomes Project dataset, we can study selective signals in genomic
regions with known pathogenic structural variants. We propose a composite test which
combines the strengths of various orthogonal purifying signal estimators, including
statistical tests, functional tests, and evolutionary tests, to amplify the overall selective
signal of a target region. Through comparisons of target regions and their
corresponding selective signals, we can functionally prioritize regions according to the
relative susceptibilities to functional disruption. We can further associate regional
susceptibilities to overlapping structural variations to effectively prioritize individual
structural variants by their contributions to both rare and common disease.
Contributors: Bartanus, Justin; Yu, Fuli
22
MODULATION OF EIF2S2 AFFECTS TUMOROGENESIS IN TESTICULAR AND
COLORECTAL CANCERS
Susan Michelle Benton
Advisor: Jason Heaney, Ph.D.-Department of Molecular & Human Genetics
Cancer is one of the deadliest diseases in the world and is the second leading
cause of death in the United States, where about 589,430 Americans are expected to
die of cancer this year. Additionally, about 1,658,370 new cancer cases are expected
to be diagnosed in 2015. Cancer requires the up-regulation of oncogenes to grow and
metastasize. The protein synthesis of several oncoproteins (e.g. Cyclin D1 and Myc) is
highly sensitive to changes in the rate of translation initiation, and key elements of the
translational machinery are up-regulated in cancer. Our preliminary data show that
partial deficiency for the beta subunit of the translation initiation factor eIF2 (Eif2s2)
suppresses testicular germ cell tumors (TGCTs) in 129 mouse models and significantly
reduces both polyp number and size in Apcmin/+ mouse model of colorectal cancer
(CRC) however the mechanisms are unknown. We hypothesize that partial deficiency
of eIF2s2 reduces available eIF2 complex. We propose that modulating the amount of
available eIF2 will affect the hallmarks of tumorigenesis including migration,
proliferation, and cell death. We will use cell lines and mutant mouse models genetically
engineered to reduce Eif2s2 expression to test the effect of Eif2s2 availability on
hallmarks of tumorigenesis. Additionally we propose that reduction of eIF2 availability
suppresses TGCT initiation and intestinal tumorigenesis by altering the protein
synthesis of tumor suppressor proteins and oncoproteins that control tumorigenesis.
We propose that genetically limiting the amount of Eif2s2 limits the pool of eIF2
thus altering translation initiation profiles. We will use cell lines and mutant mouse
models genetically engineered to reduce Eif2s2 expression determine the mechanism
by examining global and oncogene mRNA and protein expression (e.g. Cyclin D1, Myc,
and Bcl).
Contributors: Benton, Susan; Heaney, Jason
23
DETERMINING THE MECHANISM OF PD-1’S ACCUMULATION AT THE CSMAC
DURING SYNAPSE FORMATION
Grant Daniel Bertolet
Advisor: Dongfang Liu, M.D./Ph.D.-Department of Pediatrics
Introduction: PD-1 is an inhibitory surface receptor expressed in B cells, NK
cells, and T cells, and whose activity has been demonstrated to be a critical player in
the insufficiency of the immune system in eradicating chronic diseases and pathogens,
including HIV-1, hepatitis C virus, cancer, and parasitemia. Ligation of PD-1 results in
attenuation of the activating signal cascades generated by the T cell receptor (TCR)
and CD28. Important to this process is the ability of PD-1 to co-localize with the TCR
during synapse formation and ultimately accumulate in the cSMAC. How PD-1 does this
is not known.
Methods: Utilizing cell lines expressing fluorescently-tagged forms of either PD-1
or its ligand, PD-L1, and the Amnis ImageStream imaging flow cytometer, we have
assayed the ability of PD-1 to accumulate at the cell-cell interface between PD-1+
Jurkats or HEK293T cells and PD-L1+ Daudis or K-562 cells. We have also assayed
the ability of PD-1 to accumulate following actin disruption with either Latrunculin A,
Cytochalasin D, or Jasplakinolide, or following truncation of the intracellular domain of
PD-1.
Results & Conclusions: Our research has demonstrated that PD-1 can localize to
the cell-cell interface in a variety of cell lines, including the epithelial cell line HEK293T,
which lacks the TCR. These results indicate that PD-1 can accumulate independently of
the so-called ‘first signal’. However, we have also found that disrupting the actin
cytoskeleton using either monomerizing or polymerizing drugs significantly abrogates
PD-1’s accumulation. To test whether PD-1 requires the cytoskeleton for transport into
the synapse, we transformed HEK293T cells with a truncated form of PD-1 lacking its
intracellular domain. We found that in these cells, PD-1 was still able to accumulate
with PD-L1, thus indicating that cytoskeletal interaction is not a necessary condition for
PD-1’s accumulation at the cell-cell interface.
Contributors: Huang, Yu
24
THE VALIDITY OF THE PRIMARY OPEN ANGLE GLAUCOMA ICD-9 CODE AT THE
MEDVAMC
Kristin Schmid Biggerstaff
Advisor: Donna White, Ph.D./M.P.H.-Department of Medicine
Jennifer Kramer, Ph.D./M.P.H.-Department of Medicine
Administrative databases that employ International Classification of Diseases,
9th Revision, Clinical Modification (ICD-9 CM) codes can be valuable sources of
information for epidemiological, health services, and outcomes research however the
accuracy of these codes in correctly identifying the presence of these conditions is a
potentially limiting factor for studies that use administrative databases. The primary aim
of this study was to evaluate the accuracy of the ICD-9 code for primary open angle
glaucoma (POAG) at the Michael E. DeBakey Veterans Affairs Medical Center
(MEDVAMC) in Houston, Texas. We calculated the positive predictive value, negative
predictive
value,
sensitivity,
specificity
and
percent
agreement.
We identified 19,015 patients seen at the MEDVAMC eye clinic between Oct. 1,
1999 and Oct. 1, 2013 with at least one ICD-9 code for POAG. We randomly selected
229 patients from this group and reviewed the chart to determine if there was sufficient
evidence of POAG. We also identified 55,496 patients seen in the eye clinic during the
study period that did not have ICD-9 codes for POAG and randomly selected 105
patients to confirm the absence of POAG. After exclusion of all patients with missing or
incomplete data and reviewing all the eye notes during the study period for each chart,
the sensitivity was 100%, specificity 89.5%, positive predictive value 65.9%, negative
predictive value 100% and percent agreement 91.3%.
Contributors: Biggerstaff, Kristin S; White, Donna L;Kramer, Jennifer R;Frankfort, Benjamin J.
25
THE ROLE OF INNATE LYMPHOID CELLS IN ASTHMA IMMUNOTHERAPY VIA
MICRO-NEEDLE PATCH
Lynn Bimler
Innate Lymphoid Cells (ILCs) are tissue resident immune cells present in mice
and humans. ILCs are a family of effector cells which are not B or T cells, do not have
rearranged receptors or antigen specificity, and are involved in tissue repair,
homeostasis, and innate immunity. There are three types of ILCs (ILC1s, ILC2s, and
ILC3s), each with their own distinct phenotype and function. Each class of ILCs rapidly
produces a distinct group of cytokines, including IL-5, IL-13, IFN-γ, and IL-17. The
functions and cytokine profiles of ILC classes parallels the balance between TH1, TH2,
and TH17 T cells.1
While TH2 cells and eosinophils have traditionally been considered the active
cell types in asthma, ILCs have recently been implicated in the development of asthma.
ILC1s potentially have a regulatory role in asthma by inhibiting eosinophil induced
airway inflammation. ILC2s have been connected to asthma and lung disease as both
proinflammatory and inducing tissue repair. ILC3s, which produce IL-17, have been
implicated in the development of airway hyper responsiveness (AHR) in mouse models
of obesity-induced AHR.1
Our preliminary data demonstrates that allergen coated micro-needles represent
a novel and effective therapy to treat allergic asthma. Here, we propose to evaluate the
effector functions of ILCs in the skin and lung of healthy, asthmatic, and micro-needle
patch treated mice, in which asthma symptoms were reduced upon micro-needle patch
treatment, to assess the impact of micro-needle therapy on the contributions of ILCs to
allergic asthma. A better understanding of how ILCs contribute to the development of
allergic asthma and our novel micro-needle therapeutic may open the door for improved
and painless asthma treatments.
1Yu, Sanhong, Hye Young Kim, Ya-Jen Chang, Rosemarie H. Dekruyff, and
Dale T. Umetsu. "Innate Lymphoid Cells and Asthma." Journal of Allergy and Clinical
Immunology 133.4 (2014): 943-50.
Contributors: Bimler, Lynn; Smith, Jansen; Song, Amber; Aviles-Padilla, Kevin; Tri Le, Duy;
Angelo, Laura; Gill, Harvinder; Paust, Silke
26
DECIPHERING THE ROLE OF FOXI3 IN EARLY MOUSE DEVELOPMENT
Onur Birol
Advisor: Andrew Groves, Ph.D.-Department of Neuroscience
Foxi3 is a Forkhead transcription factor that is expressed in the preplacodal
region of mice. This region gives rise to craniofacial placodes, such as the neurogenic
trigeminal and epibranchial placodes in addition to otic placode, the precursor to the
inner ear. The division of embryonic ectoderm into neural plate, neural crest, preplacodal region and epidermis is achieved in part by the establishment of different
domains of transcription factors. As the pre-placodal region is induced, Gata3 and Dlx5
genes become restricted to this region, together with definitive markers of the preplacodal region such as, Six1, Six4 and their co-factors Eya1 and Eya2. A posterior
group of pre-placodal cells respond to FGF signaling from the adjacent structures and
differentiate into the otic placode.
Our lab has knocked out Foxi3 in mice and Foxi3 mutants completely lack all ear
structures. Ear induction appears to fail at the very earliest stages, as we see no
expression of markers of the otic placode, such as Pax2, Sox9 or Dlx5 induced in Foxi3
mutants. We have shown that transcription factors downstream of Foxi3 (Gata3, Eya1
and Six1) are absent in mutants, and that FGF signaling is received by the pre-placodal
region in Foxi3 mutant embryos but it fails to execute a program of otic placode
differentiation. Moreover, the neurogenic epibranchial and trigeminal placodes are also
disrupted, although not absent, in Foxi3 mutant mice. Together, our data suggest that
Foxi3 acts at multiple stages of otic placode induction and is necessary for pre-placodal
ectoderm to execute an inner ear program in response to FGF signaling, as well as
being necessary for proper cranial ganglia development.
Contributors: Ohyama, Takahiro; Groves, Andrew
27
THE ROLE OF LIGASE B IN ANTIBIOTIC RESISTANCE AND DNA REPAIR IN
ESCHERICHIA COLI
Truston J Bodine
Advisor: E. Zechiedrich, Ph.D.-Department of Molecular Virology & Microbiology
Richard Hamill, M.D.-Department of Medicine
Antibiotic resistance has become a worldwide health concern and threatens the
use of many advanced medical treatments. Gram-negative pathogens are especially
problematic because of their increasing resistance to multiple classes of antibiotics and
the dearth of new drugs under development to treat these organisms. To better
understand the genetic underpinnings of antibiotic resistance we have been collecting
gram-negative clinical isolates along with the associated clinical microbiological and
patient data. Using both clinical and laboratory-generated data, we took a
representative subset of clinical Escherichia coli isolates and grouped them into pools
base upon their antibiotic resistance phenotypes with particular attention to the
fluoroquinolone class of antibiotics. We then sequenced these pools and compared
each pool to both susceptible and resistant reference genomes to find conserved single
nucleotide polymorphisms (SNPs) associated with fluoroquinolone resistance and
susceptibility (Swick, Evangelista, Bodine et al. PloS ONE 8:e65961). One of the
conserved SNPs associated with fluoroquinolone resistance was found in a NAD+dependent DNA ligase, LigB. Ligase B is one of two NAD+-dependent DNA ligases in
E. coli and has weak in vitro ligase activity (Sriskanda, Shuman. Nucleic Acids
Research 29:4930-4934). In spite of this weak in vitro activity, ligase B is found
throughout the Enterobacteriacae family and maintains a conserved gene order and
orientation among the various enterobacterial species we analyzed. This conservation
along with its newly found tie to antibiotic resistance prompted us to investigate the role
and function of ligase B in E. coli. Deletion of ligB decreased the minimal inhibitory
concentration for only 3 of the 10 antibiotics tested. However, ligB deletion decreased
cell viability upon exposure to hydrogen peroxide and mitomycin C suggesting a role for
ligase B in DNA repair, specifically in the repair of oxidized or alkylated DNA bases. In
addition, overexpression of ligB resulted in a prolonged lag phase in growth that
resembles the growth delay seen when E. coli are exposed to the heavy metal
cadmium. Cadmium nicks DNA and ligB expression is increased with cadmium
exposure. Therefore, we hypothesized that the delay in growth seen with cadmium
exposure may be mediated, at least in part, by ligase B and that ligase B may be
important in the repair of DNA nicks. To test this hypothesis we exposed a ∆ligB strain
and its isogenic parent to increasing concentrations of cadmium and measured growth
over a 72-hour period. The ∆ligB strain showed an increased growth lag time compared
to the isogenic parent suggesting both a possible role for ligase B in the repair of nicked
DNA and that the growth delay seen with cadmium exposure is not completely due to
the increased expression of ligB.
Contributors: Bodine, Truston; Evangelista, Mike; Chang, Huan Ting; Swick, Michelle; Sucgang,
Richard; Hamill, Richard; Zechiedrich, Lynn
28
PAK1 IS A NOVEL TARGET FOR COMBINATION THERAPY IN SCA1
Vitaliy V Bondar
Spinocerebellar Ataxia type 1 (SCA1) is a dominantly inherited fatal neurodegenerative
disorder for which there is no available treatment. It is caused by the expansion of a
trinucleotide repeat that encodes a polyglutamine (polyQ) tract in ATAXIN-1 (ATXN1),
rendering the protein more stable and resulting in its toxic accumulation. Several lines of
evidence suggest that ATXN1 protein levels are crucial to SCA1 pathogenesis. 1)
Overexpression of wild-type ATXN1 is toxic in mice. 2) Loss of phosphorylation of ATXN1 at
Serine776 by a substitution to alanine reduces its stability and its toxicity. 3) Decreasing ATXN1
interacting partners that stabilize ATXN1 such as YWHAE decreases ATXN1 levels and
rescues SCA1 pathology.
Recently we discovered the kinase MSK1 phosphorylates ATXN1 at S776 thus
increasing its levels. Haploinsufficiency of Msk1 and its homolog Msk2 rescues SCA1
pathogenesis and increase survival in mice providing the first potentially druggable target to
treat SCA1. Interestingly, a new emerging strategy in many diseases such as cancer and HIV is
combination therapy. In combination therapy two or more distinct molecular pathways are
targeted resulting in improved treatment outcome. This is done in lower than typical doses
thereby reducing off targets and side effects. Such combination therapy has not yet been
accomplished in a neurodegenerative disease, providing us with an opportunity to test its
effectiveness in SCA1. We hypothesize that reduction of ATXN1 protein levels through two or
more druggable targets will safely and effectively suppress SCA1 pathogenesis. To achieve
this, we search for additional drug targets that regulate ATXN1 levels. To monitor ATXN1 levels
we engineered a human medulloblastoma-derived (DAOY) cell line with a transgene encoding a
glutamine expanded ATXN1 fused with red fluorescent protein (mRFP-ATXN1[82Q]). To
distinguish suppressors that affect ATXN1 protein levels from those that regulate transgene
transcription, internal ribosomal entry site (IRES) followed by yellow fluorescent protein (YFP)
was inserted, resulting in mRFP-ATXN1[82Q]-IRES-YFP transgene. A forward genetics
screening approach was used to identify suppressors of mutant ATXN1 using a pooled shRNA
library screen targeting over 2,500 potentially druggable human genes. shRNA screen was
performed, where cells were sorted based on lowest five percent fluorescent ratio of
mRFP/YFP. In parallel, an independent screen is performed to identify suppressors of ATXN1
in fruit fly in vivo. Through several steps of subsequent validation, measuring transgenic and
endogenous ATXN1 levels by Western blotting, we identified PAK1 as a novel ATXN1
modulator. PAK1 is a serine/threonine kinase that regulates cytoskeleton dynamics.
Knockdown of Pak1 in mouse primary granule neurons with shRNA significantly reduced Atxn1
levels. Pharmacological inhibition of upstream PAK1 activators, SRC1 and RAC1, reduces
PAK1 activity and ATXN1 levels. In SCA1 fly model, PAK1 knockdown suppress eye
degeneration and improves climbing phenotypes. Furthermore, three different PAK1 small
molecule inhibitors reduced ATXN1 levels in concentration dependent manner, providing us a
need drug target for combination therapy in SCA1. Currently, we are investigating molecular
mechanisms of ATXN1 modulation by PAK1 through direct phosphorylation at S776. In the
future, we will perform genetic interaction by generating Pak1 haploinsufficiency in SCA1
mouse model to determine whether halving Pak1 rescues behavioral and pathological deficits.
In combination with MSK1 inhibitors, this study provides an exciting new therapeutic venue to
treat SCA1.
Contributors: Tan, Qiumin; Diaz, Javier; Park, Jeehye; Lu, Hsiang-Chih; Botas, Juan; Zoghbi, Huda
29
CA2+-HANDLING IS ENRICHED FOR ALTERNATIVE SPLICING DURING
POSTNATAL MUSCLE DEVELOPMENT
Amy Elizabeth Brinegar
Advisor: Thomas Cooper, M.D.-Department of Pathology & Immunology
Alternative splicing (AS) increases proteome diversity by producing isoforms that
help define tissue-specificity. During development, subsets of AS events are highly
regulated by modulated activities of RNA binding proteins. Postnatal skeletal muscle
development, encompassing the first 4 weeks after birth in mice, is a dynamic period
with extensive changes at the cellular and molecular levels that remodel the tissue from
fetal to adult function. At birth, the number of myofibers is set, and muscle growth
occurs predominantly by myofiber hypertrophy. Other muscle features such as fiber
type specification, innervation pruning, satellite cell quiescence, and sarcomere
maturation also occur during this period. Although some muscle-specific AS has been
identified during postnatal development, very few isoforms have a known biological
function. To identify novel transcriptome changes, particularly AS events, that affect
muscle physiology, we have performed RNA-seq analysis during multiple time points of
development. RNA-seq analysis demonstrated that AS transitions predominantly occur
separately from gene expression changes, and AS is enriched specifically for Ca2+handling genes during postnatal development. To determine the functional significance
of postnatal AS in vivo, we focused on Ca2+-handling genes of calcinuerin A (CnA).
CnA is the catalytic domain of a Ca2+-dependent phosphatase with known roles in fiber
type specification and hypertrophy in muscle tissue. To determine if spliced isoforms of
CnA have an impact on Ca2+-handling, or CnA’s known roles, we are using antisense
oligonucleotides to re-direct CnA splicing in adult muscle to the embryonic pattern.
Analysis of re-directed splicing will include ex vivo force measurements, Ca2+ sparks,
fiber type composition, and downstream effects on Nfatc signaling. We anticipate that
the results will identify the role of coordinated AS transitions on Ca2+-handling and the
impact on postnatal-to-adult tissue remodeling.
Contributors: Brinegar, Amy; Xia, Zheng; Manning, Kathleen; Loehr, James; Sharpe, Joshua;
Rodney, George; Li, Wei; Cooper, Thomas
30
CHARACTERIZATION OF THE INCOMPATIBILITY OF TWO MUTATIONS THAT
INCREASE CEFTAZIDIME HYDROLYSIS IN THE CTX-M CLASS OF ΒLACTAMASES
Cameron August Brown
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
Antibiotic resistance is a growing concern in the hospital setting, with many
antibiotics becoming obsolete due to the evolution of drug-resistant bacteria. The
introduction of new and novel antibiotics in the clinics leads to the bacteria adapting to
the new antibiotics via mutations in drug resistance genes. The bacterial production of β
-lactamase enzymes that hydrolyze β-lactam antibiotics such as penicillins and
cephalosporins is a common mechanism of resistance. In addition, mutations in these
enzymes can broaden their specificity to include additional β-lactam drugs. The use of
ceftazidime, a potent antibiotic that was once effective against bacteria harboring
enzymes from the CTX-M class of β-lactamases, has led to the selection of mutations
that allow the CTX-M enzyme to hydrolyze this drug.
Two of these mutations, P167S and D240G, are often found in CTX-M
variants that exhibit increased hydrolysis of ceftazidime; however, these two mutations
have never been found together in a naturally occurring variant. It has been shown that
bacteria with an artificially created P167S:D240G CTX-M enzyme have decreased
resistance to ceftazidime compared to the single mutants. This result is surprising since
many mutations exhibit an additive effect when combined. The mechanism behind this
negative cooperativity is not known. Using circular dichroism spectroscopy and steadystate kinetics, we propose that decreased thermal stability and decreased ceftazidime
hydrolysis explain the loss of ceftazidime resistance, as well as the absence of the
double mutant in natural CTX-M variants. These results suggest that the P167S and
D240G mutations represent two mutually exclusive pathways for CTX-M enzymes to
evolve ceftazidime hydrolysis.
Contributors: Brown, Cameron; Patel, Meha; Palzkill, Tim
31
THE ROLE OF NOTCH SIGNALING IN THE VESTIBULAR SYSTEM OF THE INNER
EAR
Rogers Milton Brown, II
The inner ear is a complex and specialized vertebrate structure that mediates
hearing as well as the detection of linear and angular acceleration. The
mechanosensitive properties of the inner ear are dependent on six sensory organs
consisting of sensory hair cells and surrounding supporting cells.
It is thought that Notch signaling plays a key role in at least three stages of inner
ear development: first during neurogenesis to generate delaminating neuronal
precursors, second, to promote the prosensory domains that will give rise to the
sensory organs of the inner ear through lateral induction, and finally to establish the
pattern of hair cells and supporting cells during hair cell differentiation through lateral
inhibition. Previous studies to analyze the role of Notch in these processes targeted key
ligands or receptors in the Notch signaling pathway like Jag1, Jag2, Notch1, and Dll1.
These experiments produced a range of distinct phenotypes which suggests that when
some of these elements are removed from the pathway, other receptors or ligands may
be able to compensate in certain situations.
To address this problem, we are testing the effect of a complete loss of Notch
signaling in the inner ear by conditionally deleting RBPJ, the transcription factor through
which the Notch pathway acts, throughout the entire inner ear. By attacking the
“bottleneck” of the pathway we abolish all Notch signaling in the inner ear. We
examined the morphological development of RBPJ conditional knockouts (RBPJ CKO)
by performing paint fillings and found that as the semi-circular canal plates grow out,
they develop severe truncations. When we examined the vestibular prosensory
domains using Sox2 and Jag1 immunostaining, we found both were greatly reduced as
development progressed. To examine the specific development of the cristae and
maculae, we used RNA in situ hybridizations for BMP4 and Lfng which are expressed in
each respective organ. We found that both genes were expressed at minimal levels or
absent entirely in the vestibular system. This suggests that the RBPJ CKO represents a
more severe loss of Notch signaling in the inner ear than those previously examined.
Contributors: Brown, Rogers II; Basch, Martin; Groves, Andrew
32
STRUCTURAL AND DYNAMIC IMAGING OF PREIMPLANTATION EMBRYOS IN
THE MOUSE OVIDUCT
Jason Christopher Burton
Advisor: Irina Larina, Ph.D.-Department of Molecular Physiology & Biophysics
Infertility rates among U.S. couples are ~15% and present a major health
concern. Nearly 60% of failed pregnancies occur prior to implantation, yet our
investigations of this time period have been limited, due to restricted access to the
reproductive tract (RT) and inadequate research tools. To address this need, we are
developing novel imaging approaches that allow for non-invasive investigations of
preimplantation embryo transport and transport dynamics in vivo, using Optical
Coherence Tomography (OCT). We utilize the mouse, as it is a widely accepted model
of mammalian reproduction, and perform routine surgical procedures to expose the RT
of the live mouse to the OCT scanning head. This allows for in vivo imaging of the
preimplantation embryos as the exit the ovary and progress through the oviduct to the
uterus. Structures of the ovary and oviduct are clearly visible, including developing
follicles at different stages, corpus luteum, longitudinal folds of the mucosal epithelium,
as well as oocytes and cumulus cells suspended in the oviduct. Time-lapse studies
allow for a detailed analysis of the bulk transport of preimplantation embryos and
cumulus cells, and the movement of pre-implantation embryos through the oviduct can
clearly be seen. We have also developed a novel functional-OCT (fOCT) approach that
allows for in vivo analyses of cilia dynamics. Motile cilia are hypothesized to contribute
to the majority of preimplantation embryo transport, yet little is known about these
dynamics in vivo. Previous studies relied on dissection and exposure of the ciliated
layer to the imaging objective, altering the environment of the cilia far beyond natural
conditions. Our in vivo approach allows for non-invasive, quantitative analyses of the
motile cilia activity during different stages of preimplantation pregnancy. To the best of
our knowledge, this is the first time preimplantation embryos have been imaged in vivo
and the first demonstration of fOCT approaches to quantify cilia dynamics. Our results
indicate that OCT is capable of providing detailed three-dimensional reconstructions
and quantitative transport dynamics of the female RT. This study will provide a detailed
analysis of preimplantation embryo transport and can act as a springboard for further
investigations of morphological and dynamic causes of infertility.
Contributors: Burton, Jason; Wang, Shang; Larina, Irina
33
TEM8/ANTXR1 SPECIFIC T CELLS TARGET TUMOR CELLS AND ASSOCIATED
VASCULATURE IN TRIPLE-NEGATIVE BREAST CANCER
Tiara T Byrd
Advisor: Nabil Ahmed, M.D.-Department of Pediatrics
Robert Grossman, M.D.-Department of Neurosurgery
Background and Cancer Significance: Triple-negative breast cancer (TNBC) refers to a
subset of breast cancers that are HER2, estrogen-receptor and progesterone-receptor
negative. Associated with an aggressive phenotype and high incidence of recurrence, and
devoid of the aforementioned receptors, there are currently no targeted therapies for TNBC.
Tumor Endothelial Marker 8 (TEM8) is one of nine gene products up-regulated in the tumor vs.
normal endothelium and is overexpressed in TNBC.
Purpose: To investigate whether TEM8 could serve as a viable target for TNBC therapy.
Methods/Results:
To validate the target antigen we conducted double immunofluorescence staining of a
cohort of 6 TNBC specimens for TEM8 and the pan-endothelial cell marker (CD31). In all 6
cases TEM8 was overexpressed, when compared to normal adjacent breast tissue. TEM8
staining was not restricted to the CD31 positive cells, but also detected in tumor parenchymal
cells. The immortalized TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and
Hs578T) expressed endogenous levels of TEM8 protein as revealed by western blot.
To target TNBC using T cells we designed in silico a novel TEM8-specific chimeric
antigen receptor (CAR) molecule. This CAR consisted of a TEM8 recognition extracellular
domain single-chain variable fragment (scFv) derived from the monoclonal antibody, L2, a
connecting transmembrane chain, followed by CD28, 41BB and CD3-zeta chain intracellular
signaling domains. The TEM8 CAR-encoding DNA construct was synthesized and then
sequence verified. Retroviral transduction was used to integrate the TEM8 CAR transgene in
HEK 293T, then on primary T cells. Over 90% of primary human T cells expressed the TEM8
specific CAR, as indicated by flow cytometry.
TEM8 specific T cells recognized and killed TEM8 positive cells in standard 4 hour 51Cr
release cytotoxicity assays, effectively targeting both TNBC and tumor endothelium cell lines.
Further, TEM8 CAR T cells secreted immunostimulatory cytokines in coculture. In contrast,
there was minimal reactivity against TEM8 negative targets or by non-transduced T cells from
the same blood donor. Adoptively transferred TEM8 specific CAR T cells resulted in a
significant decline in the tumor growth of vascularized orthotopic breast cancer xenografts in a
murine model, compared to non-transduced T cells (p=0.03).
Conclusion: We conclude that TEM8 specific CAR T cells could serve as a tumor and
vascular targeted therapy for TNBC.
Contributors: Byrd, Tiara; Fousek, Kristen; Pignata, Antonella; Szot, Christopher; Bielamowicz,
Kevin; Wakefield, Amanda; Seaman, Steven; Landi, Daniel; Aware, Nikita; Sorensen, Poul;
Koch, Joachim; Wels, Winfried; Fletcher, Bradley; Hegde, Meenakshi; St Croix, Brad and
Ahmed, Nabil
34
THE ROLE OF BILE ACID HOMEOSTASIS AND GUT MICROBIOTA IN
NECROTIZING ENTEROCOLITIS
Lee Thomas Call
Advisor: Douglas Burrin, Ph.D.-Department of Pediatrics
Robert Britton, Ph.D.-Department of Molecular Virology & Microbiology
Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease which
primarily affects premature, low birth weight infants and is characterized by severe
inflammation, a disrupted intestinal barrier, and extensive necrosis. Our group has
established a unique preterm-delivered, formula-fed piglet model of NEC that
recapitulates the main characteristics of human disease. Preterm piglets are delivered
by cesarean-section at ~90% gestation and given total parenteral nutrition for the first 2
days followed by gradual introduction of enteral formula feeding from days 3 to 7.
Piglets are fed formulas matched in nutrient content but containing differing
carbohydrates: lactose, corn syrup solids, or a 1:1 mix. We have performed 16S rRNA
sequencing on samples of ileal and colonic mucosa as well as stomach, ileum, and
colon contents. Bacterial diversity is decreased in NEC vs. healthy piglets and in piglets
fed maltodextrin vs. lactose formula, coinciding with an increase in abundance of
bacteria from the genus Clostridium.
Additionally, results from non-targeted
metabolomic analysis of plasma and intestinal contents suggest that piglets with NEC
have reduced intestinal luminal bile acids and increased plasma bile acids compared to
healthy pigs. These findings suggest a cholestatic condition involving decreased bile
acid secretion into the intestinal lumen and accumulation of conjugated bile salts in the
bloodstream. This observation is interesting given that in addition to their bactericidal
effects and essential role in fat digestion and cholesterol metabolism, bile acids have
also been shown to promote intestinal barrier function and regulate inflammation via
bile acid specific receptors. Current work is focused on determining the relationship
between increased clostridia growth, disrupted bile acid homeostasis, and NEC
pathogenesis.
Contributors: Call, Lee; Stoll, Barbara; Garcia, Selina; Akinkuotu, Adesola; Olutoye, Oluyinka;
Wittke, Anja; Ajami, Nadim; Petrosino, Joseph; Burrin, Douglas.
35
THE ROLE OF NR4A IN ACUTE MYELOID LEUKEMIA
Steven Gregory Call
Advisor: Orla Conneely, Ph.D.-Department of Molecular & Cellular Biology
Acute myeloid leukemia (AML) is a disease in which differentiation of cells down
the myeloid lineage of hematopoiesis is corrupted and a population of immature blasts
predominates in the blood system. Our lab has shown that the NR4A family of orphan
nuclear receptors plays a critical tumor-suppressive role in AML. While NR4As are
highly expressed in hematopoietic stem cells (HSCs) from healthy donors, their levels
are
silenced
in
cells
from
AML patients with varying cytogenetic
backgrounds. Widespread silencing of NR4As suggests that this might be an obligate
step in AML development and that reactivation of NR4As could benefit a wide spectrum
of AML patients. Recent studies in our lab have shown that NR4As are silenced at the
elongation stage of the transcription cycle. Using a chemical genomics strategy, our
group identified the FDA-approved drug dihydroergotamine (DHE) as a potent inducer
of NR4A transcription elongation and expression in human AML cells. DHE inhibits
AML cell growth in vitro and significantly increases overall survival of xenograft AML
mouse models. We now seek to understand the mechanism by which DHE and other
drugs activate NR4A transcription elongation by investigating the signaling pathways,
transcription factors and coregulators that are mediating this response. Recent
preliminary data implicates calcium signaling pathways in DHE-mediated NR4A
induction. We are currently interrogating these pathways to identify the transcription
factors as well as specific branches of calcium signaling that are required for NR4A
induction. Understanding the pathways and transcription factors mediating drug
reactivation of NR4As will likely provide additional components that can be
therapeutically targeted in AML patients.
Contributors: Call, Greg; Duren, Ryan; Nguyen, Loc; Conneely, Orla
36
NOVEL PKG ACTIVATORS: CAN WE SOLVE AN OLD PROBLEM IN CYCLIC
NUCLEOTIDE RESEARCH?
James Christopher Campbell
Advisor: Choel Kim, Ph.D.-Department of Pharmacology
Cyclic nucleotide research began almost 60 years ago. Since then this research
has led to 7 different Nobel Prizes and several “blockbuster” drugs. However, this
progress has been made with an incomplete set of well vetted, chemical tools. The
interpretation of data from biochemical and cell based assays is dependent on a valid
understanding of the mechanism of action (MoA) for the chemical tools being used.
Unfortunately, many of the commonly used cyclic nucleotide analogs used in the study
of NO-cGMP signaling have not been well characterized and often generate
contradicting results. This experimental uncertainty is due to poorly understood MoAs,
as well as cross activation of the cAMP pathway, poor membrane permeability, and the
enzymatic and chemical degradation of the analog. Published erroneous data has
undoubtedly impaired our ability as scientists to understand the role of the NO-cGMP
signaling pathway in models of healthy and diseased states of cancer, hypertension,
and cystic fibrosis.
To address the issues described above, we have decided to employ a
structure based design approach to develop novel, isoform specific activators of cGMP
dependent protein kinase (PKG), a key member of the NO-cGMP signaling pathway.
We have solved X-ray structures of the critical, cGMP binding domains of several
isoforms of PKG and determined structure activity relationships for cGMP and
commonly used analogs. Next, we performed solvent mapping simulations to detect the
presence of other potential binding sites on the protein. From a fragment library we
identified small molecules that bind to sites distinct from cGMP’s. The small
molecule:protein complex’s thermodynamic and kinetic profile is then quantified using
state of the art biophysical instrumentation (SPR and ITC). In order to further validate
the small molecules, their interactions with PKG are simulated prior to co-crystallization.
Finally, these data will be used in the structure-guided synthesis of novel chemical
tools, which will possess a clear MoA, chemical and enzymatic stability, as well as
through a pro-drug method delivery, high membrane permeability.
Contributors: Campbell, James; Sankaran, Banumathi; Young, Damian; Kim, Choel;
37
A CD47-BLOCKING ONCOLYTIC VACCINIA VIRUS FOR CANCER THERAPY
Felicia Cao
Advisor: Stephen Gottschalk, M.D.-Department of Pediatrics
Lisa Wang, M.D.-Department of Pediatrics
Background: The prognosis for patients with advanced stage solid tumors
remains poor and
bio-therapeutics such as oncolytic vaccinia virus (VV) have the potential to
improve outcomes. While VVs have shown promising antitumor activity in early Phase
clinical studies, few patients have been cured. This lack of efficacy is most likely due to
the inability of VVs to kill all tumor cells and/or induce effective anti-tumoral immune
responses. Tumor associated macrophages (TAMs) are key players in promoting tumor
growth and creating an immunosuppressive tumor microenvironment. However, recent
studies have shown that the inherent phagocytic capacity of TAMs can be harnessed to
induce antitumor responses by blocking CD47 on tumor cells and provision of an
opsonization signal using a chimeric molecule that consists of the high affinity
ectodomain of SIRPα fused to the Fc region of IgG4 (SIRPα-Fc). We now
propose to adapt this approach to oncolytic VVs and hypothesize that an oncolytic VV
that is genetically modified to express SIRPα-Fc (SIRPα-Fc-VV) will have enhanced
antitumor activity in comparison to SIRPα-VV. Local production of SIRPα-Fc should be
superior to intermittent direct infusion of the protein, because the concentration of
SIRPα-Fc should be highest at tumor sites and it should reduce the risks of unwanted
side effects associated with systemic administration of SIRPα-Fc.
Methods/Results: We have performed preliminary studies to highlight the
feasibility of the project. We constructed lentiviral vectors encoding either SIRPα-Fc and
GFP or SIRPα and GFP. CD47-positive (OV10 315, B16 and Raji) and a CD47negative (OV10) cell line were transduced to express SIRPα-Fc or SIRPα. After
transduction, greater than 90% of cells were GFP positive and SIRPα and SIRPα-Fc
expression was confirmed by Western blot and/or FACS analysis. We showed in 48
hour coculture assays that M1 and M2 macrophages are able to efficiently kill SIRPαFc-expressing CD47-positiive cells in contrast to SIRPα-expressing CD47-positive cells.
In addition, we assessed the antitumor activity of secretable SIRPα-Fc in two animal
models, Raji lymphoma in NSG mice and B16 melanoma in immunocompetent C57/B6
mice. In both models, mice with SIRPα-Fc-expressing tumors had a survival advantage
over mice injected with SIRPα-expressing and NT tumor cells.
Conclusion: We have demonstrated that CD47-positive tumor cells expressing
SIRPα-Fc are efficiently killed by M1 and M2 macrophages both in vitro and in vivo.
Based on these encouraging findings we have generated an oncolytic VV expressing
SIRPα-Fc and are currently performing preclinical studies. Our studies have the
potential to have a major impact on the treatment of advanced solid tumors with
oncolytic VVs.
Contributors: Cao, Felicia; Rodriguez-Cruz, Tania; Hong, Bang-Xing; Song, Xiao-Tong; Gottschalk,
Stephen
38
PHARMACOLOGICAL HYPOTHERMIA BY TRPV1 AGONISM PRESERVES LONGTERM NEUROFUNCTION AND REDUCES SECONDARY INJURY IN ISCHEMIC
STROKE
Zhijuan Cao
Advisor: Sean Marrelli, Ph.D.-Department of Anesthesiology
Background: Hypothermia shows promising neuroprotection in stroke, but
current cooling strategies limit the translational applications. Transient receptor potential
vanilloid 1 (TRPV1) agonism produces stable pharmacological hypothermia (PH). We
have recently demonstrated that TRPV1-mediated PH provides significant
neuroprotection following ischemic stroke at 24 hours reperfusion. The current study
determined if this protection was sustained through one month of reperfusion and if PH
treatment could reduce secondary injury.
Methods: Sham operated (Sham), stroke with vehicle (Stroke/vehicle), and
stroke with PH (Stroke/PH) groups were designed. Stroke was induced by transient
occlusion of left distal middle cerebral artery plus common carotid artery for two hours
in C57BL/6 mice. PH (33±1ºC) was induced by subcutaneous delivery of TRPV1
agonist (dihydrocapsaicin), initiated 90 minutes after reperfusion and maintained for 8
hours. Several behavioral tests were performed from pre-stroke up to 28 days
reperfusion. Total cortical necrosis and ipsilateral thalamic injury were evaluated after
one month reperfusion.
Results: The foot fault test and negative geotaxis test were sensitive in
demonstrating extended functional deficits after stroke. Stroke/vehicle displayed more
foot faults and a longer latency to turn upwards compared to Sham, demonstrating
prolonged detectable functional deficit. Stroke/PH showed significant improvement in
function compared to Stroke/vehicle. After one month reperfusion, there was no
significant difference in cortical necrosis between stroke groups. Interestingly, a delayed
secondary injury was noted in the region of the thalamus of both groups. Within this
region, Stroke/PH (compared to Stroke/vehicle) showed a 46% reduction in lesion by
Nissl stain and significantly reduced neuron loss (NeuN) and astrogliosis (GFAP).
Conclusions: TRPV1-mediated PH provides sustained improvement in
neurological function and reduced delayed secondary injury following stroke. These
studies support the therapeutic potential for TRPV1 agonism in promoting hypothermia
in the conscious stroke subject.
Contributors: Cao, Zhijuan; Balasubramanian, Adithya; Marrelli, Sean P.
39
DETERMINING DEVELOPMENTAL SIGNALING HETEROGENEITY WITHIN GLIOMA
STEM-LIKE CELL (GSC) POPULATION
Jeffrey Clifford Carlson
The dismal survival rates for those diagnosed with malignant gliomas, in
particular Glioblastoma Multiforme GBM), have remained nearly unchanged for over
sixty years. GBM causes over twelve thousand deaths per year, affecting both children
and adults; Five year survival rates for adults with malignant gliomas are less than five
percent while malignant gliomas are the leading cause of cancer related deaths in
children. Thus, the need for novel targeted therapies for GBM is ever growing.
The difficulty in developing new and effective treatment therapies for GBM is due
in part to the presence of the Glioma Stem-like Cells (GSC) and the enigmatic nature of
the underlying developmental programs that regulate their biology. This GSC population
has been identified as a putative population that affects tumor proliferation and severity,
and relies on developmental signals such as the Notch signaling pathway to maintain
their stem-like nature. While Notch signaling has been implicated in proliferation and
tumorigenic capabilities of the GSC, little has been done in the way of characterizing its
signaling activity within the GSC over the course of tumorigenesis or the nature of its
signaling heterogeneity within the bulk GSC population. Importantly, deciphering the
underlying cellular heterogeneity within the GSC has critical implications for
understanding the biology of these populations, as it has a profound impact on cell
behavior, associated molecular processes, and tumor-resistance. Despite its central
role in tumor pathogenesis, cellular diversity remains very poorly defined in malignant
glioma, and a key “bottleneck” in the development of new therapeutics.
Our preliminary studies indicate that the Notch signaling pathway is differentially
expressed in the GSC, suggesting the existence of diverse cell populations within this
key cell compartment. Describing these distinct populations of GSC will allow further
understanding of malignant cellular development and behavior.
Contributors: Carlson, Jeffrey; Yu, Kwanha; Zhu, Wenyi; Tsang, Yiu-Huen; Grzeskowiak,
Caitlin; Scott, Kenneth; Deneen, Benjamin
40
FEEDING GASTROSTOMY IN CHILDREN WITH COMPLEX HEART DISEASE:
WHEN IS A FUNDOPLICATION INDICATED?
Jennifer Leigh Carpenter
Advisor: Mary Brandt, M.D.-Department of Surgery
Introduction: Malnutrition is common among children with complex heart disease
(CHD) presumably due to a combination of sub-optimal intake and increased energy
expenditure. Feeding gastrostomies are often used to improve the nutritional status of
such patients. Many also have evidence of gastro-esophageal reflux (GER), which
raises the question of the need for a fundoplication at the time of the gastrostomy
insertion. When a gastrostomy is indicated, we generally use the open Stamm method
to avoid abdominal CO2 insufflation that may affect venous return to the heart and
cause hypercapnia and, because GER tends to resolve in infants and young children,
we rarely perform a fundoplication at the time of gastrostomy preferring to use gastrojejunal (GJ) feeding instead for patients with clinically significant GER. Objective: The
purpose of this study was to evaluate the subsequent course of a cohort of infants and
children with CHD following open Stamm gastrostomy without fundoplication. Methods:
We reviewed our experience with all CHD patients who underwent feeding gastrostomy
placement from January 1, 2004 to April 7, 2015. We examined demographic data,
cardiac diagnoses, operative details, post-operative complications, and the need for GJ
feeding and fundoplication. Results: An open Stamm gastrostomy was performed in
111 patients. Five additional patients had a fundoplication at the time of gastrostomy
placement. Median age at surgery 37 weeks (range 3 weeks to 13.7 years); average
weight was 5.3 ±4.9 kg. Forty-eight patients (43%) had single ventricle physiology, 50
(45%) had biventricular physiology and 13 (12%) had cardiomyopathy. Thirty-four
patients (30%) experienced a total of 37 minor complications, which included tube
dislodgement after maturation of the stoma (20), superficial surgical site infection (13),
mechanical failure (3), and bleeding (1). Three patients (3%) experienced a major
complication defined as a need for return to the OR or peri-operative death (<30 days).
Only three patients (3%) required a subsequent fundoplication. At last follow-up
(median 7.2 months, range 2 days-6 years), 56 of 90 surviving patients (62%) continue
to receive gastrostomy feeds, of those 7 (13%) patients continue to require GJ feeds.
Conclusion: Children with CHD tolerate an open Stamm gastrostomy well with minimal
major complications. GER in CHD patients frequently resolves over time and patients
seldom require a subsequent fundoplication. Our experience supports very selective
use of fundoplication in infants and children with CHD who require a feeding
gastrostomy.
Contributors: Carpenter, JL; Soeken, TA; Correa, AJ; Zamora, IJ; Fallon, SC; Kissler, MJ;
Fraser Jr, CD; Wesson, DE
41
ALLELIC EPIGENOME PROJECT
Ivenise Carrero
The Epigenomic allelic imbalance project is part of the Roadmap Epigenome
Project. Epigenomic allelic imbalance can be defined as an allele-specific epigenetic
variation (Younesy et. al., 2013) that may have an impact on gene expression. These
imbalances can occur anywhere in the genome, but in heterozygous loci (“index hets”)
the allelic differences are distinguishable.
In this project we are interested in finding variants with allelic imbalance to study
impact of these allele-specific variation on gene expression. We are looking for variants
in heterozygous state that show allelic imbalance, and compare them across different
tissues and different individuals to determine if the pattern of allelic imbalance is
conserved. We are also looking for potential pathogenic variants in heterozygous state,
and determining which variants have allelic imbalance to study how epigenetics can
affect gene expression. We are conducting analysis using three different datasets
coming from: allele specific binding (ChIP-seq), expression analysis (RNAseq) and
whole-genome methylation data. The above-mentioned data is from 49 complete
epigenomes and whole genome sequencing. To look for potential pathogenic or
functional variants, we are comparing this data with the population datasets-1000
Genomes and Exome Aggregation Consortium (ExAC) to obtain variants with minor
allele frequency (MAF) less than 1%. Further to obtain potential functional variants that
are pathogenic, we are using datasets like ClinVar and Genotype Tissue Expression
dataset (GTEX). We are also using the in silico prediction tool Phast Cons to look for
evolutionary conserved variants.
We have found from the methylation dataset that 98% of the 49 epigenomes
showed a significant association between the presence of epigenomic allelic imbalance
and heterozygous variants with MAF less than 1% genome wide. Moreover, 92%
showed a high degree of association between epigenomic allelic imbalance and
heterozygous variants on evolutionary conserved regions genome wide. The fact that
these variants are more likely to have a functional effect indicates that epigenomic
footprints may provide evidence of functional effect of genomic variants.
Contributors: Onuchic, Vitor; Carrero, Ivenise; Lurie, Eugene; Pawliczek, Piotr; Patel, Ronak;
Rozowsky, Joel; Galeev, Timur; Altshuler, Robert; Kellis, Manolis; Gerstein,Mark; Milosavljevic,
Aleksandar
42
SEIZURE-INDUCED HYPERACTIVATION OF MTOR IMPAIRS MEMORY
FORMATION.
Angela Carter
Advisor: Anne Anderson, M.D.-Department of Pediatrics
RATIONALE: Epilepsy is a chronic neurological disorder characterized by
spontaneously recurrent seizures and comorbid disorders that include deficits in
learning and memory. While current antiepileptic drugs target seizures, they do not treat
the comorbidities and may exacerbate these disorders. Recent studies using acquired
epilepsy models suggest that seizures may induce learning and memory deficits as well
as hyperactive signaling of the phosphoinositide 3-kinase (PI3K) and mechanistic target
of rapamycin (mTOR) cascades. In physiological conditions, activation of these
pathways is sufficient to promote protein synthesis, dendritic spine remodeling, and
learning and memory. However it is unclear as to how the of PI3K-mTOR
hyperactivation underlies behavioral deficits. Here, we evaluate the role of a single
seizure on PI3K-mTOR signaling, dendritic structure, and behavior. In addition, we
tested whether pharmacological inhibition would restore the behavioral deficits.
METHODS: Rats were administered saline (controls) or the chemoconvulsant
pentylenetetrazole to induce a generalized seizure. To determine how a seizure affects
PI3K-mTOR activation, brain tissue was harvested at 3 time-points (1, 3, and 24 hours)
and processed for western blotting (WB) for phospho (P) AKT at T308 and P-S6 at
S240/244. Spine structure was assessed using Golgi staining and tracing. Using a
separate cohort of animals, we tested short-term (STM) and long-term (LTM) memory
using the Fear Conditioning (FC) assay. Finally, we tested whether wortmannin (Wort)
or rapamycin (Rap), PI3K and mTOR inhibitors respectively, would restore the seizureinduced learning and memory deficits.
RESULTS: P-AKT and P-S6 levels were significantly elevated at 1- and 3-hours
post seizure relative to controls (p<0.01). At 24-hours post seizure, both P-AKT and PS6 levels returned to basal levels. Analyses of spine structure reveal that seizures
result in a significantly increased length-to-width ratio and a decreased number of
mushroom spines as compared to controls (p<0.05). When tested for STM in FC, there
were no significant differences in seizure animals relative to controls (p>0.05).
However, only seizure animals exhibited significant deficits in LTM (p<0.0001). Rap did
not restore seizure-induced memory deficits, while Wort partially restored these deficits.
CONCLUSIONS: Our findings reveal that a single generalized seizure is
associated with signaling cascade alterations, changes in spine maturation, and deficits
in long-term memory. In addition, our studies show that inhibition of PI3K signaling may
restore seizure-induced memory deficits. Studies are underway to further characterize
how seizures affect other memory types and the associated molecular signaling and
morphological alterations.
Contributors: Carter, A.N., Lee, W.L., Born, H.A., Dao, A.T., Levine, A.T., and Anderson, A.E.
43
ARHGEF4 INFLUENCES REACTIVE ASTROCYTE FORMATION AND BLOODBRAIN BARRIER RE-ESTABLISHMENT IN A WHITE MATTER INJURY MODEL
Lesley S. Chaboub
Astrocytes are emerging as vital for the physiologic and cognitive functions of the
central nervous system (CNS) and their deregulation is associated with several
neurological (ALS) and inflammatory disorders (multiple sclerosis). Not only are
astrocytes involved in numerous physiological functions including blood-brain barrier
formation/maintenance and metabolic regulation, but they also play essential roles
during CNS injury. Upon injury, astrocytes become “reactive” and form the glial scar, a
structure necessary for repair, especially for the re-establishment of the blood-brain
barrier (BBB). Despite these vital functions, little is known about the formation,
regulation and activity of reactive astrocytes. Understanding mechanisms that regulate
reactive astrocytes will have important implications for multiple disorders.
In a screen to identify regulators of astrocytes formation, we recently
characterized Arhgef4 as a novel gene expressed in both glial precursor cells and
mature astrocytes. Arhgef4 is a guanine exchange factor for the Rho GTPase family of
protein and has been shown to impact cell cytoskeleton in other systems. While not
essential for CNS development, Arhgef4 is expressed in Human Multiple Sclerosis
lesions, suggesting that Arhgef4 might play a significant role during CNS injuries. We
used lysolecithin injection into the ventral white matter region of the spinal cord as a
model for White Matter Injury, and multiple sclerosis in particular. Lesions performed on
Arhgef4 germline knock-out (KO) animals appear to have delayed remyelination when
compared to heterozygous littermate animals. Since Arhgef4 is expressed in OPCs, it
was necessary to assessed whether loss of Arhgef4 KO affected oligodendrocyte
differentiation. In vitro cultures of OPCs from Arhgef4 KO and HET animals showed no
difference in differentiation potential, implying that defective remyelination is probably
due to astrocytes, and reactive astrocytes especially. We looked into BBB reestablishment after lysolecithin injection and found that blood was still present in lesions
from Arhgef4 KO animals only. Overall, lesions from KO animals are bigger and take
longer to repair.
Ongoing studies are focusing on assessing whether Arhgef4 is essential in
other CNS injuries, such as photothrombotic stroke. Further work will also center on
better understanding how Arhgef KO astrocytes differ from Arhgef4 HET. Guanine
exchange factors, such as Arhgef4, are potentially good target for therapies since they
affect critical cellular pathways while being tissue specific. This study specifically
identified Arhgef4 as a critical gene in reactive astrocyte formation during white matter
injuries.
Contributors: Chaboub, Lesley S.; Lee, HyunKyoung; Manalo, Jeanne M.; Deneen, Benjamin
44
THREE-DIMENSIONAL HEAD-DIRECTION ENCODING IN MICE
Henry Xin Cham
Advisor: J. Dickman, Ph.D.-Department of Neuroscience
Head direction cells are neurons found in the limbic system of mammals that fire
when the animal’s head is pointed at a specific direction, independent of location.
These neurons seem to encode the animal’s head orientation in the horizontal plane,
resembling an internal compass. We examined whether head direction cells in mice
encode three-dimensional directional information by manipulating the animal’s
orientation while measuring neural activity. We found that head direction cells have
preferred directions in three-dimensional space. The preferred direction is consistent in
the dark when no visual cues are present suggesting a contribution from the gravity
signal in the vestibular system.
Contributors: Cham, Henry; Shinder, Michael; Laurens, Jean; Kim, Byounghoon; Angelaki,
Dora; Dickman, J. David
45
ACTIVATION AND REGULATION OF CALCIUM-ACTIVATED CHLORIDE
CHANNELS DURING ROTAVIRUS INFECTION
Alexandra L. Chang-Graham
Advisor: Joseph Hyser, Ph.D.-Department of Molecular Virology & Microbiology
With over 100 million cases each year, rotavirus (RV) is a major cause of acute
gastroenteritis in children worldwide. RV causes secretory diarrhea, which results from
overstimulation of apical chloride (Cl-) channels on intestinal epithelial cells, and can
lead to life-threatening dehydration. Though bacterial toxins such as cholera toxin are
well studied, less is known about how RV causes diarrhea through elevation of cytosolic
calcium (Ca2+) and activation of Ca2+-activated chloride channels (CaCCs). The RV
nonstructural protein 4 (NSP4) is responsible for increasing cytosolic Ca2+ by
functioning as a viroporin that leaks Ca2+ from the endoplasmic reticulum (ER) into the
cytosol. The depletion of ER Ca2+ activates stromal interaction molecule 1 (STIM1), an
ER Ca2+ sensor which activates processes for ER Ca2+ repletion called store-operated
Ca2+ entry (SOCE). Previous studies have shown that CaCCs, such as anoctamin1
(Ano1), are strongly activated through depletion of Ca2+ from the ER, which occurs
during RV infection, but CaCC activation during RV infection has not been established.
To determine if the CaCC Ano1 is activated during RV infection, I used a halidesensitive YFP (HSYFP) to measure RV-mediated Cl- channel activation, which detects
Cl- channel activity by measuring the quench in YFP fluorescence due to iodide influx
through open Cl- channels. Using HEK293 cells stably expressing mouse Ano1
(mAno1) and HSYFP, I observed robust HSYFP quench in RV-infected cells, indicating
that mAno1 is activated during RV infection. Furthermore, HSYFP quench is attenuated
when using pharmacological inhibitors of mAno1 and SOCE, supporting that Ca2+
entry through SOCE channels is important for the activation of mAno1 and Clsecretion. These are the first direct evidence for CaCC activation in RV-infected cells.
Furthermore, the HSYFP assay represents a robust and sensitive assay to identify the
Cl- channels activated by RV infection. Since Cl- secretion is the primary cause of RV
diarrhea, the elucidation of the molecular mechanisms regulating CaCC activation is a
key to developing life-saving anti-diarrheal drugs.
Contributors: Chang-Graham, Alexandra L.
46
HEMATOPOIETIC STEM CELL PROLIFERATION AND DIFFERENTIATION IS
REGULATED BY ESTROGEN
Richard H Chapple
Advisor: Daisuke Nakada, B.A.Sc.-Department of Molecular & Human Genetics
Hematopoietic stem cells (HSCs) are a rare population of bone marrow cells that
have the ability to generate every blood cell type. These cells also have the ability to
self-renew - a process in which cell division results in additional stem cells. These two
properties cooperate to maintain life-long hematopoiesis, and are exploited in the clinic
to regenerate bone marrow in irradiated patients. Thus, understanding the factors that
govern these properties has the potential to facilitate enhanced stem cell therapies.
Our lab has recently discovered that the female sex hormone estrogen regulates HSC
function. We demonstrated that HSCs from female mice are more proliferative than
their male counterparts. We performed qPCR to determine the expression of steroid
hormone receptors, and found that HSCs exclusively express the hormone receptor
Estrogen Receptor alpha (ERα). Conditional deletion of ERα in the hematopoietic
system revealed that estrogen increases HSC proliferation in an ERα-dependent
manner. Transplantation of HSCs from estrogen-treated mice into lethally irradiated
recipients revealed that estrogen also influences lineage fate decisions following HSC
division. We observed that estrogen-treated HSCs show enhanced myeloid and
platelet reconstitution after transplantation. In order to determine the molecular
underpinnings of estrogen stimulation on HSCs, we performed RNA-Seq on HSCs from
ERα or ERαfl/fl;Mx1-Cre treated either with vehicle or estrogen. Bioinformatic analyses
were performed to identify genes that were induced by estrogen treatment, yet showed
no effect in similarly treated ERαfl/fl;Mx1-Cre animals. The results of this analysis
revealed that Thrombospondin-1 (Thbs1) is massively upregulated in HSCs after
estrogen treatment and is rescued by ERα deletion. Thbs1 is a matricellular protein
that incorporates into the extracellular matrix to facilitate cellular adhesion and stimulate
downstream signaling cascades. One biological process regulated by Thbs1 is nitric
oxide (NO) signaling, which has been shown to regulate HSC function. We confirmed
that NO signaling is activated in HSCs by staining with the cell permeable fluorescent
NO probe, DAF-2 DA. We are currently testing if Thbs1 and/or NO is sufficient to
induce the HSC phenotypes observed after estrogen treatment.
Contributors: Chapple, Richard; Lin, Angelique; Kitano, Ayumi; Nakada, Daisuke
47
PARTIALLY FOLDED HUMAN ΓD-CRYSTALLIN CAUSES PARTIAL CLOSURE OF
CIS-RING AND SYMMETRY-BREAKING IN A GROUP II CHAPERONIN
Bo Chen
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Chaperonins are a class of proteins that play an essential role in protein folding
for all cells from Bacteria, Archaea and Eukaryotes. Type II chaperonin from the
archaea Methanococcus Marapaludis (Mm-Cpn), a double-ring hexadecamer complex,
is able to assist refolding of human γD-crystallin to its native conformation. Human γDcrystallin is a lens protein, which is associated with the onset of cataract when it partially
unfolds and aggregates. Here, we used cryo-electron microscopy (cryo-EM) single
particle analysis to resolve the structure of Mm-Cpn and human γD-crystallin during the
initial recognition step. The cryo-EM map of Mm-Cpn alone showed both rings in an
open conformation that resembles the apo state. Cryo-EM images of the Mm-Cpn and
human γD-crystallin reaction mixture were classified to yield two structures: an apo one
and one presumably with substrate. The structure with putative substrate had one ring
open with 8-fold symmetry while the other ring was less open and organized as a
tetramer of dimers. While the substrate density was not directly seen, the 3D variance
analysis supports the presence of a conformationally variable substrate inside Mm-Cpn
in the map.
Contributors: Chen, Bo; Sergeeva, Oksana; Schmid, Michael; Ludtke, Steve; King, Jonathan;
Chiu, Wah
48
GENETIC INHIBTION OF mTORC2 RESECUED PTEN-RELATED AUTISM AND
SEIZURE PHENOTYPE IN MOUSE
Chien-Ju Chen
Advisor: Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
Phosphatase and Tension homolog(PTEN) is known to be a negative
regulator of PI3K/AKT/mTOR pathway. In human patients, individuals carrying PTEN
mutations display a variety of neurological disorders including epilepsy and autism.
PTEN loss-of-function leads to increases of both mTORC1 and mTORC2 signaling.
Due to the lack of specific inhibitor, how activation of the two complexes contribute to
neurological disorders associated with PTEN mutation remains unclear. To address this
question, we generated forebrain neuron-specific PTEN; raptor double knockout
(PTEN; raptor fb-DKO) and PTEN; rictor double knockout(PTEN; rictor fb-DKO) mice to
inhibit mTORC1 and mTORC2 respectively in PTEN KO background. PTEN fb-KO mice
displayed severe seizures and had short lifespan. Genetic inhibition of mTORC2, but
not mTORC1, was able to ameliorate behavioral and EEG seizure activity and double
the lifespan of PTEN fb-KO. Behaviorally, consistent with other CNS specific PTEN KO
mouse models, our PTEN-fb KO mice showed ASD-like social and cognitive deficit. In
contrast,
PTEN; rictor fb-DKO performed normally in both cognitive and social behavior
assays. Electrophysiology recording showed that while conditional PTEN fb-KO and
PTEN;rictor DKO neurons both exhibited excessive mEPSC amplitude in hippocampus,
pten;rictor DKO neurons also displayed higher mIPSC frequency compared with control
and
PTEN
fb-KO.
Molecularly, we found that among all the known downstream substrates of mTOC2,
only AKT phosphorylation is increased in PTEN fb-KO hippocampus, indicating deletion
of PTEN caused an ATK-specific change on mTORC2 signaling pathway.
In summary, our study suggests a crucial role of mTORC2 signaling in PTENloss of-function related seizure and ASD–like behavior. More works need to be done to
further elucidate the cellular and molecular mechanisms on how mTORC2 effects the
pathogenesis PTEN-related neuronal disorder.
Contributors: Chen; Chien-Ju; Beate, Dillon; Lucero, Rocco; Viana Di Prisco, Gonzalo; CostaMattioli, Mauro
49
STUDY THE ROLE OF TMPRSS9 IN INTELLECTUAL DISABILITY AND AUTISTIC
DISORDER
Chun-An Chen
Advisor: Christian Schaaf, M.D./Ph.D.-Department of Molecular & Human Genetics
Our lab has identified compound heterozygous nonsense mutations in a novel
gene, TMPRSS9, in a patient with developmental regression at 2.5 years of age,
leading to severe intellectual disability, autistic disorder, mutism, and ataxia. TMPRSS9
encodes for polyserase-1, a transmembrane serine protease. However, the
physiological role of TMPRSS9 is unknown and its endogenous substrates have not
been identified yet. We hypothesize that TMPRSS9 is essential for the development
and/or maintenance of the central nervous system, and that loss-of-function mutations
in TMPRSS9 cause developmental regression leading to intellectual disability and
autism spectrum disorder. A constitutive knockout mouse model of TMPRSS9 was
generated by removal of exon 2. This causes a partial deletion of the transmembrane
domain and a frameshift at the transition of exon 1 to exon 3. Quantitative PCR
analysis verified the knockout in all tissues tested, including brain. A battery of
behavioral tests was used to evaluate social approach, learning and memory, anxietylike responses, and motor activity of 3.5-month-old homozygous knockout mice. The
results indicate that knockout mice display deficits in both social interest (three-chamber
test) and social recognition (partition test). Amygdalae, cerebella, and hippocampi of
3.5-month-old knockout mice were dissected for identification of substrates of
polyserase-1 via a proteomics approach, TAILS (terminal affinity isotope labeling of
substrates). Validation of potential substrates will be attempted by immunostaining and
western blot of the cleaved protein products. Among the candidates, synapsin 1 (Syn1)
and vacuolar protein sorting-associated protein 13A (Vps13a) have been implicated in
decreased social investigation, based on the respective knockout mouse models.
Contributors: CHEN, CHUN-AN; YIN, JIANI; KLEIN, THEO; OVERALL, CHRISTOPHER;
SABO, ANIKO; MARTIN ANDREAS; RICHARD GIBBS, ZOGHBI, HUDA; SCHAAF,
CHRISTIAN
50
LOSS OF FRATAXIN CAUSES IRON TOXICITY, INDUCES SPHINGOLIPID
SYNTHESIS, AND PROMOTES NEURODEGENERATION VIA PDK1 IN FLIES AND
MICE
Kuchuan Chen
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Mutations in Frataxin (FXN) cause Friedreich ataxia (FRDA), and increased
reactive oxygen species (ROS) are thought to play a primary role in disease
pathogenesis. In contrast, we describe a pathogenic pathway that triggers
neurodegeneration independent of elevated ROS. Loss of frataxin homolog (fh) in
Drosophila leads to iron accumulation which in turn induces sphingolipid synthesis and
activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer
factor-2 (Mef2). We show that degeneration is effectively delayed by reducing iron
toxicity or sphingolipids/Pdk1/Mef2 pathway. Moreover, removal of Fxn in the mouse
brain triggers iron accumulation and PDK1 activation, suggesting that the mechanisms
observed in flies are conserved in vertebrates. Our results suggest that iron-induced
sphingolipid/PDK1 signaling plays a major role in FRDA pathogenesis.
Contributors: Chen KC; Lin G; Haelterman N; Ho TS; Li T; Li Z; Duraine L; Graham B; Jaiswal
M; Yamamoto S; Rasband MN; Bellen HJ
51
GFI1 IS A PUTATIVE TUMOR SUPPRESSOR IN COLORECTAL CANCER
Min-Shan Chen
Advisor: Noah Shroyer, Ph.D.-Department of Pediatrics
Background: Colorectal cancer (CRC) is the third most common and the third
leading cause of cancer death in the United States. Growth Factor-Independent 1
(GFI1) is a zinc finger transcriptional repressor implicated in the differentiation of
secretory precursors into goblet and Paneth cells in the intestinal epithelium. Previous
studies suggested that gain or loss of function of Gfi1 leads to cancer, such as
leukemia and neutropenia. However, the role of Gfi1 in CRC is still largely unknown.
Recent genome-wide association studies from The Cancer Genome Atlas have
revealed that reduced mRNA expression of Gfi1 is associated with aggressiveness in
CRC, indicating Gfi1 may have a tumor or metastasis suppressive role in CRC. Our
preliminary analysis of CRC mRNA expression highlights that Gfi1 expression is
reduced in colorectal carcinoma compared to benign adenomas and noncancerous
tissue, suggesting that Gfi1 may be involved in limiting tumor progression. Therefore,
we hypothesize that Gfi1 functions as a tumor suppressor and inhibits the metastasis of
colorectal cancer.
Methods: We analyzed the expression of Gfi1 using immunohistochemical
staining in human colon cancer tissues. In order to determine the role of Gfi1 in vitro
and in vivo, we established doxycycline-inducible Gfi1 in human colorectal cancer cells.
Cell apoptotic assay and cell viability assays were used to examine Gfi1-expressing
cells and control cells. We also performed a mouse model of hepatic metastasis of
CRC by spleen implantation of a doxycycline-inducible Gfi1 human colorectal cancer
cells in immunocompromised mice. Gfi1 is induced by the administration
of doxycycline via feeding mice doxycycline in drinking water and chow.
Results: Immunohistochemical staining indicates that Gfi1 expression is
significantly decreased in malignant colonic epithelium compared with adjacent normal
colonic epithelium. MTT assay indicates that Gfi1 expression decreases colorectal
cancer cell growth. Moreover, cell apoptotic assay shows that transient expression of
Gfi1 in human colorectal cells results in increased apoptosis. In contrast to previous
studies, we found that that re-expression of Gfi1 in CRC cells promotes tumor
metastasis to liver upon intrasplenic injection. In the future, we will analyze Gfi1
expression and the role of Gfi1 in metastatic liver tumor derived from intrasplenic
injection of CRC cells. Subcutaneous tumor model will be valuable to test whether Gfi1
promotes tumor proliferation in vivo. In addition, it is important to examine the role of
Gfi1 in colonic epithelium and immune cells. Future studies for identifying the molecular
function of Gfi1 in CRC may improve our current understanding of tumor metastasis
and benefit to therapeutic strategies.
Contributors: Chen, Min-Shan; Chen, Xi; Williams, Christopher S.; Shroyer, Noah F.
52
DE NOVO MODELING IN CRYO-EM DENSITY MAPS WITH PATHWALKING
Muyuan Chen
Advisor: Steven Ludtke, Ph.D.-Department of Biochemistry & Molecular Biology
As Cryo-Electron Microscopy(Cryo-EM) can now frequently achieve near atomic
resolution, accurate interpretation of these density maps in terms of atomistic detail has
become paramount in deciphering macromolecular structures' function. However, there
are few software tools for modeling protein structure from Cryo-EM density maps in this
resolution range. Here we present an extension of our original Pathwalking protocol,
which can automatically trace a protein backbone directly from a near-atomic resolution
(3 - 6 A) density map. The original Pathwalking approach utilized a Traveling Salesman
Problem solver for backbone tracing, but manual adjustment was still required during
modeling. In the new version, human intervention is minimized and we provide a more
robust approach for backbone modeling. This includes iterative secondary structure
identification, termini detection and the ability to model multiple subunits without prior
segmentation. Overall, the new pathwalking procedure provides a more complete and
robust tool for annotating protein structure function in near-atomic resolution density
maps.
Contributors: Chen, Muyuan; Baldwin, Philip; Ludtke, Steven; Baker, Matthew
53
THE RAC-GEF TIAM1 REGULATES SYNAPSE DEVELOPMENT AND FEAR
LEARNING IN MICE
Jinxuan Cheng
Advisor: Kimberley Tolias, Ph.D.-Department of Neuroscience
Neurons communicate with one another through specialized sites of contact
called synapses. Most excitatory synapses are located in dendritic spines. Synapses
are plastic and can strengthen or weaken in response to neuronal activity, which is
thought to be important for learning and memory. The Rho family small GTPase Rac1
is a key regulator of nervous system development. Previously, our lab identified the
Rac-GEF Tiam1, a Rac activator, as a critical regulator of synapse development in
cultured neuron. In humans, altered Tiam1 expression is linked with depression and
Down’s syndrome. However, nothing is known about its role in vivo. Therefore, we
generated Tiam1 knockout (KO) mice.
Loss of Tiam1 results in mice with reduced spine density, simplified dendritic
arbors and fewer functional synaptic transmission. These results suggest that Tiam1 is
required for dendritic and synaptic development in vivo. Mice lacking Tiam1 also exhibit
hypoactivity, decreased sensory-motor gating, defects in social behavior, as well as
enhanced fear learning and decreased depressive behavior. Tiam1 KO mice also
display increased level of NR2A and NR2B subunits from NMDA receptors, which may
result in increased synaptic plasticity in the mice, since NMDA receptors play crucial
roles in learning and memory related synaptic plasticity. Tiam1 plays an important role
in receptor endocytosis, so I’m currently investigating whether loss of Tiam1 results in
altered NMDA receptor trafficking, and how this links to enhanced fear learning.
Contributors: Cheng, Jinxuan; Niu, Sanyong; Jiang, Xiaolong; Firozi, Karen; Tolias, Kimberley
54
COMBINATION THERAPY MAXIMIZES COGNITIVE RECOVERY IN A MOUSE
MODEL OF ALZHEIMER’S DISEASE
Angie Chi An Chiang
Advisor: Joanna Jankowsky, Ph.D.-Department of Neuroscience
Antibodies against various A( epitopes have been shown to reduce amyloid
levels in animal models of Alzheimer’s Disease and many are being actively pursued in
clinical trials. Despite this progress, we know little about which species of the peptide soluble, insoluble, or both – should be targeted for maximal benefit. More specifically,
is slowing the aggregation of newly synthesized A( sufficient for cognitive recovery, or
must we also remove existing deposits to achieve full functional rescue? Using the
controllable tet-off APP transgenic model of AD, we have shown that combining two
complementary approaches for A( reduction – using passive immunization to sequester
existing peptide, while simultaneously suppressing transgenic APP to reduce further A(
production – improves neuropathological outcome over either treatment alone. In this
model, we found that both monotherapies forestalled further plaque deposition, while
combination treatment not only halted accumulation but also cleared existing deposits.
In the current study, we tested whether combination treatment and concomitant plaque
clearance provide any cognitive benefit over plaque stasis through passive
immunization or A( suppression alone.
Following 9 weeks of treatment, animals
underwent behavioral testing to examine spatial learning, working memory, and
associative memory. Our findings suggest that combination treatment produces greater
improvement in cognitive performance than either passive immunization or A(
suppression alone. These results may guide development of future therapeutics by
demonstrating that greatest cognitive benefit is attained when all forms of A( are
reduced.
Contributors: Chiang, Angie C.A.; Jankowsky, Joanna L.
55
THE NUCLEAR RECEPTOR LRH-1 MEDIATES BENEFICIAL LIPOTROPIC EFECTS
OF CHOLINE SUPPLEMENTATION AND DELETERIOUS EFFECTS OF
METHIONINE AND CHOLINE DEFICIENT DIET IN RESPONSE TO ALTERED
LEVELS OF AN ENDOGENOUS AGOINST
Sungwoo Choi
Advisor: David Moore, Ph.D.-Department of Molecular & Cellular Biology
Type 2 diabetes is a metabolic disease characterized by a high level of blood
glucose due to insulin insensitivity. This disorder is tightly correlated with increased fat
accumulation in the liver. Our lab previously reported that the synthetic LRH-1 agonist
Dilauroyl Phosphatidylcholine (DLPC) potently reduces hepatic steatosis and promotes
whole body insulin sensitivity (PMID: 21614002). Similar but less effective antidiabetic
effects were observed with another phospholipid that is not a direct LRH-1 agonist,
Dipalmitoyl Phosphatidyl choline (DPPC). Interestingly, antidiabetic effects of DPPC still
require LRH-1. This effect is reminiscent of much older studies demonstrating a
“lipotropic” effect of choline and methionine, which protected high fat fed rats against
fatty liver by increasing the methyl pool size (PMID: 16994301). Our preliminary data
shows that the lipotropic effect of choline is lost in Lrh-1 liver specific knockout (LKO)
mice. Conversely, it is well recognized that a methionine and choline deficient diet
(MCD) induces fatty liver and inflammation in rats and mice by decreasing the methyl
pool size. Previous work from our lab indicates that Lrh-1 LKO mice are more resistant
to MCD induced inflammation. This is due in part to sparing methyl pool size changes
by decreasing expression of multidrug resistance-2(Mdr-2) that excretes choline into
bile. More broadly, gene array results show a significant overlap between standard diet
(chow) fed Lrh-1 knockout mouse livers and MCD fed wild type livers, suggesting that
the MCD may decrease LRH-1 activity. Thus, we propose that LRH-1 is the target for
the beneficial lipotropic effects of choline, the deleterious effects of the MCD. The
simplest prediction is that the lipotropic treatments increase levels of an endogenous
agonist, while the MCD could either deplete such an agonist or induce levels of an
endogenous antagonist. Testing this prediction requires identification of the still
unknown endogenous LRH-1 ligands. We have developed a strategy for this based on
rapid purification of a tagged human LRH-1 expressed in mouse liver, followed by mass
spectrometric characterization of bound small molecules.
Contributors: Choi, Sungwoo; Wagner, Martin; Kim, Kang; Zhou, Ying; Kim, Misun; Lee, Jae;
Moore, David
56
THE ROLE OF CUL5 IN MODULATING DRUG SENSITIVITY IN TRIPLE-NEGATIVE
BREAST CANCER
Hsiang-Ching Chung
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
Background: Triple-negative breast cancer(TNBC) is an aggressive subtype of
breast cancer that is recalcitrant to target therapies. Previous studies in our lab
uncovered the co-dependency of TNBC survival and progression on multiple receptor
tyrosine kinases (RTKs). Notably, combined inhibition of these RTKs with tyrosine
kinase inhibitors (TKis), sunitinib and crizotinib (or S+C) confers regression on many
independent patient-derived xenograft (PDX) models of TNBC in vivo. However, not all
TNBC PDX models are sensitive to S+C. To better understand the molecular
determinants of S+C sensitivity and resistance, we performed a genetic (RNAi) screen
for modifiers of S+C response in multiple TNBC models. CUL5 was a prominent
candidate from these multiple genetic screens. We validated that knockdown of CUL5
greatly enhances cell fitness upon S+C treatment, suggesting CUL5 function is a critical
determinant of TNBC drug sensitivity. The characterization of how CUL5 controls the
response of TNBCs to this new therapeutic regimen may enable us not only to
categorize which patients should undergo this targeted therapy, but also overcome the
drug resistance and resulting in durable response.
Experimental design and methods: Competition assay and barcode abundance
qPCR assay are used to validate whether loss of CUL5 confers S+C resistance in
TNBC cells in vitro and in vivo, respectively.
Results: By competition assay and barcode abundance qPCR assay, we
validated that CUL5 depletion gives TNBC cells growth advantage in the presence of
S+C both in vitro and in vivo. Given the ability of CUL5 in regulating multiple RTK
signaling, we further showed that CUL5-depleted cells have higher activating
phosphorylation of some RTK and effector kinases, suggesting that loss of CUL5
confers S+C resistance in TNBC cells potentially by restoring important cellular survival
signaling.
Conclusion: The characterization of how CUL5 controls TNBC drug response
represents an unprecedented opportunity to investigate not only the role of
ubiquitylation in modulating drug response, but also the crosstalk between
ubiquitination and RTK signaling. Our finding implies that dampening the alternative or
reactivated signaling on which CUL5-depleted cells rely may allow us to overcome S+C
resistance in TNBC.
Contributors: Chung, Hsiang-Ching; Sun, Tingting; Nair, Amritha; Kurley, Sarah
57
ANDROGEN RECEPTOR PROMOTES TAMOXIFEN RESISTANCE THROUGH EGFR
PATHWAY ACTIVATION IN ESTROGEN RECEPTOR Α-POSITIVE BREAST
CANCER CELLS WITH LOW RHO GDIΑ EXPRESSION
Andrew Mark Robinson Ciupek
Advisor: Suzanne Fuqua, Ph.D.-Department of Medicine
Background: Microarray analysis of metastatic tumors taken from patients with
Tamoxifen (Tam) resistant estrogen receptor (ER) (+ breast cancer showed lower expression of
Rho GDI( and higher expression of the androgen receptor (AR). Based on this analysis we had
previously developed a novel model of Tam resistance (TamR) and involving shRNA
knockdown of Rho GDI( and a model of TamR involving AR overexpression. Although it is
known that AR is frequently expressed along with ER( in breast tumors, AR’s role in resistance
or metastasis is unclear. Here we explore the role of AR in these processes in the Rho GDI(
knockdown model. Materials and Methods: Viral shRNA transduction was used to knock-down
expression of Rho GDI( in the ER(-positive breast cancer cell lines MCF-7 and ZR-75-B.
Western blot analysis was used to examine activation of the AR, ER(, EGFR, and MAPK
pathways. The effects of the AR inhibitor Enzalutamide (MDV3100), the EGFR inhibitor
Gefitinib (Gef), and Tam on anchorage independent growth were measured in soft agar assays
and phosphorylation in western blot assays. In luciferase transcriptional reporter assays, cells
were transfected with both an ERE-Luciferase reporter plasmid to measure ER( activity and βgal reporter plasmid for normalization. Pairwise T-tests (corrected for multiple comparisons
using the Holm method) were used for statistical analysis of data from aforementioned assays.
Interaction between AR and EGFR was analyzed by proximity ligation assay (PLA). Reverse
Phase Protein Array (RPPA) analysis was used to look for differential protein expression
between knock down and control cells under basal conditions. Microarray analyses on cells
treated with Tam for 24 hours were used to explore pathways promoting TamR. The online
mSigDB program (http://www.broadinstitute.org/gsea) was used to identify overlap with
published gene sets, and Gene Set Enrichment Analysis was used to statistically analyze these
overlaps. Results: Both MCF-7 and ZR-75-B Rho GDI( knockdown cells overexpresed AR.
Knock down cells showed increased activation of EGFR and MAPK signaling. AR and EGFR
had increased interaction in knock down cells. Tam stimulated ER( transcriptional activity in
knock down cells was blocked by MDV3100 and Gef. Tam stimulated proliferation of knock
down cells was also blocked by MDV3100 and Gef. Discussion: Since AR was significantly
overexpressed with occurrence of TamR and increased ER( transcriptional activity and AR
agonists/inhibitors modulated these phenotypes, we hypothesize that AR may play a role in
TamR via effects on ER( transcriptional activity. EGFR signaling was increased following Tam
treatment, EGFR inhibition blocked TamR, and EGFR inhibition blocked phosphorylation of
ERα, suggesting that EGFR may collaborate with AR to promote TamR.
Contributors: Ciupek, Andrew; Rechoum Yassine; Gu, Guowei; Gelsomino, Luca; Beyer,
Amanda; Brusco, Lauren; Covington, Kyle R.; Tsimelzon, Anna; Fuqua, Suzanne A. W.
58
REDIRECTING HEME DEGRADATION IN ESCHERICHIA COLI FOR THE PURPOSE
OF MONITORING HEME UPTAKE AND BIOSYNTHESIS
Justin Ryan Clark
Advisor: Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
Nearly all bacterial pathogens must acquire the element iron from their host
during infection, as this nutrient is essential for many cellular processes that fuel life. As
such, bacteria have evolved systems that mediate the acquisition and transport of iron,
including secreted and surface proteins that bind heme, the abundant iron-porphyrin
cofactor of the serum protein hemoglobin and reservoir for 75-80% of all iron within the
host. Methods to quantitatively measure the intake, processing, and/or biosynthesis of
heme in cells are laborious, involve complex chemistry, or require the use of
radioactivity. Such drawbacks have limited our ability to interrogate the mechanistic
steps of heme transport and breakdown in bacterial pathogens. We hypothesized that
heme uptake and biosynthesis in bacterial systems could be quantitatively measured
using a fluorescent approach by redirecting intracellular heme to biliverdin IXα (BV; the
first product of heme catabolism), using human heme oxygenase 1 (HO1), thereby
allowing it to bind infrared fluorescent protein 1.4 (IFP), which uses BV as a
chromophore. We show here that a pathotype of E. coli (enterohemorrhagic E. coli, or
EHEC, a prominent cause of deadly diarrhea and kidney toxicity), when expressing the
IFP-HO1 heme sensor yield fluorescence in the near-infrared region when grown in the
presence of heme, a result that is dependent on the heme transporter ChuA and
universal membrane coupling protein TonB. In addition, the IFP-HO1 heme sensor
allows for the direct reporting on the synthesis of intracellular heme in the absence of
exogenous heme but presence of extracellular ferric iron, a phenotype that is
dependent on the biosynthesis of heme by HemE. Finally, using an E. coli pathotype
that causes systemic, bloodstream infections (extraintestinal pathogenic E. coli ExPEC), we report, for the first time, the heme sensor can be used to report on heme
metabolism in infected animals - a task IFP is particularly well suited for as it
fluorescence at a wavelength that is poorly absorbed by tissue. This sensor will allow
researchers to expand the molecular toolbox used to study iron uptake, both in culture
and now during infection.
Contributors: Clark, Justin; Nobles, Chris; Green, Sabrina; Maresso, Anthony
59
INDUCIBLE MYD88/CD40, GENETIC ADJUVANT ENHANCES IN VIVO
ELECTROPORATION CANCER VACCINE VIA CONTRIBUTIONS OF MULTIPLE
CELL TYPES AT VACCINATION SITE
Matthew R Collinson-Pautz
Advisor: David Spencer, Ph.D.-Department of Pathology & Immunology
Kevin Slawin, M.D.-Department of Urology
The use of cancer vaccines to treat malignant disease has significant potential,
however that potential has yet to be achieved in treating cancer patients. Fortunately,
pioneering biotechnologies are paving the way for more effective cancer vaccines. Our
lab has previously developed a drug inducible MyD88/CD40 (iMC) composite adjuvant
that promotes robust cytotoxic T cell priming by iMC activated DCs. However, the
transition to “off-the-shelf” cancer vaccines that are not patient specific, has presented
many obstacles to the field. We have therefore begun to address the delivery of cancer
vaccines as “off-the-shelf” therapies using in vivo electroporation (EP) of plasmid DNA
(pDNA) with iMC adjuvant as a vaccination strategy (eVac). Intradermal EP of pDNA
encoding the model antigen LacZ in mice primed LacZ-specific CD8+ T cell responses,
as mice receiving LacZ + EP had significantly greater portions of antigen responsive,
IFNγ secreting T cells than mice that received that same LacZ vector without EP.
Inclusion of the iMC adjuvant further improved the efficacy of eVac, as EP of pDNA
encoding iMC and the model antigen OVA induced a greater number of OVA-specific T
cells capable of secreting significantly higher levels of cytokines in response to antigen
as compared to mice vaccinated with OVA alone. Additionally, in B16-OVA and EG7OVA tumor bearing mice, iMC.OVA vaccination resulted in a greater tumor volume
reduction than in mice vaccinated with OVA alone.
In an effort to better understand the mechanism underlying iMC-mediate vaccine
enhancement we restricted expression of iMC.OVA to all non-hematopoietic derived
cells, and therefore eliminated direct expression in immune cells present at the site of
vaccination. Interestingly, non-hematopoietic restricted OVA expression significantly,
decreased the OVA-specific immune response in mice, however addition of iMC
partially restored this response, suggesting that iMC adjuvant functions in cells other
than immune cells present at the vaccination site. As the vaccination occurs
intradermal, it is likely keratinocytes and fibroblasts take up the pDNA and express the
vaccine. With this in mind, we examined the effect of iMC expression in murine
keratinocyte (MPEK) and fibroblast (NIH3T3) cell lines in vitro. We found that iMC
signaling in these cell types activates the secretion of several inflammatory cytokines
and chemokines including GM-CSF, G-CSF, IL-1(, CXCL2, and CCL2, factors which
may positively influence the immune environment at the site of vaccination. Overall, this
data suggests a general mechanism of in which iMC enhances eVac in part by
generating an inflammatory milieu at the site of vaccination and through undefined
downstream processes leads to an increased anti-tumor immune response.
Contributors: Collinson-Pautz, M; Decker, W; Spencer, D; Levitt, J
60
IDENTIFICATION OF POTENTIAL DRUG TARGETS FOR CICLOPIROX
Zachary Christopher Conley
New antibiotics are needed to treat the continual threat of emerging antibioticresistant bacteria. We found that the repurposed antifungal drug, ciclopirox, is equally
effective against susceptible or multidrug-resistant clinical isolates (Carlson-Banning et
al. 2013 PLoS One 8:e69646). Ciclopirox is a known iron chelator but the cellular drug
targets are unknown. In addition, ciclopirox is not affected by known resistance
mechanisms. I hypothesize that ciclopirox has a novel drug target. In an effort to identify
this novel drug target, we utilized a candidate gene approach to identify biochemical
pathways affected by ciclopirox. Minimum inhibitory concentrations (MICs) of single
gene deletions were measured and compared to the MIC of the parent strain. 17 of 103
tested genes displayed increased susceptibility to ciclopirox but not other antibiotics,
nor the iron chelator 1, 10-phenanthroline. 10 of these 17 genes corresponded to either
production of the lipopolysaccharide enterobacterial common antigen (ECA), or uptake
of the iron binding siderophore enterobactin. I found that ciclopirox induces enterobactin
production and that deletion of the galactose salvage gene encoding UDP-galactose 4epimerase, galE, significantly increased ciclopirox-induced enterobactin production.
Deletion of the galactose salvage gene encoding galactose-1-phosphate
uridylyltransferase, galT, significantly decreased production. Deletion of galE did not
have this effect on 1, 10-phenanthroline-induced enterobactin production. Additionally,
deletion of the genes necessary for ECA production did not alter enterobactin
production. Therefore, ciclopirox may kill bacteria by altering cellular iron acquisition in
addition to chelation. Ciclopirox may also increase cellular accumulation of necessary
sugar metabolites, which at high levels cause toxicity. The cell may be unable to restore
metabolite balance and thus no resistance mechanisms have been identified.
Contributors: Conley, Zachary C.; Carlson-Banning, Kimberly M.; Carter, Ashley; Chou,
Andrew; Hamill, Richard J.; Song, Yongcheng; and Zechiedrich, Lynn
61
CHARACTERIZATION OF A TRANSCRIPTION FACTORS ROLE IN POLYCOMB
GROUP PROTEIN RECRUITMENT
Michael Courtland
Advisor: Qinghua Wang, Ph.D.-Department of Biochemistry & Molecular Biology
Polycomb Group (PcG) proteins are highly conserved and involved in the
epigenetic regulation of thousands of genes related to development, cellular
differentiation, cell fate, and others. PcG proteins catalyze covalent histone marks that
are associated with repressed gene transcription. Due to their vital role in gene
regulation, PcG proteins are frequently found to be overexpressed or mutated in a
variety of cancers and as such have become promising targets for pharmaceutical
intervention. While their roles in repressing gene transcription are fairly well understood,
the mechanism by which these proteins are recruited to their target genes in
mammalian cells is unclear.
Here we focus on identifying potential transcription factors involved in
recruitment of human PcG proteins to gene targets. Polycomb and Trithorax
Recruitment Factor (PTRF) was identified by the similarity of its DNA binding domain to
that of Zeste, one Drosophila transcription factor involved in PcG recruitment. PTRF
contains an N-terminal DNA binding domain and a C-terminal coiled-coil domain. Gel
filtration chromatography has illustrated its DNA binding capabilities and structural
studies are currently underway in the hopes of elucidating the mechanistic details of
binding. Gel filtration studies of the coiled-coil domain have revealed the presence of
higher order oligomers but the exact oligomeric state is currently unknown. With these
studies we hope to determine the mechanistic basis by which PTRF recruits and
regulates PcG functions. RNA-Seq of PTRF knockdown in mouse P19 cells identified
thousands of genes that are potentially regulated by PTRF, directly or indirectly. Gene
ontology classes of up and down regulated genes include transcription factors and
transcription regulation, regulation of metabolic processes, development, and others.
ChIP-Seq and further functional studies on PTRF are needed to identify its contribution
to PcG gene regulation.
Contributors: Du, Junqing; Kirk, Brian; Gou, Yufeng
62
CROSSMODAL PERCEPTUAL ADAPTATION IMPLIES NEURONAL
CONVERGENCE OF AUDITORY AND TACTILE FREQUENCY SIGNALS
Lexi E. Crommett
Advisor: Jeffrey Yau, Ph.D.-Department of Neuroscience
We perceive temporal frequency information by audition and touch. Because
these modalities reciprocally influence each other in frequency perception, temporal
frequency channels appear to be linked across audition and touch. Auditory and tactile
perceptual channels may be tied explicitly if common neural populations support
auditory and tactile frequency processing. Adaptation paradigms have been used
previously to infer neural tuning properties in psychophysical experiments. In a series of
psychophysical experiments, we employed a crossmodal frequency adaptation
paradigm to test the hypothesis that a common frequency-tuned neural population
processes auditory and tactile frequency signals. Participants (n = 20) each performed
a tactile frequency discrimination task in 3 experiment sessions. Each session began
with an auditory adaptation period (180s) during which the participant received
prolonged auditory stimulation (adaptation conditions with bandpass noise stimuli
centered at 200 Hz or 400 Hz) or silence (control condition). After initial adaptation,
participants performed trials of a 2AFC tactile discrimination task in which they judged
which of two vibrations presented sequentially to their finger was perceived as being
higher in frequency. Vibration frequencies ranged from 100-300 Hz. We used a
generalized linear mixed effects model (GLMM) to test whether auditory adaptation
modulated tactile discrimination performance and whether this modulation was
frequency-specific. Crossmodal adaptation significantly improved tactile frequency
sensitivity when the spectral composition of the noise adaptor overlapped the tactile test
frequencies. We implemented a simple and biologically plausible model that represents
tactile frequency information with likelihood functions computed from a population of
sensory neurons. By allowing auditory adaptation to modify the model’s sensory neuron
response characteristics, our model reproduced the frequency-specific crossmodal
aftereffects. These psychophysical and modeling results support the hypothesis that
auditory and tactile signals converge on a common frequency-tuned neural population.
Contributors: Crommett, Lexi E.; Perez-Bellido, Alexis; Yau, Jeffrey M.
63
DNMT3A AND TET1 ARE COMPLEMENTARY AND COMPETITIVE IN REGULATING
GENE EXPRESSION
Sean Michael Cullen
Advisor: Margaret Goodell, Ph.D.-Department of Pediatrics
DNA methylation is an epigenetic modification that promotes stable yet
reversible gene repression. Despite the recent description of DNA methylation patterns
in a variety of tissues with advancements in next generation sequencing, the
mechanisms involved in establishing and maintaining these patterns remain unclear.
DNA methyltransferase 3a (DNMT3A), one of three enzymatically active proteins
responsible for DNA methylation, enables efficient differentiation of both embryonic and
hematopoietic stem cells (ESC & HSC). Little is known, however, regarding the specific
mechanisms through which DNMT3A impacts this important process in stem cells. We
hypothesized that DNA methylation is partly controlled by access of DNMT3A to
genomic regions as dictated by other epigenetic marks. We have addressed this
question by generating an ESC line with inducible in vivo-biotinylated-DNMT3A to
identify genome-wide localization of DNMT3A, and have compared DNMT3A
localization with sites in which DNA methylation is lost after knock-out (KO) of Dnmt3a.
Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) was performed for bioDNMT3A. We verified enrichment of DNMT3A within gene bodies and depletion within
CpG Islands, which generally exhibit high and low levels of DNA methylation,
respectively. Interestingly, DNMT3A was highly enriched at the edges of large
unmethylated canyon regions previously described by our group.
Due to the dramatic loss of DNA methylation in specific regions of Dnmt3a-/ESCs, we hypothesized that the TET methylcytosine dioxygenase protein family, known
to play a important step in DNA demethylation via a hydroxymethylation intermediate,
would be a crucial factor in directing this change. TET1 ChIP-Seq data revealed a
striking complementary binding pattern to DNMT3A. Specifically, TET1 was bound
throughout ESC canyons, but was depleted in many other genomic regions. The
promoter regions with highest TET1 binding also exhibited among the highest levels of
DNMT3A binding. Notably, deletion of Dnmt3a dramatically reduces TET1 binding
capacity genome-wide, but Tet1 KO via CRISPR specifically increases full-length
DNMT3A binding capacity at genes with the highest TET1 binding in wild type ESCs.
Furthermore, DNMT3A and TET1 serve complementary epigenetic regulatory roles
within gene groups, in coordination with histone methylation marks, to regulate gene
expression. This study further expands on our understanding of the epigenomic
molecular mechanisms necessary to regulate processes such as self-renewal and
differentiation, and establishes a novel system to elucidate the regulation of DNA
methylation in a wide range of cell types.
Contributors: Cullen, Sean*; Lin, Xueqiu*; Luo, Min; Jeong, Mira; Chen, Taiping; Li, Wei;
Goodell, Margaret
64
GENE AND PATHOGENIC VARIANT DISCOVERY FOR MENDELIAN AND
COMPLEX FAMILIAL TRAITS
Hang Dai
Advisor: Suzanne Leal, Ph.D.-Department of Molecular & Human Genetics
We provide methods and implementation of best practices to identify rare
variants involved in the etiology of Mendelian and familial complex traits using nextgeneration sequence (NGS) data. We demonstrate through case examples,
bioinformatics protocols to analyze exome and genome sequence data to elucidate
pathogenic variants that are either de novo, underlie Mendelian phenotypes or complex
traits with familial aggregation. We feature four types of commonly adopted study
designs analyzing either genomes or exomes from (1) a single affected individual (2)
multiple family members, (3) multiple families and (4) multiple unrelated individuals with
a family history of disease. For each design we illustrate the procedures to integrate
data from different sources, performing variant annotations, and selecting potentially
pathogenic variants from single or multiple exomes based on several parameters,
including but not limited to mode of inheritance, variant sharing among pedigree
members, population minor allele frequency, functional annotation and prediction,
linkage mapping data and variants/genes previously implicated in disease etiology.
Case studies were novel pathogenic variants have been discovered for nonsyndromic
hearing impairment, thoracic aortic aneurysms and dissections, autism, Moyamoya
disease, otitis media and rare autosomal recessive traits such as achromatopsia and
trichothiodystrophy are used to illustrate the protocols and best practices.. We have
also developed an easy-to-use bioinformatics software, Variant Mendelian Tools (VMT),
to implements the protocols which makes it possible for any researcher with NGS data
to efficiently hunt down and identify pathogenic variants. VMT is designed to be flexible
to accommodate regular updates from annotation databases and incorporation of new
information from a variety of sources including public databases and in-house data, e.g.
linkage regions. The analysis protocols we developed are distributed under the VMT
platform which can be readily adapted and shared for a variety of projects, owing to the
compact, human-readable syntax that VMT adopts. Our work is highly beneficial to
clinicians and researchers who aim to identify pathogenic variants from NGS data but
have minimal knowledge and experience in the use of Linux and programming
languages, and/or in annotations and variant discovery using family data.
Contributors: Dai, Hang; Wang, Gao; Peng, Bo; Santos-Cortez, Regie; Zhang, Di; Leal,
Suzanne
65
WAC REGULATES MTOR ACTIVITY BY STABILIZING THE TTT-PONTIN/REPTIN
COMPLEX
Gabriela Riva David-Morrison
The ability to sense energy status is crucial in the regulation of metabolism via
mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTTPontin/Reptin complex is responsive to changes in energy status. In energy sufficient
conditions, the TTT-Pontin/Reptin complex promotes mTORC1 assembly and
dimerization, as well as mTORC1-Rag interaction, which are critical for mTORC1
activation. Here we show that WAC is a novel regulator of energy-mediated mTORC1
activity. In a Drosophila screen designed to isolate mutations that cause neuronal
dysfunction, we identified wacky, the homolog of WAC. Loss of Wacky leads to
neurodegeneration, defective mTOR activity, and increased autophagy. Wacky and
WAC have conserved physical interactions with mTOR and its regulators, including
Pontin and Reptin, which bind to the TTT complex to regulate energy-dependent
activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin
in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating
mTORC1 dimerization and mTORC1-Rag interaction.
Contributors: David-Morrison, Gabriela; Xu, Zhen; Rui, Yan-Ning; Charng, Wu-Lin; Jaiswal,
Manish; Yamamoto, Shinya; Xiong, Bo; Zhang, Ke; Sandoval, Hector; Duraine, Lita; Zuo,
Zhongyuan; Richman, Ronald; Zoghbi, Huda; Zhang, Sheng; Bellen, Hugo
66
A FORWARD GENETIC SCREEN FOR AGGRESSION IN DROSOPHILA
Shaun Michael Davis
Advisor: Herman Dierick, M.D.-Department of Molecular & Human Genetics
Aggression is a complex social behavior that is found widely throughout the
animal kingdom. This trait can be beneficial for an organism to compete for limited
resources but excessive aggression can be costly. Both environmental and genetic
factors contribute to aggression, but the cellular and molecular mechanisms underlying
this trait remain poorly understood. To study these mechanisms we use male
Drosophila. A forward genetic screen is a powerful approach to comprehensively
dissect the genetic mechanisms involved in this phenotype. However, screening directly
for aggression is time consuming and has so far not been feasible in flies or any other
organism. To circumvent this limitation we looked at the end result of aggressive
encounters and found a positive correlation between aggression and wings with
physical damage. Furthermore, we developed a novel sensitive flight assay and showed
that flies with damaged wings have impaired flight ability suggesting that wing damage
incurred by fighting negatively affects flies. We carried out an X-linked, forward genetic
screen to identify mutants that increased wing damage through altered aggressive
behavior. Screening through ~1,400 mutants we identified 41 independent lines that
showed greater than 30% damaged wings. When tested for increased aggressive
behavior, 5 of these candidate lines showed a significant increase in aggression. All 5
candidate lines were sequenced and we further focused our analysis on the top 2 lines,
AL68 and AL147. The genomic variants were filtered to identify the unique, nonsynonymous coding mutations. Duplication lines covering the unique variants were
crossed to AL68 and assessed for aggression. Only the duplications covering the
Shaker gene, which encodes an α-subunit of a voltage-dependent K+ channel,
completely suppressed the aggression in AL68. Furthermore, a second mutation in a
similar domain of Shaker in an independent genetic background also showed an
increase in aggression. These data suggest that the regulation of neuronal function is
important for aggressive behavior. The AL147 line showed no suppression of
aggression when combined with a duplication covering a unique genetic variant. We
next mapped this locus through meiotic recombination followed by PCR mapping of
small indels and identified a 1.4 Mbp region of interest. Of the 30 duplication stocks that
cover this interval, only 1 completely suppressed the aggressive behavior of AL147.
There are no coding mutations in any of the 7 genes within this duplication, suggesting
that a regulatory mutation is causing the aggressive phenotype. Here, we have shown
that aggressive behavior can cause wing damage, which can be used to screen for
novel genetic variants, leading to further understanding of the molecular mechanisms of
aggressive behavior.
Contributors: Thomas, Amanda; Liu, Lingzhi; Campbell, Ian; Dierick, Herman
67
MISEXPRESSION OF CYCLIN D1 IN EMBRYONIC GERM CELLS PROMOTES
TESTICULAR TERATOMA INITIATION
Emily Packard Dawson
Advisor: Jason Heaney, Ph.D.-Department of Molecular & Human Genetics
Background: Testicular germ cell tumors (TGCTs) are the most frequent solid
tumor diagnosed in young men. In both mice and humans, TGCTs result from
anomalies in embryonic germ cell development. In the 129 inbred mouse model of
human TGCTs, these tumors arise during the same developmental period that male
germ cells enter G0 mitotic arrest and female germ cells initiate meiosis (the
mitotic:meiotic switch). Dysregulation of this switch associates with tumor initiation and
involves three germ cell developmental abnormalities, namely delayed G0 mitotic
arrest, retention of pluripotency, and aberrant expression of genes normally restricted to
embryonic female and adult male germ cells. One such misexpressed gene, cyclin D1
(Ccnd1), is a known regulator of the G1-S phase cell cycle checkpoint and a potent
oncogene in several tissues. Therefore, we hypothesized that misexpression of Ccnd1
promotes a pro-proliferative program that drives TGCT initiation.
Methods: To test whether components of the G1-S phase checkpoint associate
with increased tumor risk, we compared a tumor-resistant strain, FVB/NJ (FVB), and
two tumor-susceptible strains, the 129-Chr19MOLF/Ei chromosome substitution strain
(M19; 80% affected) and the 129/SvImJ inbred strain (129; 8% affected). To assess
the contribution of Ccnd1 to tumor initiation, we obtained mice harboring a Ccnd1KO
allele. The Ccnd1KO allele was then backcrossed onto the 129 inbred background for
10 generations to establish a congenic strain, and then transferred with crosses to the
M19 background. All strains harbor a germ cell-specific GFP transgene for
immunostaining experiments or fluorescence activated cell sorting (FACS) followed by
gene expression analyses.
Results: We found that Ccnd1 is aberrantly expressed in gonocytes that fail to
enter G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin
misexpressed in male germ cells during this time period. We discovered that Ccnd1
deficiency significantly reduced tumor incidence (P<0.001) and suppressed both the
proliferation and pluripotency defects critical for tumor initiation. Loss of Ccnd1
expression did not alter normal male somatic or germ cell development, implying that
the mechanisms by which Ccnd1 deficiency reduced tumor susceptibility were germ cell
autonomous and specific to the process of tumor initiation.
Conclusion: We conclude that misexpression of Ccnd1 in male germ cells is a
key component of a pro-proliferative program that disrupts the mitotic:meiotic switch
and predisposes 129 inbred males to testicular teratocarcinogenesis. These
experiments reveal the treatment potential of inhibiting CCND-kinase activity for
patients with TGCTs, a strategy which will target cancer cells without significant adverse
effects to normal tissue.
Contributors: Dawson, Emily; Lanza, Denise; Heaney, Jason.
68
THE PUTATIVE RNA BINDING PROTEIN RBM17 AND ITS ROLE IN CELL
SURVIVAL AND SPINOCEREBELLAR ATAXIA TYPE 1 PATHOGENESIS
Antonia De Maio
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder
characterized by progressive loss of balance and breathing dysfunction due to
degeneration of Purkinje cells and brain stem neurons. SCA1 is caused by expansion
of CAG repeats within the coding region of the ATAXIN1 gene (ATXN1), leading to the
production of a mutant variant of the cognate protein harboring an expanded glutamine
(polyQ) tract. Previous work in our lab defined the putative RNA binding protein,
RBM17, as an ATXN1 interactor that preferentially binds ATXN1 upon expansion of the
polyQ tract and requires phosphorylation of S776 in ATXN1, a residue critical for SCA1
pathogenesis, suggesting that RBM17 plays a key role in the disease pathogenesis.
To investigate the function of Rbm17 we generated Rbm17 constitutive knockout
mice and found this protein to have an essential developmental role, as its constitutive
ablation causes early embryonic lethality. To evaluate the regional and cellular effects
of Rbm17 loss-of-function we conditionally deleted Rbm17 exclusively from developing
cerebellum or adult Purkinje cells. These mice revealed that absence of Rbm17
severely impairs development of the cerebellum while Purkinje cells devoid of the
protein undergo progressive and fast degeneration.
Having established the critical role of Rbm17 for development and cell viability
we propose that this factor is a key component of the spliceosomal complex and that its
ablation causes aberrant splicing of still unknown RNA targets whose tight regulation is
essential for successful accomplishment of both processes.
To test our hypothesis we are using Immunoprecipitation/mass spectrometry
(IP/MS) and Crosslinking and immunoprecipitation (CLIP) methods to, respectively,
identify Rbm17 protein interactors and its RNA binding pattern throughout the
transcriptome. Using MS we analyzed the composition of Rbm17 immunoprecipitation
samples from wild-type mouse cerebellum. Our preliminary results confirmed some
previously known interactions and showed that Rbm17 belongs to the U2 splicing
complex. Currently, we are optimizing the CLIP protocol for Rbm17 to pull down the
protein and its associated RNA targets. RNA-sequencing will eventually reveal the
identity of these targets and shed light on the essential cellular network regulated by
Rbm17. The integration of these data will permit us to uncover the molecular
mechanism underlying the requirement of Rbm17 for cell survival and will help us
establish if and how Rbm17 contributes to SCA1 pathogenesis.
Contributors: De Maio, Antonia; Park, Jeehye; Gennarino, Vincenzo A.; Zoghbi Huda Y.
69
CORRELATED VARIABILITY IN POPULATION ACTIVITY: NOISE OR SIGNATURE
OF INTERNAL COMPUTATIONS?
George Hilton Denfield
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
Neuronal responses to identical visual stimuli are variable. The source of this
variability is unknown, but it is commonly treated as noise and seen as an obstacle to
understanding neuronal activity. We argue that this variability is not noise but reflects
computations internal to the brain. Little research has examined the effect of
fluctuations in internally-generated signals on neuronal responses, leaving a number of
uncontrolled parameters that may contribute to neuronal variability. One such variable
is attention, which modulates neuronal response gain. The strength of this modulation
varies over time, and we hypothesize that these fluctuations are a major source of
neuronal response variability. We devised a cued-attention task to induce varying
degrees of fluctuation in the attentional signal by changing whether the subject must
attend to one stimulus while ignoring another, or attend to multiple locations
simultaneously. Using multi-electrode recordings in primary visual cortex of macaques
performing this task, we demonstrate that attention gain-modulates V1 neuron
responses in a manner consistent with results from higher-order areas and that the
degree of attentional fluctuations indeed modulates neuronal variability. Our results
open the door to the use of statistical methods for inferring the state of these signals on
behaviorally relevant timescales.
Contributors: Denfield, George; Ecker, Alex; Tolias, Andreas
70
ALTERATIONS IN THE INTESTINAL MICROBIOME OF INFANTS ARE
ASSOCIATED WITH BREASTFEEDING PRACTICE
Chu Michael Derrick
Advisor: Kjersti Aagaard, M.D./Ph.D.-Department of Obstetrics & Gynecology
Stephen Welty, M.D.-Department of Pediatrics
Objective: Cesarean delivery (CD) has been implicated in dysbiosis of the
neonatal microbiome (totality of microbes and their genomes). However, it is possible
that colinearity of CD with breastfeeding practice may confound these findings. Formula
does not mimic the complexity of human breastmilk, which retains both
macromolecules as well as a unique microbiome. We thus sought to employ a
population-based cohort of mothers and their infants to determine if breastfeeding
impacts the neonatal microbiome, potentially negating early cesarean-mediated
dysbiosis. Study Design: A representative cohort (n=277 gravidae) was prospectively
enrolled, and a subset (n=81) consented to longitudinal sampling (3rd trimester, delivery
& postpartum). Stool swabs were collected from neonates at delivery and by 6 weeks of
age. Primary BF practices were determined in multiple interviews (breastfed-only (BF;
n=23), formula fed-only (FF; n=2), or both (BF&FF; n=34). DNA was extracted (MolBio)
and subjected to 16S and WGS metagenomics. Quality sequences were analyzed with
QIIME and causal inference (heirarchical clustering by Manhattan distance &
metagenomic biomarker discovery by LEfSe). Results: We detected a distinct intestinal microbiome profile in infants with
primary BF practice (panel A). Moreover, the infant gut microbiome did not cluster by
virtue of Cesarean delivery (A). LEfSe analysis (panel B) identified significantly distinct
taxa in unique association with both exclusive BF and BF&FF (LDA score >3.0).
Notably, the phylum Firmicutes was enriched in BF neonates (panel C; p=0.0155;
Mann-Whitney) while Enterococcus was enriched in BF&FF neonates (p=0.0179;
Mann-Whitney). Conclusion: Casual inference analysis of a robust population cohort enabled us
to detect a distinct intestinal microbiome associated with breastfeeding practice, rather
than mode of delivery. Our findings underscore the importance of incorporating
breastfeeding practices when examining the dynamics of the neonatal
microbiome. (no table selected)
Contributors: Derrick M. Chu, Jun Ma, Amanda Prince, Kathleen M. Antony, Diana Racusin,
Michelle Moller, Brigid Boggan, Kjersti Aagaard 71
HYPOMORPHIC MUTATIONS IN IFT81 ARE A RARE CAUSE OF INHERITED CONEROD DYSTROPHY
Rachayata Dharmat
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
Cone-rod dystrophy (CRD) (prevalence 1/40,000), is a progressive inherited
retinal disorder characterized by primary dysfunction and loss of cone photoreceptor
followed by rod photoreceptor death. CRD is a genetically heterogeneous disease, with
mutations in 30 genes implicated in the pathophysiology of CRD, however majority of
the autosomal recessive CRD cases remains unsolved. In this study, whole exome
sequencing based analysis of 200 CRD patient cohort identified a patient with
compound heterozygous mutations (p.R405X, p.L614P) in IFT81 (Intraflagellar
transport protein 81), a core component of IFT-B complex. The variants are extremely
rare as they are absent from all of our control databases (including ExAC). IFT complex
proteins are involved in bi-directional protein trafficking along the cilia. Functional
analysis of the identified missense mutation in cell culture and zebrafish demonstrated
their hypomorphic nature. In vivo functional rescue experiments in ift81hi409tg/hi409tg
zebrafish embryos displayed a significantly reduced rescue potential of the p.L614P
variant forms of IFT81 in comparison to wildtype. Upon overexpression of IFT81-Myc in
human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type
protein localizes to the basal body and tip of the primary cilium, whereas expression of
p.L614P variant leads to a significant reduction in the number of ciliated cells in
comparison to WT suggesting the impact of this mutation on ciliogenesis. Biochemical
studies show no apparent difference in the interaction of the IFT81 mutant variant with
IFT52 (another IFT-B core protein) in comparison to wildtype. IFT complexes in the
retina are involved in massive protein transportation from the protein synthesising inner
segment to the photoreceptive outer segment of the photoreceptor cells. This protein
transport is crucial for both photoreceptor architecture as well as phototransduction and
as a result photoreceptors are more sensitive to a decrease in function of IFT81,
thereby manifesting as CRD in our patient retina. This is the first patient report
indicating mutations in this gene can be associated with an isolated non-syndromic form
of retinal dystrophy in comparison to the more severe multi-systemic ciliopathy
previously reported for this gene.
Contributors: Dharmat, Rachayata; Zhongqi, Ge; Lui, Wei; Li, Yumei; Sui, Ruifang; Rui, Chen
(corresponding author).
72
SCHISTOSOMIASIS EGG ANTIGEN DECREASES TUBERCULOSIS-SPECIFIC CD4
T CELL EFFECTOR FUNCTION WITH CONCOMITANT ARREST OF MACROPHAGE
PHAGO-LYSOSOME MATURATION
Andrew R DiNardo
Advisor: Anna Mandalakas, M.D.-Department of Pediatrics
Epidemiologic evidence indicates people infected with M. tuberculosis (Mtb) and
helminths possess a higher risk of developing Tuberculosis (TB) disease than those
with TB alone. Helminths modulate the immune system, however their precise impact
on the mechanism of Mtb control remains unclear. We hypothesized a perturbation of
the Mtb-specific CD4 T cell response weakens the ability of macrophages to contain
Mtb. To recapitulate the effects of helminth infection, we exposed PBMC from Mtbinfected humans to Schistosomiasis soluble egg antigen (SEA) and then profiled the
functional capacity of Mtb-specific CD4 T cells via multi-parametric flow cytometry. SEA
decreased the frequency of cells producing IFN-( (6.79% vs. 3.20%; p = 0.017) and
TNF- (6.98% vs . 2.96% ; p = 0.012), with a concom ita nt re la tive incre a s e in IL-4 (p <
0.05) and in IL-10 MFI (1440 vs. 1273; p < 0.05). To test the biological significance of
this perturbation, we polarized monocyte-derived macrophages with the supernatant of
SEA-exposed, Mtb-stimulated autologous CD4 T cells and assessed their ability to
control Mtb using high-resolution confocal microscopy. We calculated a 2.19 fold
decreased co-localization amongst the lysosomes of autologous macrophages and
tomato-labeled Mtb (p < 0.05). To validate the role of the T cell cytokines most altered
by exposure to SEA, we determined that IL-4- or IL-10-polarized macrophages
possessed more intracellular Mtb than those with IFN-(/LPS (median 0.421 and 0.542
versus 0.272, respectively). Furthermore, we calculated a 1.7 and 1.4 fold decrease in
co-localization between Mtb and lysosomes in IL-10 and IL-4 polarized MDMs,
respectively, than in those of IFN-( /LPS (p < 0.001). Our work establishes a link
between the helminth-induced perturbation of Mtb-specific CD4 T cell responses and
the disruption of Mtb control by macrophages, thereby providing a mechanism for the
observation that helminth infection advances patients with Mtb infection along the
spectrum of TB disease.
Contributors: Andrew R. DiNardo*§, Emily M. Mace†, Kelsey Lesteberg†, Jeffrey D. Cirillo‡,
Anna M. Mandalakas§, Edward A. Graviss¶, Jordan S. Orange†, George Makedonas†.
73
COMPREHENSIVE SCREENING OF PIK3CA MUTATIONS REVEALS ONCOGENIC
RARE ABERRATIONS AND NOVEL TARGETING APPROACHES
Turgut Dogruluk
Advisor: Kenneth Scott, Ph.D.-Department of Molecular & Human Genetics
Tumor sequencing projects such as The Cancer Genome Atlas (TCGA) have
revealed the high complexity of cancer genomes that are comprised of both pathogenic
“driver” aberrations and neutral “passenger” events. A major effort in the cancer
research community involves discriminating drivers from passengers with the goal of
prioritizing new therapeutic targets and diagnostic biomarkers. Much of these efforts are
focused on identifying new oncogenes given that such factors have served as
successful therapeutic targets to date. To expedite oncogene discovery, I established a
target discovery pipeline involving novel high-throughput mutagenesis and molecular
barcoding (HiTMMoB) technology allowing our laboratory to engineer somatic mutations
identified by TCGA into our collection of >32,000 human open reading frames (ORFs).
Wild-type and mutant barcoded ORFs are subsequently entered into pooled in vitro/vivo
genetic screens to identify those that can drive cancer phenotypes. Since developing
HiTMMoB, I have continued using this technology for my own screens that include in
vitro/vivo driver and pharmacological screens to assess differing effects of numerous
mutations in the PIK3CA oncogene. PIK3CA is frequently mutated in breast cancer
among other tissue types and regulates cancer’s most mutated pathway, PI3K.
However, much of the focus of PIK3CA research is on the most common mutations of
the PIK3CA gene and neglects infrequent ‘tail’ mutations. I have created 24 rare
mutations of PIK3CA by utilizing HiTMMoB and entered them into few in vitro and in
vivo screens to assess the effects of the mutations on several cancer phenotypes i.e.
tumor formation, soft agar colony formation, growth-factor-independent growth and drug
resistance/sensitivity. Even though there seems to be a general correlation between
PIK3CA tail mutation frequency and ‘phenotype strength’ as expected, these
experiments interestingly revealed that some of the rarest PIK3CA mutations are still
significantly oncogenic. In addition, Reverse Phase Protein Array (RPPA) analysis
revealed novel pathway activations caused by hyperactive PIK3CA protein by which the
cells were prone to certain pharmacological agents. In summary, I have developed a
prioritization pipeline being used to study PIK3CA mutations in breast cancer along with
a variety of target screens across diverse cancer types. I utilized this pipeline to
determine true driver mutations of PIK3CA gene as well as novel drug targeting
approaches. The HiTTMoB technology together with downstream screening platforms
promise to provide the cancer research community the functional annotation on the
most promising cancer aberrations for drug development and targeting.
Contributors: Dogruluk, Turgut; Tsang, Yiu Huen; Espitia, Maribel; Chen, Fengju; Chen,
Tenghui; Chong, Zechen; Appadurai, Vivek; Dogruluk, Armel; Eterovic, Karina; Bonnen,
Penelope E.; Creighton, Chad J.; Chen, Ken; Mills, Gordon B.; Scott, Kenneth L.
74
COMBINED INHIBITION OF NON-CANONICAL HER2 SIGNALING AS A STRATEGY
TO OVERCOME RESISTANCE TO HER2-TARGETED THERAPIES
Rocio Dominguez Vidana
Background:
HER-family targeted therapies have significantly improved
outcomes for patients with HER2-positive breast cancer; however, despite the
availability of drugs directly targeting HER2, de novo or acquired resistance to therapy
remains a major clinical problem. We aim to identify the genetic networks that govern
response to the lapatinib using a forward genetics approach.
Experimental design and methods: Leveraging a series of RNAi genetic
screens, we identify a mitotic control network governing response to HER2 inhibition
both in vitro and in vivo. Genetic or pharmacologic inhibition of said network kills HER2positive breast cancer cells in combination with HER-family inhibitors.
Results: Mechanistically, we demonstrate a non-canonical role of HER2 in
governing mitotic progression. Combined inhibition of other mitotic controllers and
HER2 abolishes this critical regulation, leading to mitotic catastrophe and apoptosis.
Thus, we demonstrate an unexpected convergence of non-canonical HER2 signaling
and mitosis regulation that can be exploited to overcome drug resistance.
Conclusion: Our findings provide a strong mechanistic and pre-clinical rationale
for combined inhibition of HER2 and mitotic controllers as a treatment strategy for
HER2-positive breast cancer.
Contributors: Ronald J. Bernardi, Rocio Dominguez-Vidana, Christopher S. Bland, Kathleen A.
Scorsone, Marieke Aarts, Siddhartha Tyagi, Mayra C Orellana, Brent N. Rexer, Earlene M.
Schmitt, Martin J. Shea, Sufeng Mao, Chandandeep Nagi, Tamika Mitchell, Mitchell Rao,
Sarmistha Nanda, Tao Wang, Wenbin Liu, Susan G. Hilsenbeck, Chad Shaw, Mothaffar F.
Rimawi, Carlos L. Arteaga, Gordon Mills, Nicholas C. Turner, C. Kent Osborne, Rachel Schiff,
Thomas F. Westbrook
75
SUBNANOMETER STRUCTURE OF TRPV2 REVEALED WITH CRYOEM
Timothy L Dosey
Advisor: Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
Transient Receptor Potential (TRP) channels are a family of non-selective cation
channels conserved throughout eukaryotes and they are involved in diverse
physiological processes including vision, nociception, and insulin signaling. However,
our understanding of how TRP channels function has been impeded by the lack of
structural information. Therefore, we have focused our research on determining the
structure of TRPV2 because it can be heterologously expressed and stably purified
from yeast. With single particle cryoEM, we have produced an 8Å resolution density
map which reveals functionally important conformational differences between TRPV2
and its closest homolog, TRPV1. Furthermore, our activity assays have demonstrated
that the pore-turret domain, while not necessary for TRPV1 function, may be critical for
the calcium permeability of TRPV2. By using an electron microscope equipped with a
direct-electron detecting camera and an energy filter, our goal is to produce a nearatomic resolution map for TRPV2 in its apo-state and in its agonist-bound state. Then,
we will be in an advantageous position for understanding the gating mechanisms of
TRPV2 as well as how the pore-turret domain may control calcium permeability.
This research was funded by the Welch Foundation and a training fellowship
from the Keck Center of the Gulf Coast Consortia, of the Houston Area Molecular
Biophysics Program, National Institute of General Medical Sciences (NIGMS),
T32GM008280 and by the National Center of Macromolecular Imaging, P41RR103832.
Contributors: Wang, Zhao; Fan, Guizhan; Serysheva, Irina; Chiu; Wah; Wensel, Theodore;
76
GENETIC ANALYSIS OF A RARE SKELETAL DYSPLASIA REVEALS A NOVEL
MECHANISM FOR REGULATING THE MASTER TRANSCRIPTION FACTOR SOX9
Adetutu Egunsola
Chondrodysplasias, which primarily affect cartilage, are heterogeneous group of
skeletal dysplasias characterized by disproportionate short stature and premature
osteoarthritis. Defects in the synthesis or processing of type II collagen (COL2A1), the
most abundant extracellular matrix protein in cartilage, cause chondrodysplasias. For
example, SED (spondyloepiphyseal dysplasia) congentia and SED tarda are caused by
mutations affecting the structure and trafficking of type II collagen, respectively.
Therefore, we hypothesize that phenocopies of the SED spectrum, such as
spondyloepimetaphyseal dysplasia (SEMD) Iraqi, can be caused by genetic regulators
of COL2A1. By performing exome sequence, we identified a homozygous donor splicesite mutation within DDRGK1 in SEMD Iraqi patients, which resulted in loss of function
of DDRGK1. DDRGK1 is a poorly characterized protein required in a recently
discovered posttranslational modification called ufmylation. To assess whether Ddrgk1
has a role in skeletal development we initially knocked it down in zebrafish embryos and
found that ddrgk1 knockdown caused a craniofacial defect in zebrafish. To further
assess the in vivo function of DDRGK1 in a mammalian context, we generated Ddrgk1
knockout mice using CRISPR/Cas9 technology. Embryonic lethality of Ddrgk1-/- mice
were observed between E11.5-12.5 at which point they have defective digit formation.
These results suggest DDRGK1 might be required for chondrogenesis. We also found
that knockdown of DDRGK1 decreased Col2a1 mRNA expression and the protein
expression of SOX9, the transcription factor of Col2a1, in chondrogenic ATDC5 cell
line. To verify that sox9 is a downstream target of ddrgk1, we rescued the craniofacial
phenotype of ddrgk1 knockdown zebrafish embryos by overexpressing sox9 mRNA.
Since it been previously shown that DDRGK1 regulates IκB ubiquitination, we
hypothesized that DDRGK1 regulates the protein stability of SOX9 by affecting SOX9
ubiquitination. When we overexpressed DDRGK1 in HEK293T cells, it decreased SOX9
ubiquitination. These results validate our hypothesis that DDRGK1 increases the
stability pf SOX9 by downregulating the ubiquitination of SOX9. In conclusion, DDRGK1
directly interacts and regulates the stability of SOX9. Thus DDRGK1 loss of function
decreases COL2A1 expression by reducing the expression of SOX9, which ultimately
contributes to the skeletal phenotype in SEMD Iraqi.
Contributors: Adetutu Egunsola, Yangjin Bae, James T Lu, David Liu, Lisette Navarez, Jim
Barrish, John Hicks, Mordecai Shohat, Richard Gibbs, Eric C Swindell, Daniel Cohn, Philippe
Campeau, Brendan Lee,
77
DOUBLE STRAND BREAK REPAIR IN THE ABSENCE OF END-BINDING BY THE
KU HETERODIMER
Charlene Hannah Emerson
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
The evolutionarily conserved Ku heterodimer is a DNA end-binding (DEB)
complex involved in both telomere maintenance and non-homologous end-joining
(NHEJ). In Saccharomyces cerevisiae, Ku, comprised of Yku70/Yku80 subunits, binds
DNA ends via a channel structure. We previously generated DEB defective Ku mutants,
revealing that DEB is required for Ku’s telomeric functions. Paradoxically, we found that
these mutants demonstrated increased colony survival in an assay that probes for
imprecise NHEJ, in contrast to Ku null strains, which exhibit a drastic reduction of
colony formation. A subsequent study also generated DEB defective mutants of Ku by
deleting residues to constrict the DNA binding channel (Pfingsten et al. 2013, Cell).
Although these mutants similarly demonstrated a requirement for Ku’s DEB activity at
telomeres, they were defective for imprecise NHEJ.
To reconcile differences between the two studies, we generated additional
DEB defective Ku mutants and found that they too had increased imprecise NHEJ. We
also identified a novel region in Yku70 that is required solely for NHEJ and is predicted
to associate with residues deleted in the DEB mutants reported by Pfingsten et al. We
propose that these results reconcile the imprecise NHEJ differences observed and
support a hypothesis that Ku heterodimers solely defective for DEB can modulate
NHEJ off DNA ends.
To further test this hypothesis, we conducted experiments to explore the
mechanism by which DEB defective Ku modulates imprecise NHEJ. We found that the
Yku70-R456E-DEB defective mutant did not localize to an induced double strand break
(DSB) in ChIP assays. Additionally, we applied a next-generation sequencing platform
to simultaneously analyze 400 – 600 independent imprecise repair events, finding
distinct end-processing of DSBs in the Yku70-R456E-DEB defective strain,
characterized almost solely by small deletion events, in contrast to the small insertions
that comprised the majority of repair events in wild-type strains. Notably, an assay for
precise NHEJ activity revealed a severe precise repair defect in the Yku70-R456E-DEB
defective mutant. Epistasis experiments showed the Yku70-R456E mutation was not
conferring a gain of function. Imprecise NHEJ in Yku70-R456E-DEB defective mutant
was dependent on DNL4, similar to canonical NHEJ. The sum of our observations
supports a model in which DEB defective Ku, through interactions that occur off DNA
ends, modulates an error-prone form of NHEJ.
Contributors: Emerson, Charlene; Lopez, Chris; Ribes-Zamora, Alfred; Zaneveld, Jacques;
Chen, Rui; Bertuch, Alison
78
HUR PROMOTES TRANSLATION OF KRAS MRNA AT LATE STAGES OF HUMAN T
CELL ACTIVATION
Joseph Mario Fachini
Advisor: Joel Neilson, Ph.D.-Department of Molecular Physiology & Biophysics
A number of recent studies have utilized genome-wide approaches to
characterize the breadth of post-transcriptional gene regulation in the context of CD4 T
lymphocyte activation.
Here, we describe post-transcriptional regulation of the
expression of the KRAS proto-oncogene at late stages of the activation program. While
stimulation through the T cell antigen receptor results in a modest decrease in steadystate levels of KRAS mRNA, the mRNA is more effectively loaded to the translational
machinery, resulting in a marked increase in KRAS protein. Via reporter assay, we
map the cis-determinant of this regulation to a sub-region of the KRAS 3' untranslated
region and demonstrate a direct interaction between this region and the HuR RNA
binding protein. RNAi knockdown of the HuR gene product eliminates the increased
relative expression of a reporter under the control of the KRAS 3' untranslated region,
as does removal of predicted HuR binding sites via site-directed mutagenesis. To our
knowledge, this is the first demonstration of a direct regulatory relationship between
HuR and the KRAS proto-oncogene.
Contributors: Fachini, Joseph; Chaudhury, Arindam; Neilson, Joel
79
IMMUNOLOGICAL ROLE OF EXTRACELLUALR TRAP ASSOCIATED PROTEIN
CADA AND THE HOMOLOG CAD3 IN DICTYOSTELIUM DISCOIDEUM
Timothy Farinholt
Advisor: Adam Kuspa, Ph.D.-Department of Biochemistry & Molecular Biology
During the multicellular developmental stage the social amoeba Dictyostelium
discoideum has an innate immune system that shares features with animals. However,
little is known about the amoebal immune system. Upon starvation D. discoideum
amoebae enter development and lose the ability to phagocytize and kill bacteria leaving
them vulnerable to infection. D. discoideum utilizes specialized immune Sentinel (S)
cells to kill and remove invading bacteria from the slug. The S cells act as amoebal
neutrophils, phagocytizing bacteria, secreting antimicrobials, and releasing DNA-based
extracellular traps (ETs). How ETs are formed in animals and D. discoideum remains a
mystery. A proteomic analysis of D. discoideum ETs identified many proteins including
the 24-kDa calcium dependent cell-cell adhesion protein CadA. We hypothesize that
CadA performs an antimicrobial function during early colony formation while feeding on
bacteria and when associated with ETs during development. cadA-null amoebae
display a fivefold reduction in colony formation on gram(-) Klebsiella pneumoniae (K.p.),
compared to wild-type cells, but form colonies at near wild-type levels when grown on
the gram(+) bacterium Micrococcus luteus. CadA also appears to function during
development as disaggregated cadA-null slug cells exhibit reduced ability to kill K.p. in
suspension compared to wild type slug cells. Mutants in Cad3, a homolog of CadA
(73% amino acid identity) displayed a threefold decrease in colony formation on K.p.
compared to wild-type. We are currently utilizing GFP tagged CadA to visualize the
localization of the protein during vegetative growth on gram(-) and gram(+) bacteria. We
are also constructing a double mutant of cadA-null and cad3-null that should provide
insight into the requirement of these proteins in bacterial defense during both growth
and development. Understanding how these proteins kill bacteria will provide insight
into a possible mechanism in other animals including humans.
Contributors: Farinholt, Timothy; Zhuchenko, Olga; Kuspa, Adam
80
HIGH-RESOLUTION PREDICTION AND DESIGN OF STRUCTURALLYUNCHARACTERIZED G PROTEIN-COUPLED RECEPTOR WITH NOVEL LIGAND
BINDING SELECTIVITES
Xiang Feng
Advisor: Patrick Barth, Ph.D.-Department of Pharmacology
Ligand docking is a key component of computer-aided rational drug discovery
methods, which play a crucial role in modern drug development. It has been shown that
ligand docking can effectively decrease the number of compounds necessary to screen
while retaining the same level of lead compound discovery.
It has long been known that a simplistic rigid ‘lock-and-key’ model of ligandreceptor interaction is inadequate and incorporation of conformational rearrangement
that accounts for ’induced fit’ or ‘conformational selection’ model of ligand-receptor
interaction is required for accurately predicting ligand-receptor binding. However,
receptor flexibility remains a challenge for ligand docking applications because of the
large number of conformational degrees of freedom that have to be considered in
calculation. Besides such algorithm limitation, another factor that limits the application
of ligand docking to membrane proteins and G-Protein Coupled Receptors (GPCR) in
particular is the difficulty in obtaining accurate structural information. Although GPCRs
represent the targets of around 40% approved drugs, less than 5% have solved crystal
structures. Comparative modeling of GPCR structures can greatly increase the number
of potential drug targets that can be used in ligand docking, but currently suffers from
inaccurate ligand binding pocket modeling.
To address these two challenges simultaneously, we developed a novel protocol
in the software Rosetta to build accurate models of ligand docked GPCR complexes for
a wide range of structurally uncharacterized receptors. The new protocol not only
models explicitly receptor flexibility but also improves the accuracy of the ligand binding
site by integrating loop rebuilding and ligand docking steps. Our technique outperforms
existing methods in its ability to recover native ligand binding conformation and ligandprotein interactions in blind prediction and benchmark tests. We also show that our
approach can be used to predict the effects of receptor sequence changes on ligand
binding affinities and specificities. We also applied our technique to design structurally
uncharacterized dopamine D2 receptor variants with novel ligand binding selectivities.
Our method should prove useful in the drug discovery process of structurally
uncharacterized GPCRs and sets the stage for engineering receptors with novel
pharmacological properties.
Contributors: Feng, Xiang; Chen, Kuang-Yui Michael; Garrido, Joaquin Ambia; Barth, Patrick
81
SRC-2 ORCHESTRATES POLYGENIC INPUTS FOR FINE-TUNING GLUCOSE
HOMEOSTASIS
Tiffany Chantal Fleet
Advisor: Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Clifford Dacso, M.D./M.P.H.-Department of Molecular & Cellular Biology
Despite extensive efforts to understand the monogenic contributions to
perturbed glucose homeostasis, the complexity of genetic events that fractionally
contribute to the spectrum of this pathology remain poorly understood. Proper
maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance
carbohydrate uptake and release during the feeding-to-fasting transition is essential to
the regulation of peripheral glucose availability. The liver coordinates the expression of
gene programs that control glucose absorption, storage and secretion. Herein, we
demonstrate that SRC-2 orchestrates a hierarchy of nutritionally-responsive
transcriptional complexes to precisely modulate plasma glucose availability. Using DNA
pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an
indispensable integrator of transcriptional complexes that control the rate-limiting steps
of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as
a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify
a previously unappreciated model that helps to explain the clinical spectrum of glucose
dysregulation.
Contributors: Fleet, Tiffany; Zhang , Bin, Stashi , Erin; Jung , Sung Yun; Rajapakshe, Kimal;
Dean, Adam; Gonzales, Naomi; Foulds, Charles; Coarfa, Christian; Qin, Jun; York, Brian;
O’Malley, Bert.
82
MICROBIALLY-INDUCED EPIGENETIC CHANGE IN A COLONIC ENTEROID
SYSTEM
Tatiana Y Fofanova
Richard Kellermayer, M.D./Ph.D.-Department of Pediatrics
Ulcerative colitis (UC), a form of Inflammatory Bowel Disease (IBD)
characterized by chronic inflammatory destruction of the colonic mucosa, is estimated
to affect 593,000 people in the United States. Current UC pathogenesis models
theorize that environmentally primed, genetically susceptible individuals develop an
aberrant immune response to colonic mucosal microbiota. Recently, select microbiota
have been suggested to epigenetically modify gene expression in their hosts. Thus,
microbial components isolated from inflamed colonic mucosa may contribute to the
development and maintenance of an ulcerative colitis phenotype. Specifically, we
hypothesize that microbiota, or their metabolites, epigenetically influence stem cell
populations in the human colonic mucosa.
Animal models of this disease are limited, requiring significant manipulation
and leveraging. Thus, there is a great need for an alternative experimental model
mimicking human intestinal physiology while concomitantly allowing for the introduction
of specific enteric organisms that may maintain or augment the UC phenotype.
Intestinal enteroids, self-organizing mini-intestines cultured ex vivo from human LGR5+
intestinal stem cells, are capable of differentiating into all known colonic cell types and
present a unique opportunity for studying UC at the epithelial level. However, these
human cell lines require significant optimization prior to conducting large, sequencingbased experiments. The described research will summarize this optimization process
and report on preliminary epigenetic experiments. To address the central hypothesis,
we will present an experimental plan using Illumina-based Reduced Representation
Bisulfite Sequencing and analysis to (1) characterize the methylation phenotype in
pediatric UC patients, (2) determine the extent to which (CEs) preserve the
transcriptional and epigenetic signatures of their source tissue over time, and (3)
investigate the impact of microbial communities on the methylome by reintroducing
colitis-associated bacterial species to their (matched) primary CE cultures.
Contributors: Fofanova, Tatiana; Kellermayer, Richard; Petrosino, Joseph
83
SRC-2 AND SRC-3: POTENTIAL NEW TARGETS TO INHIBIT CASTRATIONRESISTANT PROSTATE CANCER
Christopher James Foley
Advisor: Nicholas Mitsiades, M.D./Ph.D.-Department of Medicine
Although the focus of prostate cancer treatment has primarily revolved around
inhibiting the androgen receptor (AR) signaling axis, prostate cancer almost invariably
overcomes these therapeutic approaches by developing resistance to endocrine
therapies while often maintaining a reliance upon the AR or the AR signaling axis.
Identifying which proteins may play a key role in conferring this resistance to traditional
endocrine therapies is therefore critical to developing new therapeutics and extending
survival for patients who suffer from prostate cancer. The p160 family of steroid
receptor coactivators (SRCs), which contains three master transcriptional regulators,
has already been implicated in the progression of numerous cancers – including
prostate cancer. Based on the observation that the p160 SRCs are overexpressed or
amplified in some primary prostate cancers, but that they are overexpressed or
amplified to a greater degree in more advanced prostate cancers, as well as castrationresistant prostate cancers, we conducted ChIP-Seq and microarray analysis to
determine what role the p160 SRCs may be playing in prostate cancer progression.
Through these studies, we observed that SRC-2 may be driving a more dedifferentiated
phenotype, allowing prostate cancer cells to survive in the absence of androgens, while
SRC-3 may be driving an increase in cell cycle progression, resulting in a resumption of
proliferation. Following this, we have demonstrated that a small molecule inhibitor
targeting the SRCs has been more effective inhibiting our castration-resistant model
compared to our primary prostate cancer model. Taken together, this evidence strongly
suggests that the SRCs play a key role in the transition from androgen-dependent to
castration-resistant prostate cancer, and that targeting the SRCs may be a promising
therapeutic target to both inhibit the development of castration-resistance, as well as
treating already-resistant prostate cancers.
Contributors: Lanz, Rainer; Fiskus, Warren; Rajapakshe, Kimal; Chew, Sue Anne; Geng,
Chuandong; Coarfa, Cristian; O’Malley, Bert. W; Mitsiades, Nicholas
84
TARGETING NEDD8: A NOVEL APPROACH TO TREATING NEUROBLASTOMA
Jennifer Haunani Foster
Advisor: Terzah Horton, M.D./Ph.D.-Department of Pediatrics
Background: Patients with high-risk and relapsed neuroblastoma have a poor
event free survival (EFS). There is an urgent need to develop more effective therapies
for this population. Novel chemotherapy agents, such as the NEDD8-activating enzyme
(NAE) inhibitor MLN4924, provide great possibilities for advancements in cure.
MLN4924 is a novel chemotherapy agent; it blocks the degradation of proteins that
would normally be degraded by the 26S proteasome, the major mechanism of protein
removal in the cell. MLN4924 is more specific than previous proteasome inhibitors
because it blocks the degradation of Cullin-RING ligases (CRL), narrowing the targets
to only a handful of key regulatory proteins important in cell survival. There is currently
no existing literature on the mechanism of action of MLN4924 in neuroblastoma, we
also do not know how it will interact with standard chemotherapy. This is important in
determining how to best incorporate MLN4924 into current neuroblastoma treatment
regimens.
Methods: MTT assays were used to test the efficacy of MLN4924 and to
determine drug combination effects. Flow-cytometry was used to determine cell-cycle
arrest, presence of DNA rereplication, and apoptosis after staining with Annexin V/PI.
Beta-galactosidase activity was used to detect senescence. Whole cell extracts were
analyzed by immunoblotting from cells treated with MLN4924 for proteins targeted in
neddylation.
Results: Neuroblastoma cell lines are sensitive to MLN4924, and sensitivity is
unaffected by N-myc or p53 status. MLN4924 induced cell death is not solely mediated
by senescence, and senescence is cell line dependent. After treatment with MLN4924,
p53 mutant neuroblastoma cell lines undergo rereplication and cell-cycle arrest in G2M, while p53 wild-type cell lines exhibit cell-cycle arrest in G0-G1. After treatment with
MLN4924, proteins involved in DNA rereplication are increased in p53 mutant
compared to wild-type cell lines.
Conclusion: Neddylation affects proteins involved in DNA replication. Based on
our preliminary data, the mechanism of action of MLN4924 in neuroblastoma cell lines
appears to be p53 dependent. Further studies are on-going.
Contributors: Foster J, Zhang L, Scorsone K, Barbieri E, Muscal J, Perlaky L, Jenkins G, Zage
P, Berg S, Horton T.
85
THE ROLE OF MAGEL2 AND INTERACTING PARTNERS IN NEUROBEHAVIORAL
PHENOTYPES
Michael David Fountain
Daryl Scott, M.D./Ph.D.-Department of Molecular & Human Genetics
The maternally imprinted, paternally expressed MAGEL2 gene (affected only
when inherited from the father, unaffected when inherited from the mother) located in
the Prader-Willi critical region 15q11-13 has recently been reported as the first single
gene responsible for Prader-Willi syndrome (PWS) and Prader-Willi-like (PW-like)
phenotypes. Preliminary studies identified four individuals with truncating mutations in
MAGEL2, expressing hypotonia, feeding difficulties, and intellectual disability. A
diagnosis of autism spectrum disorder (ASD) has also been described. Since then,
over 30 individuals have been identified, of which 23 have frameshifting mutations in
MAGEL2, i.e. a c.1996dupC (p.Q666fs). Two children with this mutation are cousins,
and represent the first familial case, with genotypes and phenotypes following an
inheritance pattern that is consistent with a maternally imprinted, paternally expressed
gene. While some of the patients may resemble PWS, they are also phenotypically
distinct from classic PWS. As such, the syndrome caused by MAGEL2 point mutations
has been renamed from Prader-Willi-like syndrome to Schaaf-Yang syndrome (SYS)
(OMIM #615547). Magel2 null mice were shown to have an altered social phenotype,
in particular a deficit in appreciation of social novelty. Patients diagnosed with PWS
and SYS have been described as being able to engage in social interactions and
making friends, however, there are noted deficits in the amount of time spent with
friends and interpersonal communication, which may have some similarity to what we
observe in this mouse model.
We investigated the functional network of MAGEL2, and identified the USP7
protein to be a direct interactor of MAGEL2, critically important for endosomal protein
recycling. Database queries identified seven patients with de novo heterozygous lossof-function variants, including six contiguous, non-synonymous deletions including
USP7, and one likely pathogenic nonsense variant of USP7. The patients’ clinical
phenotypes involved developmental delay/intellectual disability, ASD, seizures,
hypogonadism, hypotonia, and aggressive behavior, suggesting a molecular and clinical
spectrum of disorders, which highlights the important role of MAGEL2 and USP7 in
human neurodevelopment.
Contributors: Yin, Jiani; Chen, Chun-an; Tao, Huifang; Schaaf, Christian P.
86
A COMPREHENSIVE INVESTIGATION OF SIGNALING AXES UPON ANTIGEN
SPECIFIC ACTIVATION OF CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS
Kristen Nicole Fousek
Advisor: Nabil Ahmed, M.D.-Department of Pediatrics
Helen Heslop, M.D.-Department of Pediatrics
Background: Chimeric antigen receptor (CAR) T cells have shown promising
results for several cancers in pre-clinical models as well as in early phase clinical trials.
Throughout these studies it has been observed that first generation CAR T cells
demonstrate successful killing of tumor cells but are unable to proliferate sufficiently
while second generation CAR T cells are able to kill tumor cells as well as proliferate
and exhibit improved survival in vitro. Furthermore, third generation CAR T cells display
enhancements in cytotoxicity, proliferation, and cell survival, however, there has not yet
been a comprehensive investigation that examines the correlation of signaling patterns
with CAR endodomains and functionality thereof. Substantial knowledge on TCR
signaling exists, and the pathways downstream of the TCR/CD3 zeta chain as well as
those of the CD28, 4-1BB, and OX40 co-stimulatory molecules on canonical T cells are
well understood. In this project I propose to build upon this T cell signaling knowledge
and investigate why distinct CAR moieties confer enhanced functionalities to T cells by
analyzing the activation of phosphoproteins involved in key signaling pathways on a
molecular level. Hypothesis: I hypothesize that encounter and binding of specific target
antigens will activate CAR signaling molecules in a dose-dependent fashion and
generate distinct quantifiable differences in the activation of surrogate nodes within the
signaling pathways, culminating in correlative changes in the effector functions of CAR
T cells. Results: I have synthesized and expressed CAR sequences (with antigenic
specificity to the tumor associated antigen HER2) comprised of 9 permutations of the ζ,
CD28, 4-1BB, and OX40 signaling moieties on T cells using a retroviral system. CAR T
cells were characterized by flow cytometry, and T cell effector function was validated
through standard immunological assays such as ELISA, co-culture, and 51Cr release
cytotoxicity assays. HER2 CAR T cells of 1st, 2nd, and 3rd generation demonstrated
the ability to specifically target HER2+ tumor cells, leading to an increase in the
production of IFN-γ and IL-2 cytokines and increased lysis of tumor cells. Future
Directions: We are currently optimizing flow cytometry and Western blot assays to
investigate trends in activation of surrogate nodes (phospho- ZAP-70, Akt, TRAF2, and
others) involved in distinct signaling pathways downstream of the co-stimulatory
domains in each of these first-, second-, and third-generation HER2 CARs. In the future
we will expand our analysis to include Luminex multiplex assays.
Contributors: Fousek, Kristen, Byrd, Tiara, Bielamowicz, Kevin, Heczey, Andras, Varadarajan,
Navin, Heslop, Helen, Dotti, Gianpietro, Hegde, Meenakshi, Ahmed, Nabil
87
THE ROLE OF ORPHAN NUCLEAR RECEPTORS NR4A IN HEMATOPOIESIS
Pablo Riera Freire
Hematopoiesis is a dynamic biological process that requires tight coordination
between the differentiation and proliferation of hematopoietic stem cells (HSC) and
progenitors. Our group has previously shown that the members of the NR4A orphan
nuclear receptor family act as tumor suppressors of Acute Myeloid Leukemia (AML) and
that conditional deletion of Nr4a1 and Nr4a3 in adult mice causes death by AML with a
mean latency of 15 weeks after induction of ablation. However, the pre-leukemic
mechanisms by which the NR4As regulate HSC development remain poorly
understood. Here, we have used a tamoxifen-inducible Rosa26-Cre-ERT2; Nr4a1fl/fl;
Nr4a3-/- mouse model to examine the cellular and molecular consequences of acute
codepletion of Nr4a1 and Nr4a3 on hematopoietic stem (HSC) and progenitor cell
homeostasis in adult mice.
Acute deletion of Nr4a1/3 leads to a rapid increase (within four days) in the
frequency of the most primitive HSC and multipotent progenitor (MPP) populations.
Proliferation analysis revealed that the accumulation of HSCs and MPPs was
associated with abnormal activation of cell cycle in both populations. An RNA-Seq
analysis of HSC after acute ablation of Nr4a1/3 revealed the down-regulation of
transcriptional networks regulated by C/EBPα and IRF8, and the up-regulation of the
interferon alpha pathway, which is known to induce HSC proliferation. Analysis of longterm ablation of Nr4a1/3 at 8 weeks after deletion reveals a partially depleted HSC
compartment due to aberrant cell cycle combined with a reduced bone marrow
cellularity and an accumulation of CD48+ multipotent progenitors. We conclude that the
NR4As exert coordinate control of the HSC transcriptome, being essential for HSC
quiescence and complete differentiation of multipotent progenitors. To identify direct
targets of the NR4As in HSCs, we are currently performing ChIP-Seq analysis of NR4A
binding and analyzing the changes in histone enhancer marks upon acute Nr4a1/3
deletion.
Contributors: Freire, Pablo; Conneely, Orla
88
METASTASIS-ASSOCIATED ALTERATION OF TG2 EXPRESSION IN
OSTEOSARCOMA
Daniel G Fuja
Advisor: Jason Yustein, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma (OS) is the most common form of primary bone cancer. Age of
OS incidence follows a bimodal distribution with a higher incidence in pediatric and
adolescent patients. It is found primarily in long bones such as the femur and humerus,
with metastases most often arising in the lung or other bony sites. Metastatic spread
of OS from the primary tumor to distant metastatic sites drastically decreases survival
rate. Current treatment of metastatic OS still largely parallels that of the non-metastatic
disease, usually consisting of surgical resection of the primary tumor and adjuvant
chemotherapy.
Treatment of metastatic OS would be facilitated by a better
understanding of factors driving osteosarcoma metastasis. Metastatic progression is
often enabled through altered expression of genes affecting motility, invasive potential,
immune evasion, and other significant functions. We hypothesize that specific genetic
and molecular changes are required for OS cells to metastasize from the primary tumor
and colonize a distant, physiologically distinct site.
Because chemotherapy is begun immediately following diagnosis of OS, there is
limited access to samples of lung metastases which have not already been exposed to
chemotherapeutic regimen and, thus, some selection. In order to investigate untreated
lung metastasis, our lab has developed genetically engineered mouse models
(GEMMs) which phenocopy either metastatic or non-metastatic osteosarcoma. Using
high-throughput genome-wide microarray, we screened RNA isolated from GEMMderived primary tumors and their corresponding metastatic lesions for genes
significantly altered. These microarrays were subsequently repeated with cell lines
derived from corresponding primary and metastatic lesions. Several factors exhibited
higher mRNA levels in lung metastases than their corresponding primary tumor, both in
tissue and cell line mRNA. Statistical analysis and stringent filtering yielded highprobability candidates which were subsequently corroborated through multiple-sample
quantitative PCR (qPCR). Some cell line protein levels were also checked through
Western Blot to confirm upregulation. Corresponding data in human-derived samples
were analyzed and confirmed upregulation of several genes, including TG2. Analysis
via Ingenuity Pathway Analysis (IPA) showed important connections to immune
evasion, cell motility, and other pro-metastatic survival functions for Transglutaminase 2
(TG2). These pro-metastatic mechanisms may be required for OS dissemination,
colonization and metastatic tumor growth. Functional studies indicate that alterations in
TG2 affects cellular phenotypes which might facilitate metastatic disease through
multiple functions. in vivo experiments in progress should demonstrate the extent of
metastatic enhancement.
Contributors: Fuja, Daniel; Kurenbekova, Lyazat; Shuck, Ryan; Ghosal, Gargi; Roos, Alison;
Satterfield, Laura; Rainusso, Nino; Khan, Fatima; Trucco, Matteo; Allen-Rhoades, Wendy;
Donehower, Lawrence A.; Yustein, Jason T.
89
THE INTERACTION OF ONCOGENIC PHOSPHATASE WIP1 AND p27KIP1 IN DNA
DAMAGE RESPONSE
Kenichiro Fujiwara
Advisor: Lawrence Donehower, Ph.D.-Department of Molecular Virology & Microbiology
A mammalian cell undergoes the DNA Damage Response (DDR) after
encountering various types of genotoxic stresses, including ionizing radiation, which
results in single or double-stranded breaks in the DNA. The sensor kinases ATM and
ATR detect this damage and activate downstream effectors through phosphorylation
cascades to promote various cellular responses, including cell cycle arrest. Once DNA
repair is complete, the expression of Wild-type p53-induced phosphatase 1 (WIP1) is
induced to return the cell to a homeostatic state. WIP1 has been shown to
dephosphorylate and downregulate various DDR mediators and effectors, including
p53, CHK1, CHK2, ATM/ATR, MDM2, and H2AX.
WIP1 is amplified and overexpressed in numerous human cancers. WIP1
overexpression in cancer cells expressing wild-type p53 leads to suppression of p53
activity. WIP1 inhibition, either chemically or through the overexpression of an
inhibitory miRNA, has been shown to suppress tumor growth via induction of cellular
senescence or apoptosis.
Thus, understanding the influence of WIP1 on various
cellular pathways involved in cancer is critical, since WIP1 has been suggested to be a
potential therapeutic target.
Currently, our knowledge of the function of WIP1 is mainly limited to its role in
the DDR pathway. Preliminary data from our lab suggests that WIP1 may play a role in
cell cycle regulation. Our findings suggest that WIP1 may target p27Kip1 (also known
as CDKN1B), a known inhibitor of CDK2 and associated cyclins that directly regulate
the cell cycle. Thus, we believe that WIP1 may play a more direct role in controlling the
cell cycle than has previously been suggested.
We hypothesize that WIP1 dephosphorylates p27Kip1 after completion of DDR
to promote cell cycle progression. To investigate the role of p27Kip1 in DDR, we are
examining the ability of WIP1 to target p27Kip1 S140, a site implicated in DDR
signaling by ATM, and testing the ability of WIP1 to promote cell cycle progression.
Previous results indicate that WIP1 dephosphorylates p27Kip1 S140 in vitro, and
western blot analysis suggests that this same site is phosphorylated by ATM in HEK
293 cells. Studies are currently underway that are designed to observe p27Kip1 S140
dephosphorylation by WIP1 in cultured cells and to elucidate the mechanism for cell
cycle regulation through WIP1 modulation of p27Kip1. The significance of this study is
the potential discovery of additional cell cycle targets for cancer treatments that involve
WIP1 modulation.
Contributors: Fujiwara, Kenichiro; Donehower, Larry
90
INSIGHTS INTO SMAD4 LOSS IN PANCREATIC CANCER FROM INDUCIBLE
RESTORATION OF TGF-β SIGNALING
Paul Thomas Fullerton, Jr.
Advisor: Martin Matzuk, M.D./Ph.D.-Department of Pathology & Immunology
Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer
death in the United States. The transforming growth factor β (TGF-β) signaling protein
SMAD4 is lost in 60% of PDAC, and this has been associated with poorer prognosis.
However, the mechanisms by which SMAD4 loss promotes PDAC development are not
fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and
CFPAC1 by selection of stable clones containing an inducible SMAD4 Tet-ON
construct. After 24h of SMAD4 expression, TGF-β signaling-dependent G1-arrest was
observed in KRAS WT BxPC3 cells with an increase in the G1-phase fraction from
48.9% to 71.5%, but no response was observed in KRAS mutant CFPAC1 cells.
Inhibition of CDKN1A by siRNA eliminated the anti-proliferative effect in BxPC3,
indicating that upregulation of CDKN1A/p21 by TGF-β signaling is necessary for the
phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced
migration. Microarray analysis of gene expression at 8h, 24h, and 48h after SMAD4
expression in BxPC3 characterized the regulatory impact of SMAD4 expression in a
SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the
novel TGF-β targets identified are anthrax toxin receptor 2 (ANTXR2; 3.58x at 8h),
tubulin, beta 3 class III (TUBB3; 7.35x at 8h), cell migration inducing protein,
hyaluronan binding (CEMIP; 8.07x at 8h), interleukin 1 receptor-like 1 (IL1RL1; 0.403x
at 8h), regulator of G-protein signaling 4 (RGS4; 0.293x at 8h), and THAP domain
containing 11 (THAP11; 0.262x at 8h). The gene expression changes we observed
upon restoration of TGF-β signaling provide numerous new targets for future
investigations into PDAC biology and progression. Based on the G1-arrest we observed
after expression of SMAD4 in the KRAS WT cell line BxPC3, we hypothesize that
SMAD4 loss is an early event of tumorigenesis that removes TGF-β signaling-mediated
growth inhibition in PDACs lacking activating KRAS mutations.
Contributors: Fullerton, Jr., Paul T.; Creighton, Chad J.; Matzuk, Martin M.
91
MICROBE DERIVED HISTAMINE EFFECTS ON INTESTINAL SUBTYPES AND
SIGNALING
Robert Steven Fultz
Advisor: James Versalovic, M.D./Ph.D.-Department of Pathology & Immunology
Chronic intestinal inflammation reduces quality of life and is deleterious to
intestinal tissues. Due to the intimate relationship between the intestinal microbiota and
host intestinal tissues, probiotic therapies are an attractive approach for treating chronic
colitis, yet no such therapy has been proven effective in inducing remission and
maintenance of chronic intestinal inflammation. Currently, therapies with the TNF
directed monoclonal antibodies are indicated and effective for IBD refractory to
corticosteroids, but are prohibitively expensive for some patients and require long-term
administration [1]. In vitro, administration of the versatile, biogenic amine, histamine
reduces expression and production of the proinflammatory cytokine, TNF in murine
Kupffer cells and THP-1 human monocytoid cells [2]. The probiotic, lactic acid
bacterium, Lactobacillus reuteri is a native inhabitant of the mammalian gut and
produces and secretes histamine. Administration of L. reuteri ATCC 6475 to mice with
TNBS-induced colitis ameliorates local and systemic markers of inflammation in a
histamine-dependent manner [†]. Production and secretion of histamine by L. reuteri
ATCC 6475 in the gut is, therefore, hypothesized to reduce colitis by inhibiting TNF
production from intestinal macrophages; however the mechanism by which this occurs
in not fully elucidated. L. reuteri 6475 metabolites have been shown to suppress IL-6 in
murine bone marrow derived macrophages and this effect depends on endogenously
produced histamine. The mechanism by which histamine signaling reduces IL-6
transcription in intestinal macrophages will be elucidated by assaying phosphorylation
states of MAP kinase proteins in macrophages exposed to histamine in vitro, or L.
reuteri ATCC 6475 in vivo. Elucidation of this mechanism is essential for understanding
the probiotic functions of microbial-derived histamine and its clinical applications and
limitations.
1. Blonski W, et al. (2011) Current Opinion in Gastroenterology, 27:346-357.
2. Thomas C, et al. (2012) PLoS ONE, 7(2):e31951.
†. Unpublished data.
Contributors: Fultz, Robert
92
TEMPORAL CHANGES IN ANGIOGENIC MONOCYTES AFTER REMOTE ISCHEMIA
IN HEALTHY ADULTS
Amir Ali Gahremanpour
Advisor: Emerson Perin, M.D.-Department of Medicine
Background: Remote ischemic preconditioning (RIPC) has been shown to have
cardioprotective effects against lethal prolonged ischemia. The underlying mechanisms
of this beneficial effect are not clear. The role of monocytes subpopulations in
angiogenesis has recently been described. We sought to study the effect of RIPC on
monocytes and on plasma cytokine levels.
Methods: We randomized 15 healthy individuals (age range, 18-25 years) to
treatment (n=10) and control groups. The treatment group was subjected to transient
ischemia by performing 5 cycles of 4-min ischemia-reperfusion using a blood pressure
cuff around one thigh and the control group to sham maneuver. Peripheral blood was
collected at baseline, 6h, 24h, 48h, 72h, and 7days follow up. Cell immunophenotyping
was performed by flow cytometry, and plasma cytokines levels were measured by
Luminex multiplex assay.
Results: In the treatment group only the angiogenic monocyte (Tie2+CD14+CD16mid cells) subpopulation increased at 48 h after RIPC (523.68 ± 253.18
vs 1157.956±261.505 X 106/L, P=0.0001). There was no change in total monocyte
population. There was also a rapid decline in Granulocyte-colony stimulating factor (GCSF) at 6h compared to baseline (55.1±6.2 vs 40.2±4.7 pg/ml P=0.015), which returned
to baseline level at 24h.
Conclusion: This study provides the first evidence about the role of angiogenic
monocyte in RIPC in human. Experiments are on going to elucidate how and hypoxia
and GCSF are involved in phenotypic transformation of monocytes
Contributors: Gahremanpour, Amir; Resende, Micheline; Willerson, James T; Taylor Doris A;
Perin, Emerson.
93
NEURONAL ACTIVITY INDUCES THE BIOGENESIS OF SYNAPTIC VESICLES
THROUGH THE TRP CHANNEL
Upasana Gala
Synaptic vesicles (SVs) are the source of information relay between neurons.
Much has been studied about synaptic transmission and how synaptic vesicles fuse
with the plasma membrane at the terminals and are then recycled via endocytosis.
However, the question of their biogenesis is still largely unexplored. My aim is to identify
the trigger(s) that result in the synthesis of new synaptic vesicles. Transmission electron
microscopy (TEM) is the best way to study synaptic vesicle synthesis in detail. We
performed TEM on photoreceptor terminals of flies mutant for the α1 subunit of P/Q
type VGCC, cacophony (cac), which is required for SV exocytosis. We observed that
the mutant terminals start accumulating SVs at day 3 and this phenotype gets worse as
the flies age. Surprisingly, we find that when we raise cac mutant flies in complete
darkness and deprive the photoreceptor neurons of activity, we can rescue the SV
accumulation. This led us to hypothesize that neuronal activity triggers SV biogenesis.
Upon light stimulation of the photoreceptor neurons, several downstream activities take
place. Rhodopsin in the photoreceptor cell bodies is activated, which in turn activates
the light sensitive TRP channels. Opening of the TRP channels allows calcium influx
into the neuron. In addition, light stimulation results in depolarization of the neuron and
eventually fusion of synaptic vesicles in the terminal with the plasma membrane and
exocytosis of neurotransmitters. Upon testing these different downstream components,
we identified that rhodopsin affects SV accumulation. In the absence of rhodopsin, WT
terminals show reduced number of SVs. Interestingly, cac mutants raised on vitamin A
deficient food that mimics rhodopsin deficiency, show a complete rescue of the SV
accumulation phenotype even in the presence of light. We further observed that in
mutants with constitutively active Trp channel, SV accumulation occurs even in the
absence of light. We plan to next pinpoint if calcium influx through the Trp channel is
the signal responsible for SV biogenesis.
Contributors: Gala, Upasana; Zhongyuan, Zuo; Bellen, Hugo
94
A cAMP INDEPENDENT PATHWAY FOR CALCIUM MOBILIZATION BY BETA-2
ADRENERGIC RECEPTOR
Monica Laura Galaz-Montoya
Beta adrenergic receptors (β2-AR) are important for vascular regulation in the
heart and lung and for physiological responses to the hormones/neurotransmitters
adrenaline and noradrenaline. They are found in the nervous system and throughout
the body and are the targets of numerous widely used drugs. Their canonical signaling
pathway involves activation of adenylyl cyclase (AC) and a rise in cyclic AMP (cAMP)
levels, which leads to activation of cAMP-dependent protein kinase (PKA). Beta
adrenergic receptors have long been thought to activate distinct signaling pathways
from those downstream of G-protein coupled receptors which activate phospholipase C
(PLC) and elevate intracellular Ca2+. By monitoring intracellular Ca2+ levels in real time
using a fluorescent indicator dye we found that an endogenous receptor in HEK-293
cells responds to the adrenergic agonist norepinephrine by a delayed rise in
intracellular [Ca2+]. The response is blocked by ICI 118,551, a selective antagonist for
β2-AR, and the relative potency of agonists is isoproterenol > epinephrine >
norepinephrine, consistent with the pharmacological profile of β2-AR. Treatment with
thapsigargin (an inhibitor of the SERCA Ca2+ pump of the endoplasmic reticulum) and
chelation of extracellular Ca2+ revealed that the Ca2+ is released from intracellular
stores. The release is sensitive to inhibition of PLC with U73122 and of InsP3
receptors with 2-APB. Treatment with cholera toxin, a drug that activates Gαs, indicated
that direct activation of this G protein is not sufficient for Ca2+ release, although it
increases intracellular cAMP. Additionally, treatment with adenylyl cyclase inhibitors SQ
22536 and dideoxyadenosine did not inhibit isoproterenol-induced Ca2+ responses
whereas both inhibitors were able to suppress cAMP production by AC. Additionally, an
increase in intracellular cyclic AMP levels by treatment with the phosphodiesterase
inhibitors IBMX does not potentiate the response of adrenergic agonists. Furthermore,
treatment with PKA inhibitors H-89 and KT5720 had no effect on the Ca2+ signal, and
treatment with the cAMP analogue 8-bromo-cAMP, which selectively activates PKA, did
not trigger a Ca2+ response even though it was effective at triggering PKA induced
CREB phosphorylation. These results strongly support the conclusion that AC, cAMP
and the cAMP effector PKA are not involved in this signaling pathway. Together these
findings indicate that activation of β2-AR leads to an increase in cytoplasmic [Ca2+] by
a previously unrecognized signaling pathway. A novel mechanism for Ca2+ mobilization
by β2AR has broad implications for adrenergic signaling, and in particular for the effects
of βAR-directed drugs.
Contributors: Galaz-Montoya, Monica; Rodriguez, Gustavo; Lichtarge, Olivier and Wensel,
Theodore
95
EVOLUTION BASED SITE DIRECTED MUTAGENESIS CAN REDESIGN RECEPTOR
SIGNALLING OF G PROTEIN-COUPLED RECEPTORS
Jonathan Gallion
A genotype-based analysis of protein evolution can guide modern protein
engineering by predicting the impact specific mutations have on protein phenotype.
Evolutionary Trace (ET) performs a sequence alignment on groups of homologous
proteins and measures the correlation between genotypic perturbations and phenotype
divergence, among the evolutionary tree, to measure residue importance in protein
function. Utilizing ET we designed 28 mutations representing 9 residues with various
evolutionary importance throughout β-2-Adrenergic Receptor (β2AR), a G-protein
coupled receptor. These mutations were designed to bias receptor signaling by
targeting regions of evolutionary importance using mutations with a range of
Evolutionary Action (EA) scores, a measure of magnitude of impact on function. A
clustering analysis of the resulting signaling profile for each mutation, as measured by
six downstream assays, revealed that experimental behavior matched predictions; high
impact mutations at evolutionary important residues were most detrimental, resulting in
a universally disrupted phenotype across all pathways of β2AR. Mutations with
intermediate impact, however, differentially altered receptor signaling by preferentially
affecting one pathway over another. This study successfully demonstrates a targeted
protein redesign using computationally guided site directed mutagenesis based on
evolutionary divergences.
Contributors: Shonegge, Anne-Marie; Wilkins Angela
96
NEURAL CORRELATES OF BEHAVIORAL CHOICE IN THE MACAQUE BRAIN
STEM
Courtney Dawn Garcia
Advisor: Dora Angelaki, Ph.D.-Department of Neuroscience
Many sensory systems have been studied extensively in regard to the process of
encoding, or characterizing neural responses to known stimuli, but very little is known
about the process of decoding, production of a percept on a neural level. The vestibular
system provides an excellent model to probe the origins of perception because the
same basic forms of motion directional selectivity are seen at many levels of
processing, from afferents to cortex. Previous studies of rotational motion thresholds
have a fatal flaw. No simultaneous comparison in the same animal between behavioral
and neural detection thresholds was performed. Here we set out to determine if neural
choice related activity is exhibited at the level of the brainstem by utilizing simultaneous
recording of rotational motion perception and neural activity of the vestibular nuclei, VN,
and horizontal canal afferents in rhesus macaques. We measured behavioral and
neural sensitivity to rotational motion and choice-related activity or trial-by-trial
correlations, known as Choice Probabilities, between the animal’s rotation perception
and neural activity. From preliminary data collection of 7 VN neurons and 13 canal
afferents, the mean afferent neural threshold was found to be 5.2°/s while the mean VN
neural threshold was 4.9°/s. Two of 7 VN cells exhibited significant choice-related
activity and were the most sensitive of the recorded population with an average neural
threshold of 1.9°/s, while the VN cells without significant choice-related activity had a
mean neural threshold of 6.1°/s. The cells exhibiting significant choice-related activity
were determined to be rotation only VN cells, with no convergence of translational
motion driven afferents. The preliminary data support a hypothesis that cells encoding
the most relevant information to the task are exhibiting significant choice-related
activity.
This work was supported by NIDCD DC04260.
Contributors: Garcia, Courtney D.; Liu, Sheng; Dickman, J. David; Angelaki, Dora E.
97
PREVENTING BREAST CANCER IN POSTMENOPAUSAL WOMEN RECEIVING
COMBINED HORMONE REPLACEMENT THERAPY
Grady Manuel Gastelum
Advisor: Yi Li, Ph.D.-Department of Molecular & Cellular Biology
Polly Niravath, M.D.-Department of Medicine
Menopause occurs in women on average at age 51 and is induced by the
cessation of ovarian estrogen and progesterone synthesis. Hormone replacement
therapy (HRT) has been used to treat symptoms of menopause including hot flashes,
vaginal dryness, and osteoporosis. Originally, estrogen-only therapies were used, but
these treatments were later correlated with an increased risk for endometrial cancer.
The addition of a progestin to the treatment regimen nullified the elevated endometrial
cancer risk. However, this combination increased breast cancer risk by 24%. Since the
increased breast cancer risk is only present in combined HRT, but not estrogen-only
therapy, the increased risk is likely due to the progesterone signaling pathway.
Therefore, inhibition of carcinogenic signaling within this pathway may combat the
elevated cancer risk while maintaining its therapeutic benefits. It has been shown that
STAT5 and RANKL mRNA expression are significantly upregulated in the mammary
glands of macaque monkeys treated with combined HRT compared to estrogen-only
and control animals. Overexpression of RANKL in the mammary epithelial compartment
induces increased proliferation which results in formation of hyperplasias. Furthermore,
our lab has previously shown that STAT5, in response to pregnancy, can lead to
apoptosis evasion and accelerated tumorigenesis. Given these observations, we
hypothesize that combined HRT increases risk of breast cancer through RANKL and/or
STAT5 signaling. Therefore, pharmacological inhibition of either could mitigate breast
cancer tumorigenesis in combined HRT users. In this project we will use rodent HER2enriched and ER+ breast cancer models to study combined HRT promotion of
premalignant lesions to tumors.
Aim 1: Investigate STAT5 co-activation of PR transcriptional targets in combined
HRT-stimulated breast cancer.
Aim 2: Determine if RANKL is a critical mediator of combined HRT-stimulated
breast cancer.
Aim 3: Determine the efficacy of pharmacologically inhibiting STAT5 and/or
RANKL to prevent combined HRT-stimulated breast cancer.
If successful, the proposed research will identify STAT5 and/or RANKL as
therapeutic targets for preventing combined HRT-induced breast cancer. This insight
has the potential to reposition two FDA-approved drugs, targeting STAT5 and RANKL
signaling, for breast cancer prevention in combined HRT users.
Contributors: Gastelum, Grady; Niravath, Polly; Li, Yi
98
THE ROLE OF H3K4ME3-PROMOTED HISTONE ACETYLATION IN
TRANSCRIPTION
Leah Ashley Gates
Epigenetic modifications such as histone marks are critical regulators of gene
expression and chromatin structure. Determining the molecular mechanisms of how
these modifications impact transcription is therefore necessary to understand cell
physiology and pathologies resulting from altered gene expression. These histone
marks serve to promote the recruitment or dismissal of coregulators that can influence
the transcriptional outcome through the direct binding of effector proteins, or “readers”.
Trimethylation on histone H3 at lysine 4 (H3K4me3) stimulates both transcription and
subsequent histone acetylation selectively on histone H3 at lysine 9 (H3K9Ac). We
therefore hypothesized that H3K9Ac recruits specific reader proteins for progression
through the transcription cycle. By performing biotinylated histone peptide pulldowns
with HeLa nuclear extract followed by mass spectrometry and immunoblotting, we
identified the super elongation complex (SEC) as being recruited to H3K9Ac through
the reader protein AF9. Importantly, reduction of H3K4me3 results in the global
decrease of H3K9Ac and histone H4 acetyl marks. Additionally, loss of H3K4me3 and
H3K9Ac results in reduced AF9 occupancy on select genes, as well as reduced gene
expression. However, RNA polymerase II occupancy increases at select gene
promoters with the loss of these histone marks, indicating a defect in promoter
clearance and transcription elongation. Based on this data, we propose a model in
which H3K4me3 is required for transcription initiation and promotes acetylation of H3K9
through the recruitment of select histone acetyltransferases. This acetylation can then
recruit transcription elongation machinery via AF9. Next, we will determine the direct
functional relevance of H3K9Ac in transcription using biochemical functional assays.
Importantly, these studies will advance our understanding of how epigenetic marks can
regulate transcription at the level of RNA polymerase II elongation.
Contributors: Gates, Leah A.; Feng, Qin; Rohira, Aarti D.; Bedford, Mark T.; Sagum, Cari A.;
Jung, Sung Yun; Qin, Jun; Tsai, Sophia; Tsai, Ming Jer; Foulds, Charles E.; O’Malley, Bert W.
99
LOSS OF MIR-148 ENHANCES OVARIAN CANCER GROWTH AND METASTASIS
BY PROMOTING MTMR9 EXPRESSION
Triparna Ghosh-Choudhury
Advisor: Matthew Anderson, M.D./Ph.D.-Department of Obstetrics & Gynecology
Creighton Edwards, M.D.-Department of Obstetrics & Gynecology
Introduction: MicroRNAs (miRNAs) are endogenous, non-coding RNA transcripts that
play a critical role in regulating gene expression. Previous work has reported that levels of miR148 are an important determinant of ovarian cancer survival. However, the mechanisms by
which miR-148 contributes to ovarian cancer remain poorly understood.
Methods: Taqman miRNA assays were used to measure levels of miR-148a, miR148a*, and miR-148b. Proliferation and apoptosis were measured using MTS and Caspase Glo
3/7 assays (Promega). Migration and invasion were measured in vitro using Boyden chamber
assays (BD Biosciences). Western blot and quantitative real time PCR were used to validate
gene expression. In vivo experiments were done using novel DOPC liposomes containing
siRNA. All mice were treated intraperitoneally. Statistical significance was calculated using twotailed t-tests.
Results: We found that levels for miR-148a, miR-148a* and miR-148b were significantly
lower when flash frozen specimens of high grade serous ovarian cancers (n=10) were
compared to fallopian tube (n=3) and ovary (n=3). Transfection of TP53-null SKOV3ip1, TP53mutated (OVCAR8, OVCAR5) and TP53-wild type (HeyA8, OVCA433) ovarian cancer cells with
mimics for these miRNAs significantly inhibited proliferation, enhanced apoptosis and inhibited
both migration and invasion. Targets for miR-148 were identified by screening Level 3 ovarian
cancer data from the TCGA (n=483 specimens) using Lasso analysis with L1 normalization,
after which, potential targets were cross referenced against established target prediction
algorithms (Targetscan, RNA22). These analyses pinpointed MTMR9, a poorly understood
gene product known to regulate phosphoinositide metabolism, as a key target for miR-148
miRNAs in ovarian cancer. Enforced expression of each miR-148 transcript inhibited MTMR9
expression, leading to suppressed catalytic activity of MTMR6 and MTMR8 and enhanced rates
of apoptosis and autophagy. Consistent with these observations, we found that knockdown of
MTMR9 directly reduced proliferation and induced apoptosis in multiple ovarian cancer cell
lines. Finally, RFP OVCAR8 cells were injected via IP into mice abdomens and then monitored
for tumor growth. Two weeks later, all mice were treated with either MTMR9 siRNA or NT
siRNA. We found that the mice receiving bi-weekly treatments of siMTMR9 had significantly
less tumor and more interestingly, less to no metastasis of the ovarian cancer. The mice treated
with siNT liposomes had metastasis past the diaphragm and all along the abdominal wall.
Conclusions: Our findings indicate that at least 3 of the known miR-148 transcripts
function as robust tumor suppressors in ovarian cancer by regulating MTMR9 expression.
These observations provide a biologic basis for the association between levels of miR-148 and
ovarian cancer outcome. They also implicate novel aspects of phosphoinositide metabolism in
ovarian carcinogenesis.
Contributors: Ghosh-Choudhury, T; Wan, Y; Liu, Z; Anderson, M
100
THE COGNITIVE AND BEHAVIORAL PHENOTYPES OF INDIVIDUALS WITH
CHRNA7 DUPLICATIONS
Madelyn Arlene Gillentine
Chromosome 15q13 is one of the least stable regions in the genome, due to the
presence of low copy repeat elements (LCRs) that cluster into six breakpoints (BP1 to
BP6). These LCRs make the region vulnerable to non-allelic homologous recombination
(NAHR), resulting in recurrent copy number variants (CNVs). Between BP4 and BP5,
multiple recurrent CNVs have been observed. Large deletions of 1.5 Mb to 2 Mb have
been well established as pathogenic, with high penetrance of multiple neuropsychiatric
phenotypes observed, including intellectual disability (ID), developmental delay (DD),
autism spectrum disorder (ASD), schizophrenia, and epilepsy. Smaller deletions
encompassing only CHRNA7 have also been seen in individuals with a similar range of
phenotypes and penetrance. However, the significance of duplications spanning
CHRNA7 has been enigmatic, as gains of CHRNA7 have been found to occur at similar
frequencies in individuals with neuropsychiatric disease and controls. Using a genotypeto-phenotype approach, we evaluated 18 individuals, who had been identified to carry a
CHRNA7 duplication by clinical chromosome microarray analysis. Duplication sizes
varied from 148 kb to 3.231 Mb. Formal cognitive assessment of the 18 probands (age
2 to 14) revealed an average full scale IQ of 80.2, being significantly lower than the
average population. Research reliable testing for autism spectrum disorder was positive
in 41% of cases, as determined by the Autism Diagnostic Interview –Revised (ADIR)
and Autism Diagnostic Observation Schedule (ADOS). Additional phenotypes included
developmental delay, language and speech delay, attention deficit/hyperactivity
disorder (ADHD), and epilepsy in a subset of patients. Inheritance was determined in 13
of 18 cases, with just over half of the transmitting parents manifesting a neurological
phenotype, indicating that these duplications are incompletely penetrant. This study
suggests that CHRNA7 duplications may be pathogenic, and that affected individuals
manifest cognitive and behavioral phenotypes. However, ascertainment bias needs to
be considered in this context. A detailed neuropsychological phenotyping of individuals
who were not referred as probands, but subsequently identified to carry a CHRNA7
duplication, or individuals with a prenatal diagnosis of CHRNA7 duplication could be
considered to overcome that bias.
Contributors: Gillentine, Madelyn A.; Schaaf, Christian P.
101
LETHAL LOVE: BIOENGINEERING A BACTERIUM WITH ANTIMICROBIAL
ACTIVITY BY EXPLOITING BACTERIAL CONJUGATION TO DELIVER TOXIC
ELEMENTS
Mary Elizabeth Girard
Advisor: Christophe Herman, Ph.D.-Department of Molecular & Human Genetics
Bacterial infections resistant to antibiotics are an alarming threat as the incidence
numbers continue to rise, leaving us in need of new antimicrobial methods. We propose
to engineer an antimicrobial bacterium, or rather, a bacterium able to kill other bacteria.
With this novel antimicrobial approach, we highjack bacterial conjugation as a platform
to deliver toxic elements to bacteria. Conjugation is the horizontal transfer of genes
between bacteria via a conjugative plasmid through a sex pilus from the donor to the
recipient bacterium. This process is a very efficient mode of gene transfer and is quite
promiscuous, theoretically allowing us to target a variety of bacteria. Additionally, with
the current methodologies of bacterial genetic engineering, we are able to construct a
variety of conjugative plasmids encoding toxic genes of choice with ease. For the toxic
component of our antimicrobial platform, we envision utilizing either toxins from
bacterial toxin-antitoxin systems or the Cas endonuclease from bacterial CRISPR/Cas
systems. Both options are highly advantageous, untapped resources for potential
antimicrobials. The TA system toxins are known to be quite toxic to their bacterial hosts
in absence of the cognate antitoxin, and have thus often been suggested as putative
alternatives to antibiotics. Here, the engineered bacterium carrying the toxin-encoding
conjugative plasmid mates with a recipient, transferring and expressing the toxin, killing
the recipient bacterium. For our other choice toxic element, the CRISPR/Cas systems
are also known to be lethal to the host bacterium if targeted to cleave bacterial host
DNA. The CRISPR system is very exciting as a putative antimicrobial as the Cas
nuclease can be targeted to cleave very specific SNPs of DNA, and this specificity
allows us to target its lethal activity to bacterial pathogen SNPs. In this case, the
engineered bacterium carries a conjugative plasmid encoding the Cas nuclease
targeted to a desired pathogen SNP. Upon mating, the Cas nuclease specifically
cleaves the pathogen DNA, thereby only killing the desired pathogen.
Taken together, this antimicrobial platform combines the toxic elements of TA
systems and CRISPR/Cas systems with conjugation as a delivery mechanism,
transferred by a harmless bacterial carrier. We are initially testing this antimicrobial
strategy by performing mating assays between our engineered strains against nonpathogenic lab strains of E. coli, scoring for recipient cell viability. Preliminary data
shows this method is effective against E. coli, and next, we will be performing mating
and viability assays against a variety of bacterial pathogens. In the future, this work may
lead to the development of this system for application in biotherapeutics or
biocontainment.
Contributors: Girard, Mary; Herman, Christophe
102
OPTICAL COHERENCE TOMOGRAPHY AS AN ADJUNCT TO WHITE LIGHT
CYSTOSCOPY FOR INTRAVESICAL REAL−TIME IMAGING AND STAGING OF
BLADDER CANCER
Guilherme Godoy
Advisor: Seth Lerner, M.D.-Department of Urology
Introduction and Objectives : Optical coherence tomography (OCT) is a novel,
real-time endoscopic imaging modality that permits delineation of microarchitectural
features of bladder lesions. The purpose of this study was to assess the application of
OCT using the Niris Imaging System® (Imalux Corp., Cleveland, OH) for improving the
endoscopic diagnosis and staging accuracy of bladder lesions. A secondary objective
was to correlate white light cystoscopic impression with histopathologic stage.
Methods: We conducted an investigator-initiated, multicenter prospective trial
designed to validate our prior single-center retrospective data (Urology 72:133, 2008)
on the use of OCT as an adjunct to conventional cystoscopy. Patients with a history of
bladder cancer or hematuria were enrolled in the study. We performed white light
cystoscopy and OCT imaging of visible tumors and normal-appearing mucosa prior to
biopsy or resection, evaluated the images in real time, and subsequently compared
them with pathology results.
Results: We obtained OCT images of 75 suspicious lesions in 67 patients
undergoing bladder biopsy or transurethral resection of bladder tumor. Age of the
patients ranged from 45 to 97 years (mean, 70.3 years), with 61 men (91%) and 6
women (9%). White light cystoscopy and OCT were both able to distinguish benign
from neoplastic lesions with 100% sensitivity, 25% specificity, and 96% accuracy. OCT
correctly identified tumors confined to the mucosa (Ta or Tis) in 48 of 51 lesions with
94% sensitivity, 60% specificity, and 85% accuracy. OCT detected any level of tumor
invasion (T1 or higher) in 11 of 16 lesions with 69% sensitivity, 95% specificity, and
89% accuracy. Muscle-invasive tumors (T2) were detected in 2 of 6 lesions with 33%
sensitivity, 97% specificity, and 92% accuracy. OCT was able to differentiate malignant
from benign lesions with a PPV of 96% and NPV of 100%. PPV for muscle invasion
was 50% with a NPV of 94%.
Conclusions: OCT is a rapid, easy-to-use tool that can help differentiate Ta
and T1 bladder tumors and identify muscle-invasive lesions. This prospective study
validates our initial observations suggesting that OCT provides real-time
microarchitectural information that can aid in the clinical staging of bladder tumors.
Accurate information about stage and depth of invasion may have an impact on the
endoscopic surgical management of these lesions.
Contributors: Godoy, Guilherme; Cheng, Philip J.; Sanchez, Edward J.; Goh, Alvin C.; Slaton,
Joel W.; Scherr, Douglas S.; Lerner, Seth P.
103
THE ROLE OF BMP ANTAGONISTS, GREMLIN1 AND GREMLIN2, IN MOUSE
OVARY DEVELOPMENT
Nisha Thara Gokul
Advisor: Stephanie Pangas, Ph.D.-Department of Pathology & Immunology
The reproductive lifespan of women is thought to be determined by the ovarian
reserve. The ovarian reserve refers to the finite pool of non-growing primordial follicles
containing oocytes that is established before birth. Its depletion over time leads to
menopause. Some female reproductive pathologies, such as primary ovarian
insufficiency, are a result of reduced numbers or poor quality oocytes. Because the
ovarian reserve is established in the developing embryo, mouse models have been
valuable in understanding the signaling pathways important to ovary development. The
bone morphogenetic protein (BMP) signaling pathway is important in many stages of
oocyte development, including migration of primordial germ cells to the genital ridge,
proliferation, and possibly meiotic initiation. BMP activity can be modulated by
antagonists such as gremlin-1 (GREM1) and gremlin-2 (GREM2). Previous research
from our lab shows that mice homozygous null for Grem1 have impaired limb and
kidney development, as well as fewer oocytes and primordial follicles with delayed
progression through meiosis. We hypothesize Grem1 and Grem2 are required in the
developing mouse ovary to properly establish the ovarian reserve. To further unravel
the role of these BMP antagonists in ovarian development, we have generated a novel
mouse line homozygous null for both Grem1 and Grem2 (Grem1-/- Grem2-/-). Utilizing
a reporter mouse containing green fluorescent protein (GFP) downstream of a BMP
response element, we have identified BMP target cells in the developing mouse ovary.
Through analysis by histologic and apoptotic staining, we discovered that double
mutant ovaries have a unique phenotype from each of the single mutants. Future
studies will determine changes in ovarian structure, germ cell cyst breakdown, and
meiotic progression over time and amongst wild type, single knockout, and double
knockout Grem1-/- Grem2-/- mice utilizing confocal and light sheet microscopy.
Through these experiments, we will determine the role of the BMP antagonists Grem1
and Grem2 and the pathways that they regulate in the developing mouse ovary. These
pathways may underline female reproductive disorders and may provide clinically
relevant candidate genes for diagnosis and treatment of patients.
Contributors: James, Rebecca; Chuva de Sousa Lopes, Susana; Pangas, Stephanie
104
REGULATION OF E2F1 TRANSCRIPTIONAL ACTIVITY BY SUMOYLATION
Joshua Daniel Graves
Advisor: Weei-Chin Lin, M.D./Ph.D.-Department of Medicine
Background: The E2F family of transcription factors consists of crucial proteins in
higher eukaryotes with functions including cell proliferation, apoptosis, senescence, and
metabolism. How a handful of proteins, and in some cases the same family member
(e.g. E2F1), can carry out such opposing functions is an unresolved paradigm. Thus far,
specifying and directing activity has been largely attributed to the numerous posttranslational modifications found on E2Fs. We have demonstrated that the small
ubiquitin-related modifier 2 (SUMO2) modifies E2F1-5. Due to the high frequency of
perturbations in the Rb/E2F1 pathway in all cancer types, E2F1 is the main focus of
further study. These observations lead us to hypothesize that the SUMO-2 modification
on E2F1 regulates its transcriptional activity, consequently altering the cell cycle,
apoptotic propensity, or the DNA damage response.
Materials and Methods: Sumoylation assays were performed by nickel affinity
purifying 6XHis-SUMO# from HEK 293T cell lysates under denaturing conditions. E2F1
transcriptional activity was assayed by qRT-PCR or a dual luciferase reporter assay in
H1299 cells, with p14/ARF-luciferase and thymidine kinase-renilla constructs.
Results: More than 95% of SUMO2 conjugation on E2F1 requires two lysines of
E2F1. Overexpressing Ubc9, the sole E2 conjugating enzyme in the SUMO pathway,
greatly induces modification dependent on these two lysines. When using an E2F1specific luciferase reporter assay, Ubc9 overexpression reduced E2F1 transcriptional
activity by up to 60%. Additionally, E2F1 point mutants for sumoylated lysines
significantly rescued this Ubc9-mediated transcriptional repression. This suggests that
sumoylation specifically on E2F1 is responsible for the observed decrease in activity.
Furthermore, reduction of transcriptional activity by Ubc9 has been recapitulated at
endogenous E2F1 target genes.
Conclusions: We have identified a novel modification on E2F1 that inhibits and
potentially specifies its transcriptional activity. E2Fs1-3 have essential roles in
promoting cell growth, as is exemplified by the fact that a triple knockout mouse is
embryonic lethal. Consequently, the pathways that inhibit their activity are highly
mutated in all cancer types. By further understanding how SUMO2 addition to E2F1
inhibits its transcriptional activity, as well as how this mechanism is perturbed in cancer,
new pharmacologic targets to inhibit E2F1 dependent cell growth could be presented.
Moreover, since SUMO2 has also been observed on E2Fs2-3, the findings of this and
future work might yield a means to shut down all E2F dependent growth in cancer.
Contributors: Graves, Joshua; Lin, Weei-Chin
105
NOVEL NANO-ANTIOXIDANT TREATMENT IN A MODEL OF NON-ALCOHOLIC
FATTY LIVER DISEASE
Deric Maurice Griffin
Advisor: Robia Pautler, Ph.D.-Department of Molecular Physiology & Biophysics
Rajesh Krishnamurthy, M.B.,B.S.-Department of Radiology
Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders,
encompassing hepatosteatosis (fatty liver), non-alcoholic steatohepatitis, and cirrhosis.
NAFLD’s incidence has been increasing alongside obesity in the U.S. and is the leading
indication for liver transplant in the United States. Oxidative stress has been heavily
implicated in disease pathology, but an effective antioxidant therapy is still unavailable.
Our lab is currently investigating the efficacy of pegylated-hydrophilic carbon clusters
(PEG-HCCs), a bioengineered nano-antioxidant, as a possible antioxidant therapeutic.
Here we use ob/ob mice as an early model of NAFLD, which displays obesity,
hepatosteatosis, and elevated blood glucose, along with elevated liver damage and
oxidative stress markers. Male ob/ob and C57BL/6J (WT) mice, 5-8 weeks in age, were
fed either a normal chow (NC) or vitamin E enriched chow (vitE) in addition to receiving
weekly PEG-HCC or saline intravenous tail injections for 6-10 weeks. Ob/ob mice that
were in the PEG-HCC+NC and saline+VitE groups showed significant improvements in
aminotransferase levels, but only PEG-HCC treated ob/ob mice showed significant
improvements in non-fasting glucose, c-jun N-terminal kinase levels and oxidative
stress markers as compared to the NC+saline control group. Ob/ob and WT mice
treated with NC+PEG-HCCs or VitE+saline showed significant improvements in glucose
handling via glucose tolerance tests, while WT mice showed increased insulin
sensitivity via insulin tolerance tests, respectively. Currently, we are using histology to
evaluate any changes in liver pathology in addition to investigating the mechanism for
these observed effects by using western blotting to assess insulin and stress signaling
in the liver as it relates to oxidative stress.
Contributors: Griffin, Deric; Pautler, Robia; Tour, James
106
HIGH-THROUGHPUT FUNCTIONAL SCREENING FOR METASTASIS DRIVERS OF
LUNG CANCER
Caitlin L. Grzeskowiak
Large-scale profiling efforts by consortia such as The Cancer Genome Atlas
(TCGA) are revealing the complexity of cancer genomes, which are comprised of
causal “driver” aberrations and biologically neutral “passengers”. Most cancers acquire
one or more well-studied high frequency driver events promoting tumor growth (e.g.,
mutations/copy number changes in KRAS, TP53, EGFR, MYC). Much less is known
about the thousands of low frequency gene aberrations and their contribution to cancer
progression, particularly metastasis, which is the primary cause of cancer-related
mortality. Comprehensive biological assessment of low frequency metastasis drivers is
difficult given their large number and the fact that their activity may be influenced by the
specific biological context of a given cancer such as tissue type, microenvironment, and
the host immune system. We sought to address these challenges in the context of lung
cancer, which presents as metastatic disease in approximately 65% of patients and
carries a 5-year survival rate of <15%. To do this, we leveraged our High-Throughput
Mutagenesis and Molecular Barcoding (HiTMMoB) technology allowing (1) construction
of gene “libraries” by high-throughput, accurate modeling of somatic aberrations
(missense, nonsense, indels) or wild-type genes (representing amplifications) using our
robotics driven platform of >35,000 sequence verified human gene clones (2) a
molecular barcoding strategy that permits simultaneous DNA tagging of gene clones
through multi-fragment DNA recombineering for (3) pooled functional screening in vivo
to identify metastasis drivers that work alone or in combination. We used these
technologies to build gene libraries based on oncogenomics-guided integrations of
mutant KRAS-specific gene signatures derived from mouse and human TCGA lung
cancer datasets. The resulting barcoded libraries were delivered to non-metastatic lung
cancer cells expressing oncogenic KRAS and then implanted into immune competent
mice. Animals were sacrificed at 6-8 weeks where resulting primary tumors and
metastases were subjected to barcode enrichment analysis by next generation
sequencing to identify gene aberrations enriched within metastatic lesions. Our
screening approach identified known (MYC and SNAI2) and many novel (e.g., MBIP
and CCNE1) potent drivers of lung cancer growth and metastasis currently under
mechanistic and pre-clinical evaluation. These efforts are revealing new pathways
contributing to lung cancer aggression and our ultimate goal is to translate these
findings into the care of metastatic lung cancer patients who have few treatment
options. We have also scaled these efforts across other screening platforms,
functionalizing thousands of aberrations revealing the highest priority candidates across
diverse cancer types.
Contributors: Kundu, Samrat; Wu, Ping; Minelli, Rosalba; Creighton, Chad; Gibbons, Don L.;
Scott, Kenneth L.
107
A NOVEL MODEL OF LUNG ANTIGEN-INDUCED AUTOIMMUNE-MEDIATED LUNG
DISEASE IN MICE
Bon-Hee Gu
Advisor: Farrah Kheradmand, M.D.-Department of Medicine
Background: Chronic obstructive pulmonary disease (COPD) and emphysema
are common destructive inflammatory diseases of the lung and the 3rd leading cause of
death. Cigarette smoke is the most common cause of COPD/emphysema. However,
the pathogenesis of COPD/emphysema and mechanisms involved in smoking induced
lung conditions are not fully understood. We have previously shown that CD4 T cells in
peripheral blood mononuclear cell (PBMC) of smokers with emphysema differentiate to
T helper type 1 (Th1) and Th17 cells in response to elastin, indicating an increased
abundance of autoreactive T cells. Therefore, we hypothesized that loss of tolerance to
elastin in mice could result in lung inflammation and destruction as seen in smokers
with COPD/emphysema. Methods: Mice were subcutaneously immunized with human
elastin in complete Freund’s adjuvant (CFA) at the start of the experiment and
immunized once a week thereafter with mixture of human and rat elastin in incomplete
Freund’s adjuvant (IFA) for 6 weeks. Lung histology, microCT of lungs, and complete
systemic and immune responses were examined 6 days after the final immunization.
Results: Elastin immunized mice developed emphysema with increased lung volume
determined by microCT of the chest and enlarged alveolar spaces detected in lung
histology. Also, elastin immunized mice had increased number of macrophage and
lymphocyte in bronchoalveolar lavage (BAL) fluid, increased MMP9 expression in BAL,
increased frequency of IL-17+ and IFN-γ+ cells in the lung, and increased anti-elastin
antibody in the serum.
Conclusion and Significance: This is the first animal model of antigen-specific
autoimmune lung disease that shares features with smoking induced
COPD/emphysema. This novel animal model will be critical in dissection of the
mechanisms involved in loss of peripheral tolerance to self-antigens in smokers with
emphysema. Future studies will explore whether loss of tolerance could result in
inflammatory changes in other elastin rich organs such as the aorta and skin.
Contributors: Gu, Bon-Hee; Yuan, Xiaoyi; Corry, David; Kheradmand, Farrah
108
TRYPTOPHAN 2,3-DIXOYGENASE (TDO2) PROMOTES BREAST CANCER
PROGRESSION THROUGH NAD METABOLISM AND ARYL HYDROCARBON
RECEPTOR SIGNALING
Franklin Gu
Tryptophan catabolism is linked to malignant progression of breast cancer
through the suppression of anti-tumor immune responses. The initial rate-limiting step
in tryptophan catabolism is catalyzed by tryptophan 2,3-dioxygenase (TDO2) and
indoleamine 2,3-dioxygenase (IDO), enzymes that share low structural similarity and
are encoded on separate chromosomes. Compared to IDO, the molecular functions of
TDO2 in breast tumors, where it is overexpressed relative to normal breast tissues, are
less well characterized. Analysis of breast tumor microarray datasets revealed that
TDO2 is upregulated in high grade tumors as well as in more aggressive subtypes of
breast cancer, such as basal-like breast cancer. High TDO2 expression in breast
tumors is also associated with worse clinical prognosis. Knockdown (KD) of TDO2 gene
expression in the breast cancer cell lines HCC38 and MDA-MB-231 using shRNA
lentiviral vectors showed a role for TDO2 in promoting cell proliferation and migration. In
addition, tryptophan catabolism is known to activate aryl hydrocarbon receptor (AHR)
transcriptional activity and feed into de novo NAD synthesis, and it was indeed the case
that TDO2 KD inhibited AHR signaling and steady state NAD levels in breast cancer
cells. Interestingly, TDO2 expression was induced by glucocorticoid treatment, a
common chemotherapy co-treatment, suggesting a potential mechanism for the
development of chemoresistance. Together, this suggests that TDO represents a new
therapeutic target for aggressive subtypes of breast cancer.
Contributors: Franklin Gu, Salil Bhowmik, James Arnold, Susmita Samanta, Nagireddy Putluri,
Arun Sreekumar
Department of Molecular and Cellular Biology, Verna and Mars McLean Department of
Biochemistry, Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX.
109
THE ROLE OF DNMT3A IN CLONAL AND MALIGNANT HEMATOPOIESIS
Michael Gundry
Department of Molecular & Human Genetics/M.D.-Ph.D. Program
Mutations in the de novo DNA methyltransferase 3A (DNMT3A) gene occur in
approximately 20% of adult hematologic malignancies. The specific mechanisms or
targets through which these mutations act remains unclear. We have developed a
mouse model in which Dnmt3a is conditionally inactivated in a pIpC-inducible manner.
In homozygous mice, the hematopoietic stem cell (HSC) compartment demonstrates
enhanced self-renewal and a differentiation block upon serial competitive
transplantation. When Dnmt3a-/- cells are transplanted in a non-competitive manner, all
lethally irradiated mice die within one year, with a spectrum of diseases including, acute
myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasia, and T-cell
acute lymphocytic leukemia. These findings indicate that mutations in Dnmt3a can act
as the initiating event for mouse hematopoietic disorders, consistent with observed
clonal patterns in AML and the recent findings that a clone of DNMT3A mutant cells can
be found in the peripheral blood of many aging healthy individuals, termed clonal
hematopoiesis of indeterminate potential. Unfortunately, studies on healthy individuals
have not assessed the clonality of the HSC compartment. Based on our observations in
Dnmt3a null mice, the frequency of mutant cells in the HSC compartment should be
considerably higher than that observed bulk peripheral blood cells. We are collecting
bone marrow samples from healthy individuals undergoing total hip or knee
replacements. We will perform targeted sequencing of multiple flow sorted
hematopoietic populations from each individual to determine the clonal hierarchy of
DNMT3A mutant cells. We will also assess the RNA and DNA methylation landscape of
the different populations to determine the phenotypic consequences of loss of the
DNMT3A protein in human blood cells.
Acting as an initiating lesion in leukemic transformation, it is unclear whether
mutant DNMT3A leukemic cells are dependent on the absence of DNMT3A function.
To determine whether restoring WT DNMT3A can reverse the phenotypes observed in
normal and leukemic cells we have developed a mouse model allowing for the
sequential inactivation and restoration of WT Dnmt3a in the hematopoietic
compartment. We expect that these complementary studies in human and mouse will
allow provide a more complete understanding of the role of DNMT3A in normal and
malignant hematopoiesis and will help justify the development of novel therapeutics
targeting DNA methylation and DNMT3A.
Contributors: Gundry, Michael; Brunetti, Lorenzo; Goodell, Margaret
110
CHARACTERIZATION OF THE FUNCTIONAL ROLE OF CD56 ON NK CELLS
Justin Tyme Gunesch
Advisor: Jordan Orange, M.D.-Department of Pediatrics
Neural cell adhesion molecule (NCAM) is an immunoglobulin-like cell adhesion
molecule. In non-immune cells, NCAM is important for neurite outgrowth, cell migration,
cell-cell interactions and motility. In the context of immunology, NCAM (designated
CD56) is the major identifying marker for natural killer (NK) cells, however its ligand and
signaling function are unknown.
Human NK cells play important roles in human host defense such as mediating
cell death of virally infected or cancerous cells and shaping the outcome of transplant.
In human peripheral blood the two major populations of NK cells are CD56bright and
CD56dim. CD56bright NK cells are thought to mature into CD56dim NK cells and each
subset mediates distinct, critical effector functions that contribute to the overall defense
of the human host. Unlike T or B cells, the molecular signals leading to NK cell
development are poorly understood, however their maturation can be recapitulated in
vitro on developmentally supportive EL08.1D2 stromal cells.
Preliminary data shows that NK cell development is marked by acquisition of
motility on stroma and that this motility is mediated by CD56. Utilizing highly temporally
and spatially resolved quantitative analysis by time-lapse microscopy to track and
measure the behavior of NK cells, we show that human NK cells have both persistent
and transient interactions with stromal cells. In addition, NK cell contact with stromal
cells is marked by accumulation of CD56, L-selectin and F-actin, suggesting the
deliberate organization of a structured signaling platform. Motility is diminished when
CD56 ligation is prevented by blocking antibody.
Consequently, to assess the role of CD56 in human NK cells, the CRISPR-Cas9
system was used to delete CD56 in the human NK92 cell line. Initial FACS analysis
revealed greatly diminished expression of CD56 while expression of other adhesion
molecules was normal compared to parental NK92 cells. As was seen when blocking
CD56 on primary human NK cells, CD56-KO NK92 cells show decreased motility on
developmentally supportive EL08.1D2 stromal cells.
Therefore, we have shown a critical role for CD56 in human NK cell motility and
development. Future work will include the application of CRISPR-Cas9 mediated CD56
disruption to primary cells and elucidation of the signaling that links CD56 to NK cell
motility.
Contributors: Gunesch, Justin T.; Dixon, Amera L.; Mace, Emily M.; Orange, Jordan S.
111
FOUR-DIMENSIONAL CORRELATION BETWEEN FREQUENCY OF
DEGRANULATION AND NATURAL KILLER CELL CYTOTOXICITY
Lavesh Amar Gwalani
Natural killer (NK) cells are cytotoxic lymphocytes required for early control of
viral infections and tumor immunosurveillance. NK cells elicit their cytotoxicity
predominantly through directed secretion of lytic granules which are specialized
lysosomes containing effector molecules such as perforin and granzymes. Central to
the cytolytic function of NK cells is the formation of a mature and functional lytic
immunological synapse (IS) between an NK cell and its target cell. Activating signaling
and cytoskeletal reorganization by the NK cell enable delivery of lytic granules to the IS.
Fusion of lytic granules with the NK cell membrane results in exocytosis of granule
contents on to the target cell, a process termed degranulation. While the requirements
for transport and exocytosis of lytic granules are known, the minimum number of
degranulation events sufficient for an NK cell to kill a single target is unknown. In
addition, observed differences in efficacy of cytotoxicity between different human NK
cell lines frequently used and primary NK cells are largely unexplained. Using the pHsensitive degranulation indicator LAMP1-pHluorin in concert with sensitive detection of
target cell apoptosis we have developed a novel experimental approach to identify and
quantify individual degranulation events. We performed four-dimensional quantification
of degranulation events by confocal microscopy of live single NK-target cell conjugates
allowing visualization and measurement of the entire NK cell kill. Degranulation events
were quantified in LAMP1-pHluorin transduced human NK cell lines YTS and NK92
against their known target 721.221 cell line. Analysis of degranulation events observed
in cytotoxicity assays shows appearance of early degranulation events when 721.221
cells undergo fast killing whereas degranulation events are fewer and appear later in
slow target cell kills. The correlation of degranulation events and its parameters to NK
cell cytotoxicity will be further evaluated in other NK-target cell combinations as well as
in ex vivo human NK cells. This study provides detailed characterization of NK cell
degranulation at a single cell level, which will fill a gap in the current knowledge about
NK cell biology. The results can be used to better characterize degranulation in cases
of human NK cell deficiencies and can help engineer better NK cell immunotherapies.
Contributors: Gwalani Lavesh, Carisey Alexandre, Orange Jordan
112
MAINTAINING DISTANCE: THE E3 UBIQUITIN LIGASE ARIADNE-1 CONTROLS
NUCLEAR POSITIONING DURING MUSCLE DEVELOPMENT IN DROSOPHILA
Nele A Haelterman
Muscle fibers contain many nuclei that are evenly spaced. Failure to correctly
position nuclei leads to muscle dysfunction and diseases such as Emery Dreifuss
Muscular Dystrophy. However, the mechanisms that control nuclear spacing remain
largely unknown. Our lab performed a forward genetic mutagenesis screen on the
Drosophila X-chromosome and identified novel genes required for proper development
and function. We identified four mutations that lead to nuclear clustering defects in a
gene named ariadne-1 (ari-1). ari-1 encodes a conserved Ring-In Between-Ring E3
ubiquitin ligase. The nuclear mis-positioning appears relates to a defect in the
subcellular localization of Msp-300, which links the outer nuclear membrane to the
cytoskeleton to promote proper nuclear localization. Ari-1 mutants mimic the phenotype
of msp-300 mutants as both mutant muscles display clustered nuclei, fragmented
microtubular networks and compromised integrity of the nuclear lamina. In addition,
removing a single copy of msp-300 in ari-1 mutants does not enhance the nuclear
clustering phenotype, suggesting that both genes function in the same pathway. We
hypothesize that Ari-1 regulates the function of Msp-300, possibly by monoubiquitinating the protein and modulating its ability to connect nuclei to the muscle
cytoskeleton. We are currently performing a set of biochemical experiments that permit
unbiased identification of Ari-1’s target substrates. If our hypothesis holds up, Ari-1
would be the first protein to fine-tune Msp-300’s function in controlling nuclear
positioning and sustaining muscle function
Contributors: Haelterman, Nele*; Tan, Kai Li*; Jaiswal-Nagarkar, Sonal; Lee, Pei-Tseng; Bellen,
Hugo
113
DEVELOPING A NOVEL HUMANIZED MOUSE MODEL FOR TYPE 1 DIABETES
Emily Grace Haines
Type 1 diabetes is an autoimmune disease that affects 1 in 255 people in the
United States. This disease is estimated to shorten a patient’s life by up to 13 years,
and imposes a significant healthcare cost burden. The non obese diabetic, or NOD,
mouse is the most widely used animal model for type 1 diabetes research. However,
initiation of autoimmunity in the NOD mouse has little relevance to human disease
because the mice all carry a defined major histocompatibility complex (MHC) class II
repertoire that strongly predisposes them to develop T1D. An additional limitation of the
NOD model is that the investigator must rely on the murine immune system to
recapitulate a disease of the human immune system. Although many genetic and
environmental mechanisms have been proposed, a definitive etiology of human T1D
remains elusive, and no prevention strategies nor cures have been developed.
To identify and evaluate the initiating autoimmune events of T1D, we are
developing a novel humanized mouse model using specimens derived from patients
afflicted with T1D. Skin fibroblasts will be taken either from patients with T1D or from
healthy controls and reprogrammed in vitro to yield induced pluripotent stem cells (iPS).
Then iPS cells will be further differentiated into beta islets and hematopoietic stem cells
(HSC). HSCs will be transplanted into lymphocyte-deficient mice in order to reconstitute
a human immune system. When reconstitution is confirmed, donor-matched beta islets
will be transplanted, and recipients monitored for onset of T1D.
This novel model will allow us to investigate the interaction of the human immune
system with human beta islets at early time points. We hope to gain insight into which
cell types are responsible for early invasion of beta islets, what molecular events occur
to break self tolerance, and how these events can be reversed or prevented in human
patients.
Contributors: Haines, Emily; Heath, Blake; Tri Le, Duy; Borowiak, Gosia; Paust, Silke
114
DISCOVERY OF CHIMERIC TRANSCRIPTS INVOLVING APC AND TERT IN
PEDIATRIC HCC BY RNA SEQUENCING
Katherine Haines
Background: Hepatocellular carcinoma (HCC) is a rare pediatric liver tumor with a poor
prognosis. A characteristic DNAJB1-PRKACA gene fusion has been identified in a specific
subtype, fibrolamelar HCC (FL-HCC). In the majority of pediatric cases of non-FL-HCC, a
genetic cause has not been identified. We hypothesize that gene fusions could play a role in
the tumorigenesis of pediatric HCC. The goal of this project is to utilize RNA sequencing to
identify chimeric transcripts in pediatric HCC that could improve molecular characterization and
identify potential oncogenic drivers of this disease.
Material and Methods: We have used RNA sequencing (RNA-seq) to survey a cohort of
8 FL-HCCs, 4 pediatric HCCs, and 6 normal liver samples for chimeric transcripts. High quality
RNA (RIN: 6.6-9.7) was extracted from fresh-frozen tissue and strand-specific, poly-A+ RNAseq libraries were prepared for Illumina sequencing. Approximately 85 million paired-end reads
(42.5 million fragments) of 2 x 100 bp length were generated per sample. Fusion transcripts in
the tumor samples were detected using deFuse (v.0.6.1) on FASTQ files followed by
subtraction of fusions also called in the normal liver dataset. The remaining calls in the HCC
dataset were then filtered for the COSMIC Cancer Gene Census list to identify fusions involving
known oncogenes or tumor suppressor genes. Candidate fusions were verified using BLAST
and validated by RT-PCR.
Results: On average, 150 fusion transcripts were predicted per tumor sample using the
deFuse algorithm. Further filtering by the normal liver dataset reduced the number of calls in
the tumor datasets by ~60%. As expected, DNAJB1-PRKACA fusions were identified in the FLHCC cohort with no additional fusions detected. Filtering by the COSMIC Cancer Gene Census
list resulted in five additional fusion calls in HCC (three unique events in two tumors) two of
which were confirmed by RT-PCR. The first event is an inversion within the APC and AP3B1
genes that results in the two in-frame fusion transcripts, APC-AP3B1 and APC3B1-APC, but no
full-length APC transcript. The second event is a deletion encompassing the TERT promoter
that results in the in-frame fusion LPCAT1-TERT. An increase in TERT expression is seen in
this tumor as compared to both normal liver and other pediatric HCC tumors. Filtering for inframe fusions involving non-COSMIC genes did not reveal any fusions in the two remaining
HCCs and further analysis of these cases is ongoing.
Conclusion: The detection of chimeric transcripts by RNA-seq has allowed us to identify
two unique structural events involving known cancer genes in two pediatric non-fibrolamellar
HCCs. The chimeric transcripts found in these tumors provide further insight into the
tumorigenic events of pediatric HCC.
Contributors: Katherine Haines1, Angshumoy Roy2,3,5, Linghua Wang4, Pavel Sumazin2,5,
Kyle R. Covington4, Donna M. Muzny1,4, Vijetha Kumar3,5, Harsha Doddapaneni4, Hsu
Chao4, , David A. Wheeler1,4, Gail Tomlinson6, D. Williams Parsons1,2,4,5 , Sharon E.
Plon1,2,4,5 and Dolores Lopez-Terrada2,3,5,
1Departments of Molecular and Human Genetics, 2Pediatrics, 3Pathology & Immunology,
4Human Genome Sequencing Center, Baylor College of Medicine and 5Texas Children’s
Cancer Center, Texas Children’s Hospital, Houston, TX 77030, 6Greehey Children's Cancer
Research Institute, University of Texas Health Sciences Center at San Antonio
115
DETERMINING THE ROLE OF RAB-35 IN APOPTOTIC CELL CLEARANCE
Ryan Christopher Haley
Advisor: Zheng Zhou, Ph.D.-Department of Biochemistry & Molecular Biology
Apoptosis, or programmed cell death, curates proper development, ensures
proper neural function, and prevents autoimmunity. Apoptotic cells are engulfed via
phagocytosis; the nascent phagosome “matures” by fusing with endosomes and
lysosomes and subsequently initiates digestion. Rabs, weak GTPases that modulate
cellular activity, are essential in this process. Our lab has performed an RNAi screen of
Rabs found in the nematode C. elegans and discovered that rab-35 mutants exhibit the
ced (cell death abnormal) phenotype indicative of defects in engulfment or phagosomal
maturation. In C. elegans, RAB-35 localizes to the early endosome and has been
previously implicated in endocytosis. However, our lab has found that RAB-35 also
localizes to phagosomes during or shortly after their formation. We aim to fully
characterize the function, localization, and binding partners of RAB-35 in the process of
apoptotic cell clearance.
Contributors: Wang, Ying; Zhou, Zheng
116
ION TRANSPORT IN THE REGULATION OF HISTAMINE PRODUCTION BY
LACTOBACILLUS REUTERI
Anne Elizabeth Hall
Background: Certain strains of Lactobacillus reuteri can reduce inflammation and
tissue damage in mouse models of colitis. This anti-inflammatory capacity stems in part
from L. reuteri’s ability to produce and secrete histamine, which can act on specific
receptors present on mammalian cells. In the bacteria, histamine is generated by
histidine decarboxylase (HdcA), and released from the cell by a histidine/histamine
exchanger (HdcP). This process consumes protons, resulting in an increasingly alkaline
intracellular pH and an inside-negative membrane potential. These factors can
suppress the activities of HdcA and HdcP. However, hdcA is not constitutively
expressed, and it is unclear which signals may trigger its upregulation. Additionally, it is
unknown how L. reuteri balances its internal ion environment during histamine
production, especially in the presence of a complex external environment like the GI
tract. Recently, a proton/chloride antiporter, EriC2, was identified in L. reuteri as a
potential regulator of intracellular pH and membrane potential, and thus histamine
production. We hypothesize that proton influx via EriC2 may activate and sustain
histamine production in response to acid stress, and that chloride efflux may alleviate
the negative membrane potential induced by histamine production. Methods & Results:
To investigate how EriC2 might alter hdcA gene expression and histamine production
by L. reuteri, we used single-strand recombineering to generate protein knockout,
proton transport deficient, and transport null EriC2 strains. These mutations are stable,
and do not significantly alter the growth of the resulting strains compared to wild-type
(WT) L. reuteri. The pH-sensitive fluorophore, pHrodo Green, was used to observe
intracellular pH at stationary phase, and suggests subtle differences among the mutant
strains. RT-qPCR was used to assess hdcA gene expression in mutant and WT strains.
Alterations in host histamine receptor and ion transporter gene expression were
examined after incubation of WT and mutant L. reuteri strains with the secretory colonic
epithelial cell line T84. Conclusion: Together these data suggest that L. reuteri
histamine production influences host physiology. The beneficial effects of L. reuteriderived histamine may depend on a regulatory system mediated by ion transport in the
GI tract. A mechanistic understanding of histamine production by the gut microbiome
may enable the development of diet or probiotic-based strategies to suppress intestinal
inflammation.
Contributors: Hall, Anne; Versalovic, James
117
THE ROLE OF MAZ TRANSCRIPTION FACTOR IN GENITOURINARY
DEVELOPMENT
Meade Elspeth Haller
Advisor: Dolores Lamb, Ph.D.-Department of Urology
Genitourinary (GU) birth defects comprise some of the most common yet least
studied congenital malformations and range in severity from conditions such as
undescended testes (cryptorchidism), ventrally misplaced urethral meatus
(hypospadias), and renal agenesis to highly complex malformations such as bladder
exstrophy epispadias complex (BEEC) and ambiguous genitalia. Genomic copy number
gains and losses (CNVs) frequently result in congenital malformations of the GU tract,
and can be identified using array comparative genomic hybridization (aCGH).
CNV overlap mapping revealed over 30 patients with GU defects harboring
CNVs in the syndromic genomic region, 16p11.2 – the most common pathogenic CNV
hotspot in humans. The only gene covered collectively by all the mapped 16p11.2
CNVs in patients with GU defects is MYC-associated zinc finger (MAZ), which encodes
a transcription factor. In situ hybridization and immunohistochemistry staining on mouse
embryos showed robust expression of Maz in the developing GU tract. Prospective
CNV qPCR screening of genomic DNA from 258 patients with GU defects revealed an
incidence of MAZ copy variation in 6% of GU-abnormal patients, an enrichment
compared to the general population at <1% (Tucker et al, 2013).
As predicted based on the similarity of its consensus sequence to that of WT1,
knockdown of MAZ in human embryonic kidney (HEK293) cells results in differential
expression of several WNT pathway transcripts including morphogens
WNT3A/4/5B/7B/8A/11. Knockdown of MAZ in HEK293 and RWPE1 normal prostate
cells results in suppressed proliferation and deregulation of cell cycle genes.
Preliminary animal studies suggest roles for Maz in kidney development as well as
testicular descent. Once thought to be a simple housekeeping gene, MAZ encodes a
transcription factor that influences WNT signaling and cell cycle progression, and is
likely to contribute to the congenital urogenital malformations associated with 16p11.2
dosage variation.
Research supported by NIH grants T32DK007763 and R01DK078121
Contributors: Haller, Meade; Lamb, Dolores J.
118
MICRORNAS REQUIRE ACTIVATION TO STABLY BIND THE MRNA 3’UTR
Mark Patrick Hamilton
Department of Molecular & Cellular Biology/M.D.-Ph.D. Program
Advisor: Sean Mcguire, M.D./Ph.D.-Department of Molecular & Cellular Biology
MicroRNAs (miRNAs) are a small (~22 nucleotide) class of noncoding regulatory
RNAs that initiate mRNA degradation by targeting the Argonaute protein (AGO) to
mRNA 3’ untranslated region (3’UTR). AGO-miRNA binding is dependent on the large
mutiprotein RNA-induced silencing complex (RISC) to function. GW182 is the other
known essential RISC component. In the cell AGO can exist in an active high
molecular weight (HMW) complex or an inactive low molecular weight (LMW) complex.
Crosslinking immunoprecipitation of AGO coupled to high-throughput sequencing
(AGO-CLIP-seq) allows global visualization of Argonaute binding to both miRNAs and
mRNAs. By curating over 140 AGO-CLIP-seq libraries produced by our laboratory and
others, we find the majority of the miRNA pool is depleted from 3’ UTRS in most nontumorigenic tissues and cell lines. The AGO-miRNA complex redistributes onto the
mRNA 3’UTR in tumorigenic cell lines. AGO loading onto the 3’UTR exists over a 244fold range, and this range is consistent in human data, mouse data, in vivo derived
data, and data generated in our lab.
In the RWPE1 cell line, where the miRNA pool is unbound to the 3’UTR, AGO
exists exclusively in inactive low-molecular weight complexes. The mechanism of this
inactivity is failure to express GW182. Re-expression of GW182 is sufficient to drive
HMW AGO complex formation. Finally, we find that the HMW AGO complex is entirely
dependent on the nascent protein pool by way of GW182 protein instability. These
findings are important because they re-characterize miRNA activity, suggesting miRNAs
require global activation in order to suppress mRNAs. These findings also position
GW182 as a rheostat that can globally adjust miRNA binding activity through titrated
expression levels. We believe the notion that global miRNA binding is titratable may
have general application in a number of fields.
Contributors: Hamilton, Mark; Hartig, Sean; Coarfa, Cristian; McGuire, Sean
119
THE FUNCTION OF GLYCOSYLATION-RELATED GENES IN DROSOPHILA
LARVAL MESODERMAL TISSUE
Seung Yeop Han
Advisor: Hamed Jafar-Nejad, -Molecular Medicine- UT Houston
Protein glycosylation is a common post-translational modification which can
affect a multitude of processes, including protein folding, trafficking, stability, cell
surface localization, and ligand binding. Between the functions of glycosylation-related
genes (GRGs) are tightly related and ordered. Mutation in a number of human GRGs
causes various types of congenital disorder of glycosylation (CDG). However, the
relationship between the glycosylation of proteins and the clinical features of the
disease and the pathological mechanism of CDG are poorly understood. Mutations in
human N-glycanase 1 gene (NGLY1) have been reported in a rare form of a disorder of
deglycosylation called NGLY1 deficiency. Patients with this disorder show global
developmental delay, movement disorder, gastrointestinal dysfunction, and small
hands/feet. To understand the pathophysiology of NGLY1 deficiency, we have
characterized mutations in the Drosophila NGLY1 homolog, PNGase like (pngl). We
have found that pngl is functionally conserved between human and fly, and plays an
important role in the regulation of Drosophila BMP signaling. Specifically, loss of pngl
impairs the mesoderm-to-endoderm Dpp signaling during the larval midgut
development. pngl mutants show defects in the gastric caeca which are thought to
secrete water and enzyme to aid in digestion, and an impairment in the acid-secreting
zone in the larval midgut. Moreover, pngl mutants show food accumulation at the end of
the larval stage in a Dpp-independent manner.
Our data indicate that pngl is required in the mesoderm to regulate midgut
development and to ensure the survival of the animal. One way to understand the
function of Pngl at a molecular level is to identify other GRGs that have a similar lossof-function phenotype or those which when knocked down (KD) can suppress the pngl
mutant phenotypes. To this end, we performed GRGs screening using Drosophila
dsRNA lines and a mesodermal driver. Among the 361 GRGs encompassing families of
glycosyltransferases and glycoside hydrolases in Drosophila, we selected 155 genes
which have clear human homologs. We obtained 237 RNAi lines for each gene and
performed KD experiments in the mesodermal tissues using the UAS-GAL4 system.
We established screening strategies to find GRGs whose KD mimics pngl mutant
phenotypes. We dissected larvae feeding with the pH indicator dye, bromophenol blue
and investigated phenotypes of mesodermal specific KD of GRGs larvae. We found
that a total of 66 GRGs caused midgut defect phenotypes, and 9 gene families showed
partial phenocopies of the pngl mutant phenotypes. Furthermore we will perform the
pngl phenotype suppressor-screening assay and investigate their potential rescue upon
alteration of GRGs expression.
Contributors: Han, Seung Yeop; Galeone, Antonio; Jafar-Nejad, Hamed
120
STUDY OF MECHANISM OF PERSISTENCE OF POLYOMAVIRUS SV40 IN HUMAN
B LYMPHOCYTES
Lindsay Jeanette Harrigal
Advisor: Janet Butel, Ph.D.-Department of Molecular Virology & Microbiology
Polyomaviruses are small, DNA tumor viruses that can be detected in a variety
of tissues and bodily fluids, indicating viral dissemination within the host. The humanassociated polyomavirus simian virus 40 (SV40) has been detected in human
peripheral blood mononuclear cells, in human tonsils, and in non-Hodgkin lymphoma,
suggesting that SV40 may be lymphotropic in humans. Results with SV40-infected
human B cell lines show that viral genomes are maintained at low copy numbers during
cell passage through 28 days in culture and that expression of the viral replication
protein T-antigen (T-ag) can be detected in some cells. SV40 infection seems to be
non-lytic and non-productive as infected human B cells remain viable and proliferate in
culture. No mechanism for SV40 genome maintenance in human cells is currently
known. With SV40 infection appearing to be non-productive and viral integration into
cellular chromosomes being a rare event, we hypothesized that T-ag binds to the origin
of replication (ori) in the viral genome (which it does during viral replication) and tethers
the viral DNA to host cell components, allowing segregation of the viral genome during
lymphocyte cell division. The SV40 genome tethering model was tested by performing
transfection experiments with the human B cell line DG75. Transient DNA transfections
used electroporation, a plasmid that mimicked the SV40 genome in size and presence
of a viral ori, plus encoding a reporter gene, and a second plasmid that encoded only
SV40 T-ag. Results suggested that the presence of DNA encoding T-ag increased
retention of SV40-like DNA, an observation designated the “T-ag effect.” An alternative
approach to examine T-ag effects on SV40 DNA retention involved constructing a
human B cell line to have constitutive expression of T-ag protein. A wild type T-ag gene
was introduced into DG75 cells via lentiviral transduction followed by drug selection.
The viral DNA-positive selected cells were designated D-Tag cells. The D-Tag cell line
is being used to examine the effect of constitutive T-ag expression on long term
maintenance of SV40-like DNA in human B lymphocytes. Initial results suggest that
more non-integrated, viral DNA persists in infected D-Tag cells when T-ag protein is
expressed.
Contributors: Harrigal, Lindsay J.; McNees, Adrienne L.; Wong, Connie; Butel, Janet S.
121
ROLE of MAPT IN REGULATING HYPER-EXCITABILITY IN THE MATURE CNS OF
KCNA1 KO MICE
Asante Hatcher
Advisor: Jeffrey Noebels, M.D./Ph.D.-Department of Neurology
Epilepsy is a heterogeneous group of neurological disorders characterized by
neuronal hyper-excitability and recurrent, sometimes lethal, seizures. Although there
are many anti-epileptic drugs (AED) currently on the market, they are ineffective in
controlling epilepsy in nearly 30% of cases. Moreover, the molecular mechanisms
underlying the onset, spread, and persistence of epilepsy have yet to be elucidated for
the full spectrum of the disorder. Recent findings have demonstrated that germline
knockout of the microtubule organizer MapT encoding the tau protein reduces seizure
activity and lethality in several genetic models of epilepsy, including the Kcna1
potassium channel knockout mutant mouse. Using a tamoxifen-inducible cre strategy,
we will remove tau in the mature CNS of adult Kcna1 mice. This will allow us to
ascertain the presence of a critical developmental window for MapT loss in seizure
protection, and provide a better understanding of the mechanisms by which MapT
regulates hyper-excitability in the developing and mature CNS.
Contributors:
122
RSV FUSION PROTEIN SEQUENCE VARIABILITY AMONG GENOTYPES AND
CONSENSUS GENERATED SUBGROUPS
Anne M Hause
Advisor: Pedro Piedra, M.D.-Department of Molecular Virology & Microbiology
Introduction: Respiratory syncytial virus (RSV) is a leading cause of lower
respiratory tract infections among infants and young children. The fusion (F) protein is a
promising vaccine target as it is relatively conserved among RSV-A and RSV-B
subgroups and induces neutralizing antibodies. Antigenic site II of the F protein is the
targeted site of palivizumab, a monoclonal antibody used prophylactically for high-risk
infants. Candidate vaccines, based on the historical GA1 genotype of RSV-A, might
provide variable levels of protection dependent on subgroup and genotype of the
infecting virus.
Objective: To determine variability of the F gene of RSV-A and RSV-B isolates.
To develop a consensus F gene for RSV-A (Fa) and RSV-B genotypes (Fb).
Methods: The G and F genes of RSV isolates (n=118) stored at BCM were
sequenced. The F and G sequences of 118 isolates from GenBank were obtained.
Together, these sequences were used to generate phylogenetic trees based on the G
gene. The F sequences were then used to create a consensus sequence for each of
the 7 RSV-A genotypes and 4 RSV-B genotypes identified in the phylogenetic trees.
Genotype consensus sequences were then used to generate an overall consensus
sequence of F protein for Fa and Fb. We defined amino acid variation as the
percentage of nonsynonomous changes.
Results: We genotyped169 RSV-A sequences (GA1=10, GA2=94, GA5=36,
GA7=5, NA1=10, SAA1=2, ON=12) and 67 RSV-B sequences (BA=41, GB1=3,
GB3=10, GB4=13). Among the genotype consensus sequences of each subgroup,
there is approximately 98% nucleotide conservation. However, the sequence identity
decreases to approximately 93% when comparing isolates from contemporary to
historical genotypes. There is approximately 90% amino acid and 80% nucleotide
conservation between Fa and Fb consensus sequences. The F protein domains of Fa
and Fb had the following amino acid variation: signal peptide (64%), no annotation
domain 2 (23%), antigenic site ø (17%), transmembrane domain (12%), cytoplasmic
domain (12%), antigenic site II (5%), and antigenic site IV (0%).
Conclusions: Although the F protein is generally well conserved between
genotypes of each subgroup, there is variability in domains that contribute to the overall
structure of the protein. Differences in protein domains could contribute to changes in
quaternary structure or accessibility to antigenic sites. We hypothesize that a bivalent
vaccine (Fa & Fb) based on consensus of F proteins will provide broader protection
among all circulating genotypes when compared to a monovalent RSV-F (Fa) vaccine
derived from historical virus (GA1 genotype).
Contributors: Hause, A; Avadhanula, V; Piedra, P.
123
A PRACTICAL GUIDE TO STUDY DESIGN, SAMPLE SIZE REQUIREMENT AND
STATISTICAL ANALYSES METHODS FOR RARE VARIANT DISEASE
ASSOCIATION STUDIES
Zongxiao He
In this article we conducted comprehensive empirical analyses to assess the impact of
various practical issues on the outcome of rare-variant association (RVA) studies, the choice of
RVA methods and sample size requirement for well powered RVA designs. We developed a
simulation framework using real world data from NHLBI-Exome Sequencing Project (ESP) as
well as exome sequence data which was simulated using state-of-the-art demographic models
with purifying selection modeled after the empirical distribution of functional variants in ESP.
Our simulated data is highly consistent with real world data distribution of singleton, doubleton
and tripleton variants as well as the cumulative minor allele frequency (MAF) of variants for
Europeans and Africans. Using resampled genotypes from ESP European American
sequences, we evaluated relative power of 10 RVA methods by analyzing 16,568 genes across
the genome, and we demonstrated that a method most powerful for one gene is not necessarily
the most powerful for another, simply due to differences in the genomic sequence context, i.e.,
gene specific MAF spectrum and distribution of functional variants, rather than phenotypic
model assumptions. Using simulated data of European samples we evaluated impact of
phenotypic model, missing data, non-causal variants and choice of empirical MAF cutoff in RVA
analysis. We found that the assumption of variable effects model favors variable threshold tests
(e.g. VT) greatly, but the power gain of weighted burden tests (e.g. WSS) are marginal
compare to the constant effect model. In the presence of strongly protective variants, SKATO/SKAT are consistently the most powerful tests, but they perform poorly when protective effect
is mild compare with detrimental effect. The impact of non-causal variants and missing data are
more significant than the choice of RVA methods, and the enrichment of functional variants is
most crucial to the success of most RVA methods. Our exome-wide sample size estimation
suggested that the number of samples which need to be studied are highly dependent on gene
size and the number of variant sites, for example under the assumption of moderate effect size
for causal variants, i.e., odds ratio 2.0, for genes with short coding region lengths (~400bp),
>90,000 samples are required to achieve a power of 80% to detect an association using an
exome-wide significant level of α = 2.5×10-6 while for average sized genes (~1,400bp), the
required sample size is >50,000. For quantitative traits, sampling of subjects having phenotype
values more extreme than the highest and lowest 5% in very large cohort is recommended. We
also found that RVA studies using exome array data is cost effective strategy as long as a few
thousands additional samples are readily available for genotyping to compensate for the power
loss compare to using sequence data. The high quality simulated exome data generated from
this study as well as the comprehensive simulation framework we developed are good
resources for future practical and methodological researches on design and methods for RVA
studies.
Contributors: Wang, Gao; Zhang, Di; Dai, Hang; Leal, Suzanne.
124
A TAIL-LIKE ASSEMBLY AT THE PORTAL VERTEX IN INTACT HERPES SIMPLEX
TYPE-1 VIRIONS
Corey W Hecksel
Latent Herpes Simplex Virus type 1 (HSV-1) infections are a special public
health concern as asymptomatic viral shedding is believed to be responsible for over
70% of primary infections and has been shown to increase the rate of HIV transmission
in patients with concurrent infections. HSV shares a common evolutionary origin with
tailed bacteriophages, which contain a DNA packaging motor, or portal, at one vertex of
the icosahedral particle. The bacteriophage tail is a complex structure that assembles
at the portal vertex and provides the mechanism for delivering the viral genome into the
infected cell. Using electron cryo-tomography (cryo-ET), we have identified a previously
unsuspected tail-like structure at the portal vertex (termed PVAT) in the HSV-1 virion.
The location of this tail-like structure suggests that it is important for virion architecture
and may play a role in herpesvirus infection.
Contributors: Hecksel, Corey; Schmid, Michael; Rochat, Ryan, Bhella, David; Chiu, Wah; Rixon,
Frazer
125
PRIOR PARITY LOWERS THE APOPTOTIC BARRIER TO TUMORIGENESIS IN THE
BREAST
Sarah Hein
Regardless of the age of primiparity, during and immediately following any
pregnancy there is an increased incidence of breast cancer. A late aged pregnancy not
only increases the severity of the relative risk of tumorigenesis immediately surrounding
a pregnancy, but leads to long term increased breast cancer incidence. Conversely, an
early aged pregnancy leads to an overall reduction in the lifetime risk of breast cancer,
and has been associated with a reduced proliferative rate in the parous breast. As
increasing numbers of women choose to delay childbirth, it is vitally important to
understand how a late aged pregnancy promotes breast cancer so that effective
prevention strategies may be developed. Although many studies have sought to model
parity’s effect on precancerous tissue from carcinogenic and molecular means, no
study has previously determined the effect of a fully involuted parous gland on later
oncogenesis.
We have utilized the RCAS-TVA viral vector system to introduce the oncogene
ErbB2 into parous (Par) and age-matched nulliparous (NP) mice, generating discrete
precancerous lesions and tumors in an otherwise normal background. Surprisingly, we
find that parity promotes tumorigenesis in the fully involuted mammary gland. When
evaluating early precancerous lesions, we find similar load between Par and NP mice.
However, at an advance precancerous lesion time point we find significantly increased
lesion. In these advance lesions parity decreases apoptosis compared with NP controls.
Proliferation of the advanced lesions is similar between groups. To evaluate whether
the parity phenotype is intrinsic or extrinsic to the mammary epithelium, we intraductally
transplanted oncogene-initiated cells from NP donor mice into NP and Par hosts. We
find that the parous environment is still capable of accelerating lesion growth. Further
study is needed to fully elucidate the mechanism of parity promotion of breast cancer
after the completion of involution.
Contributors: Hein, Sarah; Gastelum, Grady; Muscarella, Aaron; Haricharan, Svasti; Huang,
Shixia; Chan, Keith; Zhang, Xiang; Li, Yi
126
A CHOLINERGIC BASAL FOREBRAIN FEEDING CIRCUIT MODULATES APPETITE
SUPPRESSION
Alexander Michael Herman
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Irregular food intake, either consuming too much or too little, is a primary cause
of obesity and other eating and metabolic disorders. Highly coordinated by the brain,
food intake must be balanced with energy expenditure in order to maintain proper body
weight homeostasis. To date, insight into the neural control of feeding has largely
focused on signaling mechanisms classically associated with the hypothalamus, the
major center in the brain which regulates body weight homeostasis. However, the role
of non-canonical signaling mechanisms in regulating feeding behavior has been largely
uncharacterized. Acetylcholine has long been proposed to influence feeding behavior,
but this idea has been largely based on anecdotal evidence due to functional similarity
between acetylcholine and nicotine, the addictive component in tobacco that acts as an
appetite suppressant. Although this connection has not been formally validated, it is
noteworthy that nicotine is an exogenous agonist for acetylcholine receptors,
suggesting that endogenous cholinergic signaling may play a role in normal
physiological regulation of body weight homeostasis. However, it remains unclear if
cholinergic neurons in the brain regulate food intake, and how their circuits function to
modulate feeding. Here, we report that cholinergic neurons of the basal forebrain
potently influence food intake and body weight. While impairment of cholinergic
signaling increases food intake and results in severe obesity, enhanced cholinergic
signaling decreases food consumption. Accordingly, we found that this cholinergic
population regulates appetite-suppressing POMC neurons of the hypothalamus.
Together, our data pinpoint the cholinergic basal forebrain as a major modulatory
center underlying feeding behavior, highlighting a new role of acetylcholine in
modulating food intake, which may provide insight towards the development of effective
treatments to manage eating disorders.
Contributors: Herman, Alexander M.; Ortiz-Guzman, Joshua; Kochukov, Mikhail; Garcia,
Isabella; Quast Katie; Carlson, Jeffrey C.; Selever, Jennifer; Tong, Qingchun; Arenkiel,
Benjamin R.
127
COMPLIMENTARY GENOME-WIDE SCREENING TO CHARACTERIZE THE
GENETIC BASIS OF DOXORUBICIN-INDUCED CARDIOTOXICITY
Matthew Craig Hill
Advisor: James Martin, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
Modern chemotherapeutic regiments have significantly prolonged the survival
time for cancer patients. However, several of these efficacious oncological treatments
carry with them severe cardiotoxic side effects that cause irreversible heart disease.
The most well-known cardiotoxic compound is the anthracycline member, Doxorubicin
(Dox). To gain insight into the molecular mechanisms underlying Dox toxicity, we
carried out complementary genome-wide loss- and gain-of-function CRISPR-Cas9
based screens. Top hits from both screens included genes previously reported to
confer Dox-resistance and cardioprotection. The loss-of-function screen uncovered
several genes expressed in cardiomyocytes, not previously recognized to be involved in
Dox toxicity, which may play a role in Dox-induced cytotoxicity within the myocardium.
Among these genes are Mef2a, Apobec2, Vldlr, and Lats1. The gain-of -function screen
also identified several novel mediators of Dox-resistance, many of which are normally
expressed at low levels in the adult human heart. Our results highlight the multifaceted
nature of Dox-induced cardiotoxicity, and provide novel molecular targets for potential
cardioprotective therapeutic intervention.
Contributors: Hill, Matthew; Zhang, Min; Martin, James
128
USE OF CRISPR FOR GENE THERAPY OF AUTOSOMAL DOMINANT RETINITIS
PIGMENTOSA
Tyler John Hilton
Retinitis Pigmentosa (RP) is a degenerative disease affecting cells in the retina
that causes progressive loss of vision, and impacts approximately 1 in 4,000 people
worldwide. RP results from heterogeneous genotypes, with many genes linked in a
causative or associated manner to the disease phenotype. Among these genes,
mutations in rhodopsin (RHO), which is essential to the vision pathway in rod
photoreceptor cells, are the most common cause of autosomal dominant RP (ADRP).
Conventional gene-supplementation therapy cannot be used to treat these forms of RP
because the RHOmutations act in a dominant fashion. With the use of zinc-finger
nucleases (ZFN), previous lab findings have shown that it is possible to create doublestrand DNA breaks in RHO gene in vivo, which are then repaired through the nonhomologous end-joining pathway (NHEJ). The potential therapeutic effect of the NHEJ
pathway is achieved by the generation of frameshift mutations, which may lead to
knockdown of toxic protein products. ZFN have limited sequence targeting capability,
but the recently developed CRISPR/Cas9 system should provide the flexibility needed
to target most RHO mutations and achieve knockdown. We hypothesize that
introducing targeted double-strand breaks using the CRISPR system, and
supplementation of the RHO gene, can be used to treat ADRP. We have designed
sgRNA targeting different regions of the RHO gene in the first and second exon, as well
as the promoter, which may provide different options for knockdown of RHO expression
in vivo. We are testing the sgRNA activity in vitro through Survey nuclease assays, high
resolution melting analysis, and sequencing to evaluate sgRNA on and off target activity
prior to packaging the sgRNA and Cas9 constructs into recombinant adeno-associated
virus for in vivosubretinal injection in mouse models for ADRP.
Contributors: Hilton, Tyler J.; Wilson, John H., Wensel, Theodore G.
129
ESTROGENIC NEURONS IN THE MEDIAL AMYGDALA PREVENT STRESSINDUCED HYPERTENSION
Antentor Othrell Hinton, Jr.
Advisor: Yong Xu, Ph.D.-Department of Pediatrics
Background: Psychological stress contributes to the development of
hypertension in humans, and estrogens prevent stress-induced hypertension with
unknown mechanisms. Stress-induced hypertension is associated with increased
neural activity in the medial amygdala (MeA), a brain region expressing abundant
estrogen receptor-α (ERα).
Hypothesis: Thus, we hypothesize that estrogens prevent stress-induced
hypertension partly through actions on ERα expressed by MeA neurons.
Methods: We used DREADD technology to selectively activate SIM1 neurons in
the MeA in conscious mice and tested effects of MeA SIM1 neural activity on blood
pressure (BP). In addition, Cre-LoxP system was used to genetically remove ERα from
SIM1 neurons in female mice; in parallel, we stereotaxically injected AAV-Cre into the
MeA of ERαlox/lox female mice to delete ERα from all MeA neurons. We tested effects
of estrogen replacement (vs. estrogen depletion) on stress-induced hypertension in
these mutant mice and their wild type littermates. Finally, we used both c-fos
immunoreactivity (in vivo) and slice electrophysiology (ex vivo) to examine cellular
activities of MeA neurons in wild type mice or in mutant mice lacking ERα from SIM1
neurons, with estrogen depletion or supplement, at basal or stressed condition.
Results: We showed that selective activation of MeA SIM1 neurons increased
basal BP and potentiated hypertensive responses provoked by psychological stress
(restraint) in conscious mice. While estrogen replacement prevented stress-induced
hypertension in wild type female mice, deletion of ERα from SIM1 neurons, or deletion
of ERα from the MeA attenuated these anti-hypertensive effects of estrogens during
stress. We also demonstrated that estrogens protect against increased neural activity
under stress, and that MeA neurons lacking ERα showed increased excitability
compared to wild type MeA neurons. Conclusion: Our results indicate that the
antihypertensive effects of estrogens are partly mediated through ERα in the MeA
neurons.
Contributors: Hinton, Jr., Antentor Othrell. 1,*, Yan, Xia 1, Xu, Pingwen 1, He, Yanlin 1,
Henderson, Alex 1 Reynolds, Corey L. 2 and Xu, Yong 1,3,^
1Pediatrics, 2Physiology 3Molecular and Cell Biology, Baylor College of Medicine, Houston, TX,
77030 *,first author; ^,corresponding author
130
UPREGULATION OF EGFR SIGNALING IS CORRELATED WITH TUMOR STROMA
REMODELING AND TUMOR RECURRENCE IN FGFR1-DRIVEN BREAST CANCER
Xue Bin Holdman
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
Introduction: Despite advances in early detection and adjuvant targeted
therapies, breast cancer is still the second most common cause of cancer mortality
among women. Tumor recurrence is one of the major contributors to breast cancer
mortality. However, the mechanisms underlying this process are not completely
understood. In this study, we investigate the roles of stroma on tumor dormancy and
recurrence in a pre-clinical mouse model of breast cancer.
Methods: To elucidate the mechanisms driving tumor recurrence, we employed a
transplantable Wnt1/inducible fibroblast growth factor receptor (FGFR) 1 mouse
mammary tumor model and utilizing an FGFR specific inhibitor BGJ398 to study the
recurrence after treatment. Histological staining was performed to analyze the residual
tumor cells and tumor stroma. RPPA was performed to compare primary and recurrent
tumors to investigate the molecular mechanisms leading to tumor recurrence.
Results: Treatment with BGJ398 resulted in rapid tumor regression, leaving a
nonpalpable mass of dormant tumor cells organized into a luminal and basal epithelial
layer similar to the normal mammary gland, but surrounded by dense stroma lacking
myeloid derived tumor suppressor cells (MDSCs) and tumor vasculature. Following
cessation of treatment the tumors recurred over a period of one to four months. The
recurrent tumors displayed dense stroma with increased collagen, tenascin-C
expression, and MDSC infiltration. Activation of the epidermal growth factor receptor
(EGFR) pathway was observed in recurrent tumors, and inhibition of EGFR with
lapatinib in combination with BGJ398 resulted in a significant delay in tumor recurrence
accompanied by reduced stroma, yet there was no difference observed in initial tumor
regression between the groups treated with BGJ398 alone or in combination with
lapatinib.
Conclusion: These studies illustrate the correlation between the stromal
microenvironment and tumor recurrence and the importance of EGFR signaling
pathway in this process.
Contributors: Holdman, Xue; Welte, Thomas; Rajapakshe, Kimal; Coarfa, Cristian; Mo,
Qianxing; Huang, Shixia; Hisenbeck, Susan; Edwards, Dean; Zhang, Shawn; Rosen, Jeffrey.
131
PATIENT AND PARENT CHARACTERISTICS DIFFER BY CARE MANAGEMENT
TYPE IN PEDIATRIC IRRITABLE BOWEL SYNDROME
John M Hollier
Advisor: Hashem El-Serag, M.D./M.P.H.-Department of Medicine
Background: Irritable bowel syndrome (IBS) is more costly when managed by
tertiary pediatric gastroenterology (GI) compared to primary pediatric care. However,
factors contributing to tertiary GI referral in pediatric IBS remain poorly defined. This
study determined whether patient or parental characteristics were associated with
tertiary GI referral in pediatric IBS patients.
Methods: A retrospective sample of pediatric subjects meeting pediatric Rome III
IBS criteria was assembled. Baseline socioeconomic and clinical measures were
gathered. Subject outcomes were stratified by tertiary pediatric GI referral vs primary
pediatric care management. Univariate and multiple logistic regression analysis
identified the most important factors in predicting the study outcomes.
Results: Of 239 participants, 153 were referred to tertiary pediatric GI, and 86
were managed in primary care. Multiple logistic regression analysis identified child selfassessment of abdominal pain duration and percentage of people living in poverty
(ascertained by zip code) as the strongest predictors of tertiary GI referral. In
multivariate logistic regression model adjusted for demographics and IBS subtype,
these factors remained statistically significant.
Conclusions: Socioeconomic status and clinical symptoms are associated with
tertiary GI referral. Identification of facilitators promoting tertiary GI referral could assist
in the design of interventions to reduce healthcare overutilization for functional
gastrointestinal disorders.
Contributors: Hollier, John; Czyzewski, Danita; Self, Mariella; Weidler, Erica; Smith, O’Brian;
Shulman, Robert
132
CLINICAL PROTEOMICS FOR PROSTATE CANCER
Matthew Valle Holt
Advisor: Jun Qin, Ph.D.-Department of Biochemistry & Molecular Biology
Christopher Peters, Ph.D.-Department of Biochemistry & Molecular Biology
As one of the most prevalent types of cancer for men, the need for effective and
non-invasive diagnostics of prostate cancer is paramount. In addition, characterization
of the disease is key for patient quality of life. To this date, the only technique capable
of diagnosing and characterizing prostate cancer accurately is an invasive tissue biopsy
followed by immunohistochemistry and analysis by a pathologist. Thus we seek to
create a diagnostic method that is capable of detecting the disease early, and
characterizing its prognosis. Urine is truly the least invasive and most readily attainable
sample source. Many prostatic proteins, cells and small vesicles such as exosomes,
are readily detected in urine and contain a wealth of information about potential
prostatic cancers as well as other ailments. We hypothesize that there are significant
protein markers present in the urine of prostate cancer patients which are capable of
addressing the aforementioned challenges.
We developed a streamlined mass spectrometry based workflow, with which we
are able to identify and quantify around 2000 proteins in less than 3 hours. With this
capability, we were able to detect outlier proteins whose abundances increased due to
physiological changes such as the stress associated with trans-continental travel, and
innate immune response to the common cold. We have used this method to analyze
over 400 samples from 17 healthy individuals to establish the normal range of proteins
in urine. To explore the feasibility of identifying urine biomarkers, we have measured 12
urine samples from biopsy positive prostate cancer patients. We found that, in addition
to elevated PSA, multiple proteins are elevated by several orders of magnitude above
the normal range (many of them are 100 to 1000 fold higher than the upper limit of the
normal range), making them potential biomarkers for prostate cancer.
Contributors: Holt, Matthew; Leng, Wenchuan; Wang, Yi; Qin, Jun;
133
LOCAL ANCESTRY INFORMED RARE VARIANT ASSOCIATION STUDY
IDENTIFIES GEGNES ASSOCIATED WITH WHITE BLOOD CELL COUNT IN
AFRICAN AMERICANS
Stanley Eugene Hooker
With the advent of next generation sequencing there is great interest in studying
the involvement of rare variants in complex trait etiology. For many complex traits
sequence data is being generated on DNA samples from admixed populations such as
African Americans and Hispanics to elucidate rare variant associations. Analyses of
admixed populations present special challenges due to spurious associations arising
from ancestry specific variants.. However using information on admixture and local
ancestry can also be highly beneficial and increase the power to detect associations in
these populations. Here we present a rare variant (RV) association test for admixed
populations incorporating a local haplotype sharing (LHS) method. Our method is
capable of using information on local ancestry to boost power of rare variant
association analysis without having to reconstruct haplotypes, which is otherwise
computationally intensive and error prone. The method was evaluated in simulation
studies and was then applied to association analysis in African Americans (AA) from
the National Heart Lung and Blood Institute - Exome Sequencing Project (NHLBI-ESP).
We have identified significant associations of novel genes with the white blood cell
count (WBC) phenotype. These genes, FLG and OR6N2, are located in the 1q21
region and the 1q23 region, regions previously known to be associated with WBC in
AA. No significant associations were found for the other 6 traits analyzed. This study
shows the importance of utilizing local ancestry when analyzing RV in admixed
populations.
Contributors: Hooker, Stanley; Li, Biao; Wang, Gao T.; He, Zongxiao; Zhang, Di; Peng, Bo;
Leal, Suzanne M.
134
EFFECT OF DNA VECTOR LENGTH ON CELL TRANSFECTION
Benjamin Daniel Hornstein
Gene therapy requires the delivery of nucleic acid to replace, regulate, or correct
genes to treat diseases. Viral vectors deliver DNA efficiently to cells, but are often
unsuitable for therapeutic applications because their integration into the host genome
can disrupt or dysregulate essential genes or proto-oncogenes. Nonviral vectors do not
have these issues, but many human cell lines are refractory to transfection with
plasmid. Our laboratory developed minimized nonviral vectors that can be as small as
250 bp, called minivectors. Minivectors are devoid of bacterial sequences, and are
unlikely to be silenced. Published and preliminary data show that smaller vectors have
improved transfection efficiency, but we sought to determine whether this trend
continues to the physical lower limit of DNA length. I hypothesized that DNA vectors
with lengths below a certain threshold would not have improved transfection efficiency
relative to larger vectors.
To test how vector length affects transfection, I created DNA vectors
expressing shRNA against GFP. The lengths were 383, 735, 1,026, 1,869, 2,844,
3,913, 4,265, and 4,556 base pairs (bp). I tested transfection efficiency by measuring
GFP knockdown in HeLa cells stably expressing GFP. Flow cytometry and fluorescence
microscopy were used to quantify the data. We found that smaller vectors required less
mass to be transfected in order to elicit similar knockdown as larger vectors. However,
because of the differences in molecular weight, our results also showed that smaller
vectors require more molecules to be transfected in order to elicit similar knockdown as
larger vectors. Because each clinical application is different, understanding the effect of
vector size on transfection is necessary for calculating the amount of DNA vector
needed to elicit the appropriate expression level for any given application. The ultimate
goal of this project is to find the optimal minivector size for transfection so we can
design a nonviral gene therapy vector that maximizes expression while minimizing
toxicity from transfection vehicle and vector.
Contributors: Roman, Dany; Arevalo-Soliz, Lirio Milenka; Engevik, Melinda; Zechiedrich, Lynn
135
DENOMINATOR DOESN’T MATTER: STANDARDIZING HEALTHCAREASSOCIATED INFECTION RATES BY BED DAYS OR DEVICE DAYS
Molly Jane Horstman
Advisor: Barbara Trautner, M.D./Ph.D.-Department of Medicine
Objective: To examine the impact on infection rates and hospital rank for
catheter-associated urinary tract infection (CAUTI), central line-associated bloodstream
infection (CLABSI), and ventilator-associated pneumonia (VAP) using device days and
bed days as the denominator.
Design: Retrospective survey from October 2010 to July 2013
Setting: Veterans Health Administration medical centers providing acute medical
and surgical care.
Patients: Patients admitted to 120 Veterans Health Administration medical
centers reporting healthcare-associated infections.
Methods: We examined the importance of using device days and bed days as
the denominator between infection rates and hospital rank for CAUTI, CLABSI, and
VAP for each medical center. The relationship between device days and bed days as
the denominator was assessed using a
Pearson correlation and changes in infection rates and device utilization were
evaluated by an analysis of variance.
Results: A total of 7.9 million bed days were included. From 2011 to 2013,
CAUTI decreased whether measured by device days (2.32 to 1.64, P= .001) or bed
days (4.21 to 3.02, P=.006). CLABSI decreased when measured by bed days (1.67 to
1.19, P=.04). VAP rates and device utilization ratios for CAUTI, CLABSI, and VAP were
not statistically different across time. Infection rates calculated with device days were
strongly correlated with infection rates calculated with bed days (r =0.79–0.94, P< .001).
Hospital relative performance measured by ordered rank was also strongly correlated
for both denominators (r= 0.82–0.96, P<.001).
Conclusions: These findings suggest that device days and bed days are equally
effective adjustment metrics for comparing healthcare-associated infection rates
between hospitals in the setting of stable device utilization.
Contributors: Horstman, Molly J; Li, Yu-Fang; Almenoff, Peter L; Freyberg, Ron W; Trautner,
Barbara W
136
VALIDATION OF CRYO-EM STRUCTURES TO IDENTIFY MOLECULAR MACHINES
FOR DRUG DESIGN
Corey F Hryc
Currently, high-resolution biophysical techniques have been used to resolve
atomic features (1-2.5 Å) of proteins and nucleic acids. This data has been successfully
utilized for developing biological inhibitors and engineer drug delivery methods. The
Protein Data Bank archives approximately 7,300 structures at 3-5 Å resolution and this
type of data has been increasing exponentially each year. More specifically due to the
recent advances in cryo-EM, there has been an explosion in number of structures
solved at this resolution range, particularly for mammalian membrane channels and
membrane viruses. These complexes, such as Chikungunya virus, human γ-secretase
complex, hemocyanin isoform 1, and the IP3 receptor lack X-ray structures for
comparison and thus, have not been properly validated.
This lack of validation posts an uncertainty for medicinal chemists to use for
serious drug design. Essentially, this has halted the translational aspect of this
research, repressing these structural findings from drug development. Validating this
data will potentially allow for specific domains, intersubunit interfaces and binding
pockets to be confidently analyzed, and results may be used to target individual amino
acids, inhibiting various biological processes. Over the past few years I have used
model systems (plant and bacterial viruses) to develop and refine the cryo-EM structure
validation pipeline.
Our new validation method, which includes analyzing independent data sets and
models in addition to model-based map validation, can be used in conjunction with fitto-density and stereochemistry scores to assess data uncertainty. Understanding the
level of certainty not only validates our structures, but it also helps identify adequate
inhibitors and novel drug targets. Validation will assure reliability, push the limit in
structural findings, and allow for results to quickly translate into the drug discovery
pipeline.
Contributors: Hryc, Corey; Chen, Dong-Hua; Schmid Michael; Afonine, Pavel; Adams Paul;
Chiu Wah
137
INVESTIGATION OF RNA SPLICING DEFECTS IN DROSOPHILA MODELS OF
TUAOPATHIES
Yi-Chen Hsieh
Advisor: Joshua Shulman, M.D./Ph.D.-Department of Neurology
At autopsy, Alzheimer’s disease (AD) and related tauopathies are defined by the
presence of insoluble, cytoplasmic aggregates comprised of the microtubule-associated
protein tau, termed neurofibrillary tangles (NFTs). We recently revealed that numerous
core components of the U1 spliceosome, including U1-70K and SmN (SmB paralog in
brain), are abnormally enriched in AD detergent-insoluble proteome. Independently, we
discovered that the core components of the U1 spliceosome robustly interact with the
neurotoxicity of human Tau (hTau) in the transgenic animals. We hypothesize that
hTau leads to the mislocalization of SmB, U1-70K, and perhaps other spliceosomal
components from the nucleus to the cytoplasm, leading to the alterations in neuronal
transcriptome and gene expression profiles, and ultimately resulting in
neurodegeneration. We are currently investigating the interactions between splicing
factors and hTau in the brains of Drosophila. In fly brains, the cytoplasmic
phosphorylated hTau aggregates co-localize with SmB (the fly ortholog of human
SmN), and the transgenic expression of hTau disrupts the expression of spliceosomal
proteins. We are also characterizing adult nervous system phenotypes induced by lossof-function in SmB and related splicing factors. The loss-of-function for the core
spliceosomal component SmB leads to several neurodegenerative phenotypes,
including reduced survival, progressive locomotor impairment and decreased neuronal
cells, in adult flies. We will also assess splicing of neuronal genes in the brains of hTau
transgenic flies. In sum, our preliminary studies support our hypothetical model and
begin to reveal a novel mechanism for Tau-induced neurodegeneration mediated by
disruptions of the RNA splicing machinery, possibly leading to deleterious changes in
global gene expression and/or splicing.
Contributors: Hsieh, Yi-Chen; Powers, Martin; White, Janson; Seyfried, Nicholas; Lah, James;
Levey, Allen; Shulman, Joshua
138
NK CELLS CONVERGE LYTIC GRANULES TO PROMOTE CYTOTOXICITY AND
PREVENT BYSTANDER KILLING
Hsiang-Ting Hsu
NK cell activation triggers a tightly regulated series of events leading to
degranulation of lytic granules. Prior to degranulation, preformed lytic granules rapidly
converge from dispersed cytoplasmic locations to the microtubule-organizing center
(MTOC), and then polarize to the immunological synapse. We have previously
identified that granule convergence requires LFA-1 signaling, Src family kinase
activities, and dynein motor function, but does not require commitment to NK cell
cytotoxicity. Despite these advances the purpose of lytic granule convergence in NK cell
cytotoxicity remains unknown. Using Drosophila S2 cells, which unlike mammalian cells
do not express human NK cell ligands for adhesion and activation receptors, we
governed NK cell signal inputs to precisely control granule convergence (as measured
by the distance of granules to the MTOC) and degranulation. Specifically, engagement
of LFA-1 induces convergence of granules, but not degranulation, whereas ligation of
CD16 triggers non-directional degranulation; the combination of the two promotes
targeted degranulation. The average distance of lytic granules to the MTOC was
significantly higher in NK cells conjugated with S2-IgG cells (S2 cells labeled with antiS2 IgG antibody to activate CD16) compared to S2-ICAM1 (S2 cells expressing ICAM-1
as ligand for LFA-1) or S2-ICAM1-IgG cells (3.22μm, 2.45μm and 2.18μm respectively,
p<0.0001, One-way ANOVA), indicating diffuse subcellular granule localization in IgGactivated NK cells. Using imaging flow cytometery, the accumulation of lytic granules at
the lytic synapse was determined in over 200 conjugates. NK cells conjugated with S2IgG cells showed significantly lower synaptic lytic granule localization and degranulation
compared to S2-ICAM1-IgG cells, suggesting non-directional lytic granule release
without LFA-1 signaling. Using these cell combinations we utilized an ultrasoundassisted microwell system to enforce live single NK cell and multiple target cell contacts
to determine killing efficiency. NK cells showed ~30% lower lysis of S2-IgG cells when
compared to S2-ICAM1-IgG cells (p<0.05, t-test). NK cells triggered by S2-ICAM1 cells
alone resulted in granule convergence without cytotoxicity. More importantly, NK cells
activated by S2-IgG cells caused ~20% higher non-specific killing of neighboring
unlabeled “bystander” S2 cells compared to S2-ICAM1-IgG cells (p<0.01, t-test). These
single cell investigations demonstrate that the purpose of NK cell lytic granule
convergence is to improve the efficiency of targeted lytic granule secretion and prevent
collateral damage to neighboring tissue.
Contributors: Hsu, Hsiang-Ting; Viswanath, Dixita; Mace; Emily; Orange, Jordan
139
NOVEL APPROACH TO ASSESS THE FUNCTIONAL AND CLINICAL IMPACT OF
GENETIC VARIATIONS
Teng-Kuei Hsu
Whole genome sequencing uncovers a plethora of genetic alterations that are
often with unknown functional and clinical impact. A major challenge is to estimate the
consequences of these alterations. As a complement to traditional computational tools
that follow machine learning, statistical and biophysical modeling approaches and
trained with numerous available data, we sought to assess the impact of novel
mutations from a phylogenomic perspective, namely by relating specific genotype
variations to specific speciation events. Here we defined the overall change in fitness
due to a mutation as a novel biological quantity called the Evolutionary Action (EA) of
the mutation and that factors in the size of the mutation plus the site at which it occurs.
In our retrospective data analysis, this approach correlated with the loss of protein
function, separated the disease-associated mutations from the benign and matched the
morbidity of monogenic disorders. This approach was also tested by independent
judges at the two most recent international CAGI contests and consistently achieved
top ranking. In practice, this approach allowed us to stratify patients into subgroups by
the impact of mutations in TP53, such that tumors harboring greater deleterious impact
TP53 mutations were more invasive and patients with such mutations associate with
the poorest clinical outcomes. We also evaluate the functional impact of somatic
mutations from the TCGA and identify novel cancer genes by detecting the functional
impact bias on cancer mutations during tumorigenesis. Together, these results show
“Evolutionary Action” (EA) is a novel approach to assess the functional and clinical
impact of genetic variations, identify novel therapeutic targets, and may eventually play
a role in the stratification of cancer patients for precise treatment selection.
Contributors: Katsonis, Panagiotis; Lichtarge, Olivier
140
SPECTRIN CYTOSKELETON REGULATES PERIPHERAL NERVOUS SYSTEM
NODE OF RANVIER ASSEMBLY AND AXON INTEGRITY
Yu-Mei Huang
Advisor: Matthew Rasband, Ph.D.-Department of Neuroscience
Spectrins are a family of cytoskeletal proteins, that provide structural
support of the cell membrane, link membrane-associated proteins to actin and serve as
platforms for cell signaling. Spectrins consist of α and β subunits, forming
heterotetramers to function as a complex. Among the spectrins, αII-spectrin is the only
α-spectrin expressed in the nervous system. αII-spectrin is also implicated in
neurological disorders, including traumatic brain injury, spinal cord injury,
neurodegenerative diseases and West syndrome. Moreover, embryonic lethality of
constitutive knockout mice with nervous system malformation highlights the importance
of αII-spectrin in the nervous system.
To investigate the potential function of αII-spectrin in the brain, we
generated αII-spectrin conditional knockout (cko) mice. Using Nestin-cre mice, we
generated αII-spectrin cko mice to examine the function of αII-spectrin in the central
nervous system (CNS). Mutant animals had profound neurological phenotypes and died
perinatally. Loss of αII-spectrin results in dramatic reduction in all brain β-spectrins.
To specifically interrogate the spectrin function in neurons and to bypass
the perinatal lethality observed in Nestin-cre; αII-spectrin cko mice, we generated
sensory neuron specific αII-spectrin cko mice using advillin-cre. Peripheral sensory
neuron KO mice had abnormal gait and motor incoordination while nociception
remained intact. We found that dorsal root nerve conduction was significantly reduced
in cko mice. Immunohistochemistry showed that mutant mice had fewer nodes of
Ranvier. Moreover, paranodal junctions, flanking nodes, were extensively disrupted. We
also found widespread axon degeneration in advillin-cre cko mice. Consistent with
axonal injury, we observed ATF3 in cko dorsal root ganglia (DRG) neurons beginning at
P10 and increasing with age. Remarkably, ATF3+ neurons are mostly large diameter
neurons; small TRPV1+ neurons did not have the injury marker. This difference could
account for the motor coordination defects caused by deficits in proprioception while
nociception remains unaffected.
Contributors: Huang, Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel; Lalonde, Joshua;
Noebels, Jeffrey; Rasband, Matthew
141
DOXYCYCLINE-INDUCIBLE CRISPR-CAS9/CAS9N MICE FOR CANCER
MODELING
Yung-Hsin Huang
Cancers are often caused by specific gene mutations. Recent discoveries
involving the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)associated RNA-guided endonuclease Cas9 have made eukaryotic genome editing
feasible. Cas9 utilizes single-guide RNA (sgRNA) to target genomic DNA sequences,
creating mutations or precise corrections with homology-directed repair. Genome
editing not only allows us to correct mutations but also enables us to recreate
humanized mutations in murine models, which further facilitates cancer research.
However, the Cas9 sequence is difficult to deliver due to its large size, and prolonged
expression of the Cas9 protein may lead to off- target mutations. Therefore, kKnockingin the Cas9 sequence under an inducible promoteor mitigates thosese two factors
mentioned above. Nevertheless, even in limited duration of Cas9 expression, off-target
mutations might occur due to non-specific SgRNAsgRNA even when Cas9 is expressed
for a limited time. To solve overcome this obstacle, Cas9 nickase (Cas9n), which is
Cas9 D10A point mutation, with a combination of two SgRNAs can increase specificity
up to 1000 times. Hence, oOur goal is to generateing a doxycycline-inducible
Cas9/Cas9n murine model that enables the establishment of , leading to establishing
humanized mutations for cancer modeling.
In murine embryonic stem cells (mESCs) containing reverse tetracyclinecontrolled transactivator in ROSA loci, Cas9 and Cas9n sequences were engineered
into Hprt loci via inducible cassette exchange. Chimera mice were generated by
injection of iCas9 and iCas9n mESCs into blastocyst and then bred to C57BL/6J. Under
10mg/kg doxycycline treatment for 5 consecutive days, hematopoietic progenitor cells
were purified and we confirmed their Cas9/Cas9n expression.
In both mESCs and hematopoietic progenitor cells upon doxycycline treatment,
Cas9/Cas9n expression was confirmed through Western Blots and Cas9/Cas9n
functionality was also confirmed by surveyor assay under transduction of sgRNA
targeting Tet2 and Dnmt3a.
An inducible Cas9/Cas9n system will allows for temporal control of Cas9/Cas9n
expression, decreasing off target effects. This system would also decreases the
complexity of genome editing with Cas9, only requiring only the introduction of sgRNA.
The inducible Cas9 mouse model described in this abstract enables us to generate
humanized mutations in mice and provides a screening platform for plausible cancer
therapeutic targets
Contributors: Yung-Hsin Huang, Y. Daniel Lee, Sunny Sun-Kin Chan, Michael Kyba, Margaret
A. Goodell
142
SMALL WONDERS: CARBON NANOTECHNOLOGY TO TREAT AUTOIMMUNE
DISEASES
Redwan Huq
Advisor: Christine Beeton, Ph.D.-Department of Molecular Physiology & Biophysics
Autoimmune diseases, such as multiple sclerosis, are mediated by a type of
white blood cell—T lymphocytes. Current treatments for these diseases are broad
immunosuppressants associated with life-threatening side effects, necessitating the
development of new therapeutic strategies. The inflammatory microenvironment in
these diseases generates large quantities of harmful reactive oxygen species (ROS).
However, low levels of intracellular ROS act as signaling molecules necessary for T
lymphocyte activation. Therefore, intracellular ROS represent attractive targets for
modulating T lymphocyte activity and for treating autoimmune diseases.
Carbon nanoparticles can scavenge ROS with higher efficacy than dietary and
endogenous antioxidants. The affinity of carbon nanoparticles for specific cell types
represents an emerging tactic for targeted therapy. Here, we show that nontoxic
poly(ethylene)-glycol-functionalized hydrophilic carbon clusters (PEG-HCCs) are the
first carbon nanoparticles to be preferentially internalized by T lymphocytes over other
splenic immune cells. We use this selectivity to attenuate T lymphocyte function in vitro
without affecting major functions of macrophages, an immune cell subset crucial for
physiological activation of T lymphocytes. We demonstrate the effectiveness of PEGHCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity
and in ameliorating experimental autoimmune encephalomyelitis, an animal model of
multiple sclerosis. Our results suggest that the remarkable selectivity of PEG-HCCs for
T lymphocytes is a novel and promising route for treating T lymphocyte-mediated
autoimmune diseases without inducing broad immunosuppression.
Contributors: Redwan Huq, Errol L.G. Samuel, Mark R. Tanner, Thomas Lee, Rajeev B.
Tajhya, William Sikkema, Fatima S. Khan, Taeko Inoue, Robia G. Pautler, Paul C. Porter,
David B. Corry, James M. Tour, Christine Beeton
143
DISCOVERY OF NOVEL PHAGE-DISPLAYED PEPTIDE REAGENTS FOR THE
DETECTION OF NOROVIRUS
Amy Marie Hurwitz
Background: Norovirus (NoV) infections are the most common cause of nonbacterial gastroenteritis and lead to about 21 million new cases and cost $2 billion per
year in the United States alone. Existing diagnostics have limited feasibility for point-ofcare applications, so there is a clear need for more reliable, rapid, & simple-to-use
diagnostic tools for detecting NoV.
Methods: In this study, phage display technology was used to screen a phage
library displaying over a billion randomized 12-mer peptides for those that bind to NoV
virus-like particles (VLPs) composed of the major capsid protein, VP1. Since NoV
strains classified in genogroups GI and GII cause the majority of human outbreaks, we
focused on the prototypical Norwalk strain (genotype GI.1) to identify binding reagents.
Phage populations were characterized by deep sequencing after three and four rounds
of selection for NoV binding and computational analysis was performed to identify
amino acid motifs involved in binding GI.1 VLPs.
Results: Deep sequencing of the selected phage populations revealed
thousands of unique peptides with possible binding to GI.1. The peptides occurring
most commonly increased in fractional frequency from round three to four, as did the
overall binding affinity of each population for GI.1 VLPs. A computational algorithm
developed for sequence analysis identified a consensus motif, “YRSWXP,” from the
sequences obtained after round four of selection. Phage ELISAs and solid-phase
peptide arrays confirmed the ability of this motif to bind specifically to GI.1 in the context
of two 12-mer peptides.
Conclusions: The work reported here provides evidence for the use of phage
display technology and deep sequencing for the identification of novel peptides that
detect NoV. This method enables rapid identification of lead clones that bind new
pathogens and can be further developed with directed evolution strategies to produce
optimized protein binders. Further, using phage-displayed peptides directly as detection
reagents provides advantages including the ease and low cost of phage propagation
and purification, and high avidity provided by the phage structure. Ultimately, this work
will lead to the establishment of novel tools for rapid and sensitive diagnosis of NoV
infection with specificity for a broad range of clinically relevant NoV strains.
Contributors: Hurwitz, Amy; Huang, Wanzhi; Atmar, Robert; Palzkill, Timothy
144
THE NATURAL HISTORY OF PEDIATRIC ULCERATIVE COLITIS IN THE ERA OF
BIOLOGIC THERAPY
Faith D Ihekweazu
Advisor: Richard Kellermayer, M.D./Ph.D.-Department of Pediatrics
Background: The clinical outcomes of pediatric ulcerative colitis (UC) are not well
known. Most of the population-based studies were conducted outside of the United
States and prior to the introduction of biologic (anti-tumor necrosis factor α) agents in
the standard treatment of pediatric UC. We aimed to describe the natural history of
pediatric UC in the era biologic therapies.
Methods: We conducted a retrospective review of 152 pediatric patients at Texas
Children’s Hospital with a new diagnosis of UC between January 2003 and December
2009. The patient records were followed through July 2014. Localization of disease at
diagnosis, use of steroids, immunomodulator therapy or biologic agents, presence of
extraintestinal manifestations, and need for surgery at a minimum five year follow up
were noted. A follow-up phone call was made to patients who were lost to follow-up
within five years of diagnosis to assess these clinical outcomes. Only patients with a
minimum of 5 years of follow up or need for surgery within the first 5 years of diagnosis
were included in the final analysis.
Results: We identified 106 pediatric UC patients with a minimum of 5 years of
follow up or colectomy within the first 5 years of diagnosis. Mean age at diagnosis was
10.7±4.1 years, with an average length of follow up of 6.5±1.9 years. Eight percent (8%)
presented at diagnosis with ulcerative proctitis (E1), 12% had left sided disease (E2),
48% had extensive UC (E3). Biologic medications were used in 36% of patients, 27%
were treated with immunomodulators, 94% received 5-aminosalicylic acid therapy, and
90% were exposed to steroid therapy throughout the follow up period. Extra-intestinal
manifestations occurred in 26 patients (25%). In respect to all patients, 4% had arthritis,
11% arthralgia, 10% had primary sclerosing cholangitis, 2% had autoimmune hepatitis,
and 1% had aphthous stomatitis. Cumulative rate of colectomy was 20% at 5 years,
with 8% progressing to colectomy within 1 year of diagnosis. Gender, race, extent of
disease at diagnosis and extraintestinal manifestations did not have statistically
significant impact on progression to colectomy in this cohort.
Conclusion: This is the largest pediatric cohort of UC patients with 5 year clinical
outcomes. Compared to previous studies from the same geographic region in the prebiologic era, pediatric UC patients in our cohort presented with more extensive disease
and required a higher rate of colectomy; however, these rates are similar to other
worldwide studies in the pre-biologic era. This work emphasizes the need for novel
preventative and therapeutic measures to combat pediatric UC.
Contributors: Ihekweazu, Faith; Fofanova, Tatiana; Ajjarapu, Avanthi; Kellermayer, Richard
145
A VISUAL AND CURATORIAL APPROACH TO VARIANT PRIORITIZATION AND
DISCOVERY IN GENOME-WIDE DIAGNOSTICS
Regis Aaron James
Advisor: Chad Shaw, Ph.D.-Department of Molecular & Human Genetics
Genome-wide data are increasingly important in the clinical evaluation of
human disease. However, the large number of variants observed in individual patients
challenges the efficiency and accuracy of diagnostic review. Recent work has shown
that systematic integration of clinical phenotype data with genotype information can
improve diagnostic workflows and prioritization of variants. We have developed visually
interactive, analytically transparent analysis software that leverages existing disease
catalogs, such as the Online Mendelian Inheritance in Man database (OMIM) and the
Human Phenotype Ontology (HPO), to integrate patient phenotype and variant data into
ranked
diagnostic
alternatives.
Our
tool,
“OMIM
Explorer”
(http://www.omimexplorer.com), extends the biomedical application of semantic
similarity methods beyond those reported in previous studies. The visual approach
collapses high-dimensional phenotype and genotype data from an individual into a
graphical format that contextualizes the patient within a low-dimensional disease map.
The map proposes a differential diagnosis and algorithmically suggests potential
alternatives for phenotype queries. Visual interactivity affords the user the ability to
filter and update variant rankings through interaction with intermediate results. The tool
also implements an adaptive approach for gene discovery based on patient
phenotypes. We retrospectively analyzed pilot cohort data from the Whole Genome
Laboratory at Baylor College of Medicine, demonstrating performance of the tool and
workflow in the reanalysis of clinical exomes. Our tool assigned to clinically reported
variants a median rank of 2, placing causal variants in the top 1% of filtered candidates
across the 47 cohort cases with reported molecular diagnoses of exome variants in
OMIM Morbid Map genes. This integrative paradigm can likely improve the quality and
efficiency of genomic medicine by more effectively utilizing available phenotype
information, catalog data, and genomic knowledge.
Contributors: James, Regis; Campbell, Ian; Chen, Edward; Boone, Philip; Rao, Mitchell;
Bainbridge, Matthew; Lupski, James; Yang, Yaping; Eng, Christine; Posey, Jennifer; Shaw,
Chad
146
ARV1 DELETION ALTERS FAT STORAGE VIA ADIPOSE-EXTRINSIC
MECHANISMS
Kelsey Elizabeth Jarrett
Advisor: William Lagor, Ph.D.-Department of Molecular Physiology & Biophysics
ARE2 Required for Viability 1 (ARV1) is a transmembrane protein of the
endoplasmic reticulum (ER) that is believed to promote intracellular sterol transport in
yeast. Deletion of ARV1 in yeast causes lipid accumulation in the ER, altered
membrane structure, and constitutive activation of the unfolded protein response. This
protein is conserved in mammals, and human ARV1 is known to rescue the growth and
viability defects in ARV1 deficient yeast. In previous work, we found that germline
deletion of Arv1 in mice results in a 25% reduction in body weight, greatly reduced fat
mass, increased energy expenditure, and improved glucose tolerance. In the present
work, we sought to test the hypothesis that loss of ARV1 activity in white and brown fat
is responsible for the metabolic changes we observed. To test this, we utilized two
different adipose-selective Cre drivers: Adiponectin-Cre and Ap2-Cre. Adiponectin-Cre
mediated deletion was robust and highly specific to the white and brown fat, and had no
impact on fat storage or blood glucose. In contrast, Ap2-Cre resulted in a substantial
53% decrease in Arv1 mRNA in the brain (p < 0.05), accompanied by reduced fat mass
and markedly improved glucose tolerance. Due to strong leaky expression of Ap2-Cre
in the brain, we utilized Nestin-Cre to delete Arv1 in the nervous system (NKO). The
NKO mice mirror the phenotypes of the whole-body Arv1-KO mice with decreased body
weight and fat mass as well as spontaneous death of 30-50% of mice by 20 weeks of
age. In addition, we also observe seizures in these mice. The data suggest that
mammalian ARV1 function is primarily important in non-adipocyte tissues, and our
preliminary studies suggest that ARV1 contributes to metabolic rate and fat storage via
the central nervous system.
Contributors: Gupta, Rajat; Billheimer, Jeffrey
147
IDENTIFICATION OF DIRECT TARGETS OF THE ATOH1 TRANSCRIPTION
FACTOR OF COCHLEAR HAIR CELLS
Hsin-I Jen
Age-dependent hearing loss is mainly caused by loss of hair cells in the cochlea.
Unlike humans and other mammals, adult birds can regenerate their hair cells from
surrounding supporting cells, thus restoring hearing. Inducing trans-differentiation of
supporting cells into hair cells in the adult mammalian cochlea may be a pathway to
hearing restoration.
Atoh1 is a bHLH transcription factor that is necessary and sufficient for hair cell
formation. Ectopic overexpression of Atoh1 can trans-differentiate supporting cells into
hair cells in embryonic and neonatal mice, but not in mature animals. To successfully
induce trans-differentiation in the adult cochlea, it is important to decipher the
mechanisms that prevent Atoh1-induced hair cell formation.
One possible mechanism that prevents Atoh1 induced trans-differentiation in
adults may be the inability to activate Atoh1’s target genes. To address this problem,
we first need to identify Atoh1 target genes in the cochlea. We analyzed and compared
hair cell RNA-seq data from the cochlea and Atoh1 ChIP-seq from cerebellum and
intestine. We used in situ hybridization to validate the expression of hair cell genes in
the inner ear. We confirmed 10 of these genes as being direct Atoh1 targets in the
cochlea by ChIP-PCR. The identification of candidate Atoh1 target genes is a first step
in identifying gene regulatory networks for hair-cell development and may inform future
studies on the potential role of Atoh1 in mammalian hair cell regeneration.
We further analyzed the active epigenetic mark (H3K9ac) on Atoh1 targets in
both P0 hair cells and supporting cells. As we expected, Atoh1 target genes in neonatal
hair cells have H3K9ac enrichment. In contrast, Atoh1 target genes in neonatal
supporting cells do not have H3K9ac enrichment. We will further examine poised
histone marks and DNA methylation of Atoh1 targets in the hair cells and supporting
cells in neonatal and mature animals. These will help us to decipher the possible role of
epigenetic changes in preventing Atoh1-induced supporting cells to hair cell transdifferentiation in adults.
Contributors: Jen, Hsin-I; Cai, Tiantian; Groves, Andrew
148
QUANTITATIVE REAL-TIME MONITORING OF GLUTATHIONE
Xiqian Jiang
Advisor: Jin Wang, Ph.D.-Department of Pharmacology
Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells.
Together with its oxidized partner (GSSG), GSH maintains the cellular redox
homeostasis, regulates protein functions through S-glutathionylation, and acts as a
signaling molecule to directly activate gene expression. All these important functions
are regulated by the intracellular concentration and distribution of GSH. Currently, very
few method could provide reliable information about the dynamics of GSH
concentration changes and crosstalk between GSH concentration differential in
different cellular compartments. Previously, we developed the first ratiometric
fluorescent probe (ThiolQuant Green, TQG) for quantitative imaging of intracellular
GSH by introducing reversible Michael Addition. However, the slow reaction kinetics
between GSH and TQG has limited its application in live monitoring of GSH dynamics.
Through quantum mechanical calculation and experimental modification, we
significantly improved reaction kinetics and optical profile of our probe. The new probe,
designated as ThiolQuant Green Real-Time (TQG-RT), has a time resolution below
40s, which provides the first ever molecular tool for monitoring transient changes of
GSH within cells upon various stimulations. In a pilot experiment, we observed minutescale GSH fluctuations after H2O2, GSH-ester and EGF treatment in TQG-RT loaded
HeLa cells. We were also able to apply our probe in hard-to-transfect cells such as
primary cells and macrophages. We could even achieve in vivo GSH mapping in C.
elegans. In summary, we developed the first fluorescent probe that enable quantitative
real-time imaging of GSH in living cells and animals. We envision that our GSH probes
will enable unprecedented opportunities to study GSH dynamics and revolutionize our
understanding of the physiological and pathological roles of GSH in cells and
organisms.
Contributors: Jiang, Xiqian; Chen, Jianwei; Song, Xianzhou; Cheng, Ninghui; Wang, Meng;
Wang, Jin
149
LOSS OF AMP-ACTIVATED PROTEIN KINASE LEADS TO PROTEIN
HYPOACETYLATION IN MLL-AF9 INDUCED LEUKEMIA
Yajian Jiang
Energy homeostasis is fundamental for cells to survive, proliferate and function.
Among all the metabolism gene networks, AMP-activated protein kinase (AMPK) is the
central regulator controlling various catabolic processes including fatty acid oxidation,
lipid synthesis, ketonegenesis and glucose metabolism. The kinase is activated by
elevated AMP:ATP ratios in the cell and leads to increased ATP production by
stimulating catabolic pathways and inhibiting anabolic pathways. Energy homeostasis is
also key for tumor cells and some tumor cells even acquire higher metabolism capacity
because they have to adapt to the poor-nutritious, hypoxic local environment such as
the bone marrow. Controversial results have been published about the roles of AMPK in
different cancer cells. Some suggested AMPK is tumor suppressor whose stimulation
can inhibit cancer cell growth and prolong survival, while others revealed AMPK
inhibition has a protective effect in cancer treatment.
We previously demonstrated AMPK deletion largely decreased the growth
potential of MLL-AF9 induced leukemia cells and prolonged the survival of leukemic
mouse. AMPK deletion increased oxidative stress and DNA damage in leukemia cells.
Interestingly, I discovered that protein acetylation levels are reduced in AMPK-deficient
AML cells, including histones H3 and H4. Protein acetylation affects protein
stabilization, localization and function. The modification is achieved by
acetyltransferases transfering the acetyl group from the major donor acetyl-CoA to
specific amino acid and it could also be removed by deacetylases. I found that the
acetyl-CoA level is reduced in AMPK deficient leukemia cells. Other groups have
reported HATs (histone acetyltransferase) and HDACs (histone deacetylase) can be
targets of AMPK phosphorylation in non-hematopoietic cells. Taken these results into
consideration, our working hypothesis is that AMPK deficiency causes protein and
histone hypoacetylation in MLL-AF9 induced leukemia by reducing the acetyl-CoA
levels and possibly directly affecting the function of acetyltransferases and
deacetylases. My work may reveal the overlapping gene networks between metabolism
and protein acetylation and raises the possibility of combining AMPK modulators and
histone acetylation modulators to treat leukemia.
Contributors: Yajian Jiang, Ayumi Kitano, Richard H. Chapple, Angelique Lin, Rebecca L.
Murdaugh, Yusuke Saito, Daisuke Nakada
150
TORC2: A NOVEL TARGET FOR TREATING AGE-ASSOCIATED MEMORY
IMPAIRMENT
Jennifer Leigh Johnson
As our population ages, cognitive decline and dementia are becoming more
prevalent. The fact that memory declines as a function of age indicates that there must
be crosstalk between components of these two processes. Yet, the molecular
mechanisms underlying these inevitable processes are not fully understood. Although
the serine/threonine kinase mechanistic target of rapamycin (mTOR) has long been
implicated in aging, its role in brain aging remains unclear. Given the evolutionary
conservation of mTOR between fruit flies and humans, we investigated the specific role
of the new mTOR complex 2 (mTORC2) in age-related memory impairment in both the
fly and rodent. We show that the activity of mTOR complex 2 (mTORC2) declines with
age in the brain of both fruit flies and mice Interestingly, treatment with a small molecule
that activates mTORC2 restores mTORC2 activity and rescues long-term memory
(LTM) deficits in both aged mice and flies. In addition, we found that pharmacologically
activating mTORC2 or promoting actin polymerization enhances long-term memory. In
contrast to the current approaches to reverse memory loss that have primarily focused
on changes in gene expression at the epigenetic and transcriptional level, our data
suggests a novel, evolutionarily conserved mechanism for restoring memory that is
dependent on structural plasticity. Although we are only beginning to understand the
unique functions of mTORC2 in the brain, therapeutic approaches targeting the
mTORC2 signaling pathway may have a profound impact in the prevention and
treatment of age-related neurological diseases.
Contributors: Johnson, Jennifer; Huang, Wei; Roman, Gregg; Costa-Mattioli, Mauro
151
NEUROLEPTICS AND BREAST CANCER RISK
Alyssa N Johnston
Background: Psychiatric medications are among the most widely prescribed
class of drugs in the world. The main mechanism of action for both typical (class 1) and
atypical (class 2) antipsychotics is the blockade of the post-synaptic dopamine D2
receptors which normally suppress prolactin (PRL) production and secretion; a
blockade of these receptors cause serum PRL levels to rise. Epidemiological studies
have associated dopamine antagonist use with an increased breast cancer risk while
others have shown a significant effect on serum PRL levels in psychiatric patients on
antipsychotics. PRL is known most for its role in activating PRL-PRLR-JAK2-STAT5
signaling pathway. Breast cancer arises from precancerous lesions but is prevented
from progressing by apoptosis and thus breaking the apoptosis barrier in breast cancer
is important to halt its progression; pSTAT5 suppresses the apoptosis barrier in cancer
initiation. We hypothesize that these mechanisms are responsible for neuroleptics
effect on increasing breast cancer risk.
Experimental design and methods: Dopamine blocking neuroleptics are known
to cause an increase in serum PRL and there is significant epidemiological evidence
that correlates antipsychotic use with an increase in breast cancer risk To test whether
antipsychotics could increase breast cancer risk by accelerating the progression of preexisting early lesions, we utilized our previously reported RCAS-caErbB2 virus to infect
MMTV-tva transgenic mice. Following oncogenic infection, mice were randomized into
two groups: 5mg/kg daily treatment of pimozide or the diluent control for two weeks. We
also performed an independent experiment with a class 2 antipsychotic, Risperidone, to
confirm a board spectrum effect from the antipsychotics.
Results: We found that the early lesion number and burden was higher in the
pimozide-treated mice than in the control. In addition, we found that pimozide treatment
significantly lowered apoptosis in early lesions. Serum PRL was elevated in treated
mice as well as extensive alveologeneis and production of β-casein. We detected
elevated pSTAT5 levels in normal ducts and early lesions in mice treated with pimozide.
Similar results were found in Risperidone treated mice. Surprisingly, we found that the
percentages of pSTAT3+ cells were significantly increased both in normal mammary
epithelium and in these early lesions in Pimozide and Risperidone treated mice.
Conclusion: These data suggest that neuroleptic agents that block dopamine
receptors can activate PRL-JAK2-STAT5 signaling in preexistent precancerous cells to
suppress apoptosis and promote progression to cancer. Further, our unexpected
pSTAT3 findings allow us to believe that neuroleptic treatment may promote early
lesion progression via activating both STAT5 and STAT3 to severely lower the
apoptotic barrier. We believe we have discovered a novel mechanism as to how
dopamine-antagonizing neuroleptic treatment may impact breast tumorigenesis.
Contributors:
152
THE MUTATION SPECTRUM OF A LARGE BRAZILIAN INHERITED RETINAL
DISEASE COHORT
Evan Michael Jones
Inherited retinal dystrophies (IRDs) affect approximately 1 in 2,000 individuals.
These disorders range from most severe cases of congenital blindness to more delayed
cases of progressive visual and retinal degeneration in adulthood. A significant amount
of clinical and genetic heterogeneity among these disorders makes diagnosis difficult
without comprehensive sequencing of potentially causative genes. IRDs, moreover, can
manifest in autosomal dominant, autosomal recessive, and X-linked inheritance
patterns, adding to the complexity. Cohort studies in multiple populations have
highlighted distinct genetic effects based on population. Here we characterize the
molecular cause of IRDs in a large Brazilian cohort, a prominent world population that
has largely remained unstudied. We report on the molecular diagnosis of 231
individuals in an available cohort of 1,492 Brazilians affected by IRDs. Through targeted
capture and next-generation sequencing of 224 known retinal disease genes, we
identify the leading molecular causes of retinitis pigmentosa (RP), Leber’s congenital
amaurosis (LCA), Stargardt’s disease, and Usher syndrome among numerous other
IRDs in our cohort. The solving rates for IRDs found within our cohort are slightly higher
than generally reported rates in the current literature. This is likely in part a result of
improved access to reported pathogenic variants in numerous publicly accessible
databases. Roughly 35% of pathogenic variants in our cohort have been previously
reported to cause retinal disease. The other 65% are novel variants likely to be
pathogenic as either putative loss-of-function variants or predicted damaging variants in
a gene associated with the expected diagnosis. Notably, 11% of RP individuals were
found to have pathogenic or likely pathogenic variants in GPR98. This is a much higher
percentage than what is seen in other populations and may highlight the presence of a
founder mutation. Furthermore, in LCA individuals the most common genes were CRB1
(12%) and ABCA4 (12%) with less than 1% of individuals affected by CEP290 variants,
differing significantly from that observed in Caucasian populations. These findings
highlight the need for a precise and population-based molecular diagnosis of IRDs.
With the development of current gene therapies for diseases such as LCA, an accurate
molecular diagnosis is essential for appropriate genetic counseling and informed
treatment recommendations.
Contributors: Jones, Evan M.1; Soens, Zachry1; Sampaio, Shirley A.2; Sena, Isadora F.G.3;
Magalhaes, Kenia C.S.3; Li, Yumei1; Xu, Shan1; Simoes, Renata T.3; Porto, Fernanda B.O.2,
4; Chen, Rui1
153
LOSS OF THE FREM/FRAS PROTEIN COMPLEX CAUSES CONGENITAL
DIAPHRAGMATIC HERNIAS IN HUMANS AND MICE
Valerie K. Jordan
Advisor: Daryl Scott, M.D./Ph.D.-Department of Molecular & Human Genetics
Congenital diaphragmatic hernia (CDH) occurs when the abdominal viscera
enters into the thoracic cavity through an abnormal opening in the diaphragm. This lifethreatening birth defect affects 1 in 3,000 newborns and accounts for approximately 8%
of all major congenital anomalies. In approximately 20% of human CDH cases, the
herniated viscera are encapsulated by a thin layer of membranous tissue referred to as
a hernial sac. We have previously shown that isolated sac CDH in humans and mice
can be caused by recessive mutations in FREM1, which encodes an extracellular matrix
protein. In the extracellular matrix, FREM1 forms a self-stabilizing ternary complex with
two related proteins—FREM2 and FRAS1—which plays an important role in cell
adhesion and intercellular signaling.To determine if mutations in FREM2 and FRAS1
can also cause CDH, we performed exome sequencing on 69 patients with CDH. In
this cohort we identified five individuals who were homozygous or compound
heterozygous for mutations in FREM/FRAS genes. Two of these individuals had sac
CDH and the third had both CDH and a diaphragmatic eventration—an abnormal
elevation in the diaphragm similar to sac CDH. Three of the alleles involved were also
found to be significantly enriched in our CDH cohort compared to ethnically matched
controls. In support of FREM2’s role in the development of CDH, we also found that
13% of Frem2ne/ne mice have anterior sac CDH. These hernias are identical to those
seen in FREM1-deficient mice and form when the migrating muscular components fail
to meet at the midline behind the sternum. We conclude that mutations in FREM1,
FREM2 and FRAS1 increase the risk of developing sac CDH and that loss of the
FREM/FRAS protein complex inhibits the migration of the diaphragmatic musculature
leading to the formation of sac hernias.
Contributors: Beck, Tyler; Kim, Bum Jun; Lupski, James; Shalini, Jhangiani; Tomasz, Gambin;
Hernandez-Garcia, Andres; Gibbs, Richard; Boerwinkle, Eric; Scott, Daryl.
154
THE SIMPLEST NATURAL REOVIRUS AND ITS EVOLUTIONARY TRAJECTORY
Jason T Kaelber
Fako virus is a novel mosquito-specific dsRNA reovirus with 9 genomic segments, the
fewest of any reovirus. While most reoviruses have two or three capsid layers, Fako virus is
single-shelled. The 4.5Å structure of this virus reveals a streamlined capsid organization with
fewer peptides per asymmetric unit than any other reovirus. The innermost shell of FAKV
displays a “pseudo T=2” symmetry with 120 subunits arranged with one dimer per asymmetric
unit. The fold of the core protein is so conserved as to be recognizable in all reoviruses and two
other families. In the turreted reovirus subfamily (Spinareovirinae), a clamp protein decorates the
exterior of the major capsid protein. Unique among the Spinareovirinae, the Fako virus clamp
does not obey “pseudo T=2” symmetry and is present in only 60 copies per virion. Fako virus’s
“clamp” protein has no statistically significant sequence homology to any other protein, yet
structural analysis reveals 10 out of 13 secondary-structural elements are shared with cytoplasmic
polyhedrosis virus. Future work will reveal the mechanism by which this clamp selects only one
of two similar binding sites on the surface of the major capsid protein dimer.
Ancestral trait reconstruction was performed to infer the character states of nodes within
the subfamily’s phylogeny, allowing me to propose a model for the natural history for singleshelled reoviruses. The common ancestor of all turreted reoviruses had a second shell and 120
clamp proteins per virion. Fako virus emerged by serial loss-of-function events. The second shell
became dispensable in a 10-segmented insect-infecting common ancestor of three genera: Fako
virus experienced a deletion of the gene, while in cytoplasmic polyhedrosis virus, the outer shell
gene was exapted to form intracellular occlusions, and in Rice ragged stunt virus, 70% of the
shell quasi-equivalent sites are unoccupied. This highlights the role of reductive evolution among
reoviruses.
Additionally, we determined the atomic structure of human picobirnavirus by
cryoelectron microscopy, revealing similarities in capsid configuration between these two
dsRNA virus families. Despite the lack of sequence homology, underlying structural similarities
between the various dsRNA virus families point to a common origin of the extant dsRNA
viruses.
Contributors: Kaelber, Jason T; Auguste, A Jonathan; Hryc, Corey F; Collier, Aaron M; Tao,
Yizhi J; Weaver, Scott C; Chiu, Wah
155
INHIBITION OF HEXOSAMINE BIOSYNTHETIC PATHWAY PROMOTES
CASTRATION-RESISTANT PROSTATE CANCER
Akash Kumar Kaushik
Prostate cancer is the top most diagnosed cancer in male population in the
United States. Although, response to treatment of androgen dependent (AD) localized
prostate cancer is significantly improved, recurrent tumors invariably develop into lethal
castration-resistant prostate cancer (CRPC). The precise molecular alterations driving
the progression of CRPC are not clearly understood. Using a novel network-based
integromics approach, here, we show distinct alterations in the Hexosamine
Biosynthetic Pathway (HBP) to be critical for sustaining the castrate resistant state. We
found expression of the HBP enzyme glucosamine-phosphate N-acetyltransferase 1
(GNPNAT1) to be elevated in androgen dependent (AD) PCa while relatively
diminished in CRPC. Genetic loss of function experiments for GNPNAT1 in CRPC-like
cells led to increased proliferation and aggressiveness, in vitro and in vivo. This was
mediated by specific cell cycle genes regulated by the PI3K-AKT pathway in cells with
full length androgen receptor (AR) or by SP1 regulated expression of ChREBP
(carbohydrate response element binding protein) in cells containing AR-V7 variant.
Strikingly, addition of HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to
CRPC-like cells reduced the expression of cell cycle genes and attenuated tumor cell
proliferation, both in vitro and in animal studies, while also demonstrated additive
efficacy when combined with enzalutamide in vitro. These findings are particularly
significant given that the CRPC-like cells tested, inclusive of those containing AR-V7
variant, are inherently resistant to enzalutamide. These observations demonstrate the
therapeutic value of targeting altered metabolic pathways in CRPC.
Contributors: Kaushik, Akash; Shojaie, Ali; Panzitt, Katrin; Sonavane, Rajni; Venghatakrishnan,
Harene; Manikkam, Mohan; Zaslavsky, Alexander; Putluri, Vasanta; Vasu, Vihas; Zhang,
Yiqing; Lloyd, Stacy; Szafran, Adam; Dasgupta, Subhamoy; Stossi, Fabio; Li, Hangwen;
Samanta, Susmita; Cao, Xuhong; Tsouko, Efrosini; Khan, Ayesha; Huang, Shixia; Frigo,
Daniel; Chan, Lawrence; Edwards, Dean; Kaipparettu, Benny; Mitsiades, Nicholas; Weigel,
Nancy; Mancini, Michael; Ittmann, Michael; Chinnaiyan, Arul; Putluri, Nagireddy: Palapattu,
Ganesh; Michailidis, George; Sreekumar, Arun
156
HIPPOCAMPAL PLACE CELL CODING IN A MOUSE MODEL OF RETT SYNDROME
Sara Elizabeth Kee
Rett Syndrome (RTT) is a neurological disorder affecting primarily girls,
characterized by pervasive learning disability, along with motor and autonomic
dysfunction. RTT is caused by loss of function mutations to the X-linked gene MECP2,
which encodes a transcription regulator that binds to methylated CpGs. The Mecp2+/mouse model recapitulates most of the phenotypes seen in the human disease,
including learning and memory deficits. A candidate for the neural circuit mediating
some of these behaviors is the hippocampus (HP), which is a brain area essential for
learning and memory. In particular, spatial memories are thought to be represented by
the activity of hippocampal neurons. The synchronization, or finely tuned timing, of
hippocampal neuronal activity is required for intact memory encoding and consolidation.
Therefore, we are interested in studying the hippocampal activity of the Mecp2 mouse
model in naturally behaving animals. Specifically, we will discern the circuit properties
that lead to the spatial learning and memory deficits. Our hypothesis is that, improper
synchronization of hippocampal neuronal firing activity leads to failure in memory
consolidation and poor spatial memory representation. Our preliminary data suggests
that there is a deficit in place cells coding caused by deficits in spatial memory
consolidation in this model of RTT.
Contributors: Kee, Sara; Ji, Daoyun; Zoghbi, Huda
157
AN INTEGRATIVE LASSO-BASED MODEL CONTAINING CIRCULATING
CYTOKINES AND CHEMOKINES AND INITIAL METASTATIC STATUS IMPROVES
THE PROGNOSTIC PREDICTION OF OSTEOSARCOMA PATIENTS
Aaron Josef Kelly
Advisor: Tsz-Kwong Man, Ph.D.-Department of Pediatrics
Introduction: Osteosarcoma (OS) is the most common malignant bone tumor in
children and adolescents. Although only 20% of patients during diagnosis have
detectable metastatic lesions using conventional imaging techniques, many later
relapse, contributing to dismal outcomes. Unfortunately, no biomarkers other than initial
metastatic status are currently used during patient diagnosis. Identification of molecular
biomarkers that provide prognostic information beyond initial metastasis will facilitate
better risk-stratification at diagnosis to determine the most appropriate treatment option
to improve patient outcomes. Circulating cytokines/chemokines present an attractive
opportunity for clinically used biomarkers since blood contains both tumor and host
factors and only minimally invasive procedures are needed. Furthermore, chemokines
and cytokines have been implicated in a host of pathogenic mechanisms in different
solid tumors, indicating that discovery of biomarkers may lead to greater understanding
of OS pathogenesis and identification of therapeutic targets.
Methods: We employed Luminex bead assays to measure the concentrations of
76 cytokines/chemokines in a cohort of 233 patient serum samples from the Children’s
Oncology Group (COG). A Lasso-based CoxPH model with cross-validation feature
selection was trained on a subset of this cohort (116) and tested on the remaining
patient samples (117) to determine if a subset of proteins could give stronger predictive
power with respect to overall survival than just metastasis alone. The selected model
was that which minimized the partial likelihood deviance. The model was then validated
on an independent cohort of 37 OS plasma samples collected from Texas Children’s
Hospital.
Results: A Lasso model containing initial metastatic status and 11
chemokines/cytokines was trained on the initial COG cohort, which significantly
outperformed metastatic status alone (LR p-value=0.009). A cutoff chosen using
martingale residuals derived from training cohort was used to risk-stratify patients in the
test COG cohort with respect to model output values, resulting in patients more
significantly stratified than by metastasis alone in both overall (met-dx p-value=0.02,
mod p-value=0.005) and event-free survival (met-dx p-value=0.002, mod p-value=4.4e5). The model remained significant for overall survival when applied to the independent
cohort (p=0.01).
Conclusion: In conclusion, we have shown that a panel of chemokines/cytokines
can add predictive power to the already used clinical covariate in OS prognosis and
may have the potential to more effectively stratify patients for risk-based therapeutic
options. Interestingly, all three ligands of one such therapeutic target, the CXCR3 axis,
are present in our model, indicating that this axis may play a crucial role and
intervention may be possible.
Contributors: Kelly, Aaron; Flores, Ricardo; Li, Yiting; Perlaky, Lazslo; Wang, Lisa; Lau, Ching;
Man, Tsz-Kwong
158
CIRCADIAN DYSFUNCTION INDUCES LEPTIN RESISTANCE IN MICE
Nicole Michelle Kettner
Advisor: Loning Fu, Ph.D.-Department of Pediatrics
The Leptin neuroendocrine feedback loop plays a key role in energy
homeostasis in vivo, which has been widely believed to be driven by external food cues.
Here we report that the hierarchical circadian clock, but not external food cues,
maintains Leptin endocrine function under normal physiological conditions. We have
found that the rhythmic expression of leptin controlled by the endogenous adipose clock
is sufficient to drive the circadian rhythm of plasma Leptin, and that the potentiation of
the hypothalamic energy homeostasis centers by the central clock determines the
response of the central nervous system to circulating Leptin. We demonstrated that in
adipose, the molecular clock controls leptin expression by rhythmically modulating the
activity of C/EBPα, the most potent transcription factor of leptin. Circadian misalignment
abolishes the central and peripheral coordination in wild-type mice, which not only
disrupts the adipose clock to suppress leptin expression but also desensitizes arcuate
POMC neurons to the rise in endogenous serum level of Leptin as well as to exogenous
Leptin administration, leading to the inhibition of the Leptin-mediated STAT3-POMC
activation. Together, these events suppress energy expenditure but promote body
weight and fat gain independent of all previously identified obesity risk factors, such as
diet choice, the amounts of food-intake and/or physical activity, and gene mutations.
Thus, our studies demonstrate that chronic circadian disruption is a novel and
independent risk factor for Leptin resistance, a hallmark of obesity in humans.
Contributors: Kettner, Nicole M.; Mayo, Sara A.; Hua, Jack; Lee, Choogon; Moore, David D.;
Fu, Loning
159
TRANSLATIONAL CONTROL BY p-eIF2α REGULATES CELLULAR AND
BEHAVIORAL CHANGES INDUCED BY DRUGS OF ABUSE
Sanjeev Khatiwada
Drug addiction is a major global mental health problem, costing the US over
$700 billion annually in healthcare costs, crimes, and lost productivity. However, the
biological elements that predispose individuals to drug addiction are not well
understood. The goal of this study is to understand specific molecular and cellular
mechanisms targeted by drugs of abuse to cause maladaptive learning and memory
and reinforce compulsive drug-seeking behavior, and how these changes can be
reversed.
To that end, we found that cocaine reduces phosphorylation of eukaryotic
translation initiation factor (p-eIF2() in the ventral tegmental area (VTA)—a key reward
center in the brain—in mice. In addition, a single sub-threshold dose of cocaine reduces
p-eIF2( in the VTA of adolescent mice (<5 weeks) but not in adult mice (3-5 months),
and this molecular phenotype mirrors addiction-related cellular and behavioral changes.
Interestingly, other drugs of abuse, including nicotine, ethanol, and methamphetamine
also reduce p-eIF2( in the VTA, indicating that p-eIF2( may be a common target for
multiple drugs of abuse.
To investigate whether modulating p-eIF2( level in the VTA alters susceptibility to
the effects of cocaine, we used genetic and pharmacological approaches to either
increase or decrease p-eIF2( levels and found that these manipulations render mice
more resistant and more vulnerable, respectively, to cocaine-induced cellular and
behavioral changes.
Since p-eIF2( specifically regulates the synthesis of OPHN1—a key protein
responsible in reducing synaptic strength, we investigated its role in cocaine-induced
changes in synaptic transmission and addiction-related behavior. We found that
cocaine reduces OPHN1 level in the VTA and that genetic reduction of OPHN1 in the
VTA of mice increases their susceptibility to addiction-related cellular and behavioral
effects of cocaine.
Thus, our data suggest that cocaine-induced reduction in p-eIF2(-mediated
translation of OPHN1 in the VTA could mediate a risk for drug addiction.
Contributors: Khatiwada, Sanjeev; Huang, Wei; Di Prisco, Gonzalo; Placzek, Andon; CostaMattioli, Mauro
160
LOSS OF ARGININOSUCCINATE LYASE LEADS TO NITRIC OXIDE DEFICIENCY,
ENDOTHELIAL DYSFUNCTION, IMPAIRED ANGIOGENESIS, AND HYPERTENSION
Jordan Kho
The urea cycle functions to detoxify ammonia from protein breakdown into urea.
Catalyzing the fourth reaction of urea cycle in the liver, argininosuccinate lyase (ASL) is
the only enzyme in the body able to synthesize endogenous L-arginine, a substrate for
the synthesis of urea and other biologically important metabolites including nitric oxide
(NO). Deficiency of ASL leads to argininosuccinic aciduria (ASA), the second most
common urea cycle disorder (UCD). In addition to hyperammonemia, the classic
manifestation of all UCDs, subjects with ASA can present with more complex clinical
manifestations such as hypertension that is not typically observed in other UCDs. Our
recent studies suggest that beyond ureagenesis, ASL has a cell-autonomous role in
maintaining NO homeostasis in other non-hepatic tissues. We hypothesized that loss of
ASL in endothelial cells alone is sufficient to cause vascular dysfunction, leading to
hypertension.
To test this hypothesis, we generated a mouse model with endothelial-specific
deletion of Asl (Asl cKO). We found that Asl cKO mice develop hypertension. This
hypertension was secondary to endothelial-specific NO deficiency as demonstrated by
abnormal relaxation of aortic rings and correction with treatment with an NOSindependent NO supplement. To further evaluate the consequences of ASL deficiency
on human vasculature, we utilized the cell reprogramming technology to generate
endothelial cells with patient-specific mutations. Human induced pluripotent stem cells
(hiPSCs) were derived from fibroblasts of both ASA and healthy subjects, and further
differentiated into endothelial cells. Interestingly, we discovered that ASA hiPSCs
differentiated less efficiently into endothelial cells as compared to control hiPSCs.
Furthermore, ASA hiPSCs-derived endothelial cells have impaired angiogenesis as
shown by their reduced capacity to form capillary-like structures on Matrigel in vitro and
blood capillaries in vivo upon subcutaneous injection within Matrigel into
immunodeficient mice. Our study using a novel mouse model and hiPSCs-derived
endothelial cells from patients with a rare Mendelian form of hypertension supports the
hypothesis that structural and functional abnormalities in endothelial cells contribute to
pathogenesis of hypertension. Lastly, our study provides the first proof-of-principle that
hiPSC-derived endothelial cells can be used as a model system to study genetic forms
of systemic hypertension and highlights the utility of this technology in exploring the
pathogenesis of other vascular diseases.
Contributors: Kho, Jordan; Tian, Xiao-Yu; Wong, Wing Tak; Bertin, Terry; Jiang, Ming-Ming;
Kim, Jean; Bissig, Karl-Dimiter; Nagamani, Sandesh; Lee, Brendan
161
TRANSCRIPTIONAL PROGRAMS GOVERNING SYNAPTIC PLASTICITY IN THE
ADULT BRAIN
Cynthia J Kim
The adult mammalian brain exhibits remarkable plasticity. Although basic brain
structures are established in embryogenesis, specialized areas of the adult brain
continually integrate new neurons throughout life. This astonishing reserve of new
synaptic connections is hypothesized to contribute to normal circuit function, but the
mechanisms of circuit plasticity remain largely unknown. The mouse olfactory bulb (OB)
features lifelong neuronal integration and synaptogenesis and thus serves as an ideal
model system to examine how new cells rewire the adult brain. Developing interneurons
in the adult OB undergo extraordinary genetic changes that together determine whether
they survive selection and how they will integrate into olfactory circuitry. Viral tracing
experiments in our lab uncovered a local population of interneurons in OB that secrete
the neuropeptide corticotropin releasing hormone (CRH). We discovered that
developing neurons upregulate expression of the CRH receptor (CRHR1) as they
mature and synaptically integrate. Gain and loss of function experiments further
showed that local CRH signaling is required for survival, and is both necessary and
sufficient for production of synaptic machinery. These findings suggested that CRHR1
activation initiates cell intrinsic programs of survival and synaptogenesis. To determine
the identity of these transcriptional programs, we profiled gene expression changes
associated with either low or high levels of CRHR1 activity in developing neurons, and
found that CRH signaling dynamically regulates expression of the brain-specific
Homeobox-containing transcription factor Brain-5 (BRN5). To test whether expression
of BRN5 is sufficient for survival and synaptogenesis, we targeted developing neurons
to overexpress BRN5, and found that neurons overexpressing BRN5 in early stages of
development displayed a higher propensity toward survival. Moreover, we found that
neurons that overexpressed BRN5 early in development exhibited greater success at
recruiting strong functional synaptic connections in the olfactory circuit. These findings
suggest that the transcriptional programs directed by BRN5 influence neuronal survival
and synaptic assembly. We are currently developing a model of conditional loss of
BRN5 in adult-born neurons to test whether it is required for survival and/or
synaptogenesis. Together, our studies reveal a novel pathway by which local
neuropeptidergic signaling in adult brain circuits activates specific genetic changes that
govern cell and circuit plasticity.
Contributors: Kim, Cynthia J; Garcia, Isabella; Arenkiel, Benjamin R
162
TUMOR-INDUCED HETEROGENEITY OF MYELOID CELLS AND THEIR ROLE IN
BREAST CANCER PROGRESSION
Ik Sun Kim
Advisor: Xiang Zhang, Ph.D.-Department of Molecular & Cellular Biology
Background: Tumors evolve immunosuppressive microenvironment to subvert
anti-tumor immunity. Among cancer promoting inflammatory effectors, tumorassociated myeloid cells (TAMCs) represent a heterogeneous group that predominantly
orchestrates tumor-induced immunosuppression as well as many other hallmarks of
cancer. It has become increasingly clear that these immunosuppressive mechanisms
elicited by TAMCs are a major barrier to effective anti-tumor therapies. What remains
poorly understood, however, is why and how individual tumors evolve to employ
different mechanisms to suppress anti-tumor immunological response.
Experimental Design and Methods: To characterize the immune system in the
context of heterogeneous breast cancer, we utilized p53-null syngeneic mammary
tumor models that highly resemble different human breast cancer subtypes. Various
lymphoid and myeloid cells have been profiled by multi-color flow cytometry in blood,
bone marrow, and primary tumor at different stages of tumor growth.
Results: We have demonstrated heterogeneous immunosuppression phenotype
across p53-null tumor models conferred by various expansion and differentiation of
CD11b+ Gr1+ myeloid cells. Interestingly, these myeloid cells exhibited inter- and intratumoral heterogeneity in terms of the initial expression of surface antigens, cellular
plasticity upon infiltrating tumors, and functional roles in tumor progression.
Conclusions: This work highlights the dramatic phenotypical and functional
heterogeneity of TAMCs among different subtypes of breast cancer. Identification and
intervention of such tumor-specific immune aberrations will likely provide a rationale to
classify patients for immunotherapies.
Contributors: Kim, Ik Sun; Welte, Thomas; Liu, Jun; Yu, Cuijuan; Wang, Hai; Gao, Xia; Zheng,
Duan; Tian, Lin; Muscarella, Aaron; Lo, Hin Ching; Kurley, Sarah ;Kim, Sejin; Bu, Wen;
Nguyen, Tuan; Liao, Lan; Xu, Jianming; Li, Yi; Lewis, Michael; Rosen, Jeffrey; Zhang, Xiang
163
MITOCHONDRIAL AUTOPHAGY PROTEINS BNIP3 AND NIX ARE REQUIRED FOR
MEMORY B CELL SURVIVAL
Srikanth Kodali
Advisor: Jin Wang, Ph.D./D.V.M.-Department of Pathology & Immunology
Infectious diseases, ranging from smallpox to polio to emerging strains of
influenza, have posed a threat to public health throughout history. Vaccines protect
against pathogenic infections by generating long-lived immune memory cells specific for
the immunizing pathogen. Memory B cells are immune memory cells that respond to
pathogen re-exposure by rapidly generating antibody-secreting cells against pathogens.
Memory B cells have very long lifespans, persisting for decades in humans, allowing
them to mediate pathogen-specific antibody responses years after vaccination.
Recently, it has been shown that autophagy, a process in which a portion of cytoplasm
is sequestered by autophagosomes for degradation, is required for the long-term
survival of memory B cells. Moreover, promoting autophagy has been shown to improve
memory B cell responses against viral infections. However, the molecular mechanisms
for the protection of memory B cells by autophagy have not been completely resolved.
Our objective is to elucidate the molecular mechanisms by which autophagy protects
memory B cells against cell death. In long-lived, quiescent cell types such as
cardiomyocytes and neurons, dysfunctional mitochondria may accumulate over time,
resulting in increased ROS production, decreased ATP production, and release of proinflammatory mitochondrial DNA and pro-apoptotic proteins. Mitochondrial autophagy,
the selective autophagy of mitochondria, can serve as an essential quality control
mechanism for clearance of defective mitochondria in long-lived cells. We find that two
BH3-only Bcl-2 family proteins that participate in mitochondrial autophagy, Bnip3 and
Nix, are critical for memory B cell survival. We also find that memory B cells have
increased ATP levels compared to their naïve counterparts, suggesting an increased
metabolic demand that memory B cells with dysfunctional mitochondria may have
trouble meeting. We will test the hypothesis that autophagy is required for quality
control of mitochondria to protect the survival and function of memory B cells.
Contributors: Kodali, Srikanth; Jang, Albert; Chen, Min; Wang, Jin
164
REPURPOSING GERMLINE EXOMES OF THE CANCER GENOME ATLAS
DEMANDS A CAUTIOUS APPROACH AND SAMPLE-SPECIFIC VARIANT
FILTERING
Amanda Michele Koire
Program in Structural and Computational Biology and Molecular Biophysics/M.D.-Ph.D.
Program
When seeking to obtain whole-exome or genome sequencing data, the time and
expense required to produce a patient cohort make data repurposing an attractive
option. The first step in repurposing is setting some quality baseline for the data so that
conclusions are not spurious. This is difficult because there can be variations in quality
from center to center, clinic to clinic and even patient to patient. Here, we assessed the
quality of the whole-exome germline mutations of TCGA cancer patients using patterns
of nucleotide substitution and negative selection against impactful mutations. We
estimated the fraction of false positive variant calls for each exome with respect to two
gold standard germline exomes, and found large variability in the quality of SNV calls
between samples, cancer subtypes, and institutions. We then demonstrated how
variant features, such as the average base quality for reads supporting an allele, can be
used to identify sample-specific filtering parameters to optimize the removal of false
positive calls. We concluded that while these germlines have many potential
applications, users should assess the quality of the available exome data prior to use
and perform additional filtering steps.
Contributors: Koire, Amanda; Katsonis, Panagiotis; Lichtarge, Olivier
165
IN VIVO REDUCTION OF HEPATITIS B VIRUS ANTIGENEMIA AND VIREMIA BY
ANTISENSE OLIGONUCLEOTIDES
Robert Layne Kruse
Advisor: Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
Fasiha Kanwal, M.D.-Department of Medicine
Current treatments for chronic hepatitis B virus (HBV) infection include interferon
and a small number of nucleos(t)ide analogues acting as reverse transcriptase
inhibitors. These therapies often do not reduce HBV surface antigen (HBsAg)
production, however, allowing HBV infection to persist chronically. In this study, we
evaluated antisense oligonucleotide (ASO) therapy against HBV, which inhibits viral
replication and viral antigen production by binding to viral RNAs. We identified and
characterized a lead candidate ASO in vitro and subsequently tested the drug in vivo
using HBV mouse models.
ASO administered weekly into HBV transgenic mice reduced all serum and liver
viral markers in a dose and time-dependent manner. In dose escalation studies, HBsAg
decreased ≥ 2 logs without apparent liver toxicity. After a single dose of ASO, serum
HBsAg decreased by 2 logs in a week and returned to baseline 4 weeks after ASO
injection. We also tested ASO treatment in a hydrodynamic model of HBV infection,
where HBV is replicated from episomal plasmids, analogous to natural HBV replication
from covalently closed circular DNA. ASO treatment reduced serum HBsAg and viremia
up to ≥ 3.5 logs in mice injected with plasmids encoding different HBV genotypes,
demonstrating ASO effectiveness against episomal DNA-mediated HBV gene
expression and the major HBV genotypes A-D.
Conclusion:
These
preclinical results
demonstrate
that antisense
oligonucleotides are very effective in reducing viral antigenemia as well as DNA viremia
and thus, may constitute a novel therapeutic approach for chronic HBV infection.
Contributors: Kruse, R; Billoud, G; Chen, L; Carrillo, M; Wieland, S; Bissig, KD
166
TO WNT OR NOT TO WNT: TCF3 IN SKIN CARCINOGENESIS
Amy Tsu Ku
Advisor: Hoang Nguyen, Ph.D.-Department of Molecular & Cellular Biology
Abdul Diwan, Ph.D.-Department of Pathology & Immunology
The transcription factor Tcf3 is one of the embryonic stem cell signature genes
that are upregulated in multiple aggressive cancer types, including non-melanoma skin
cancer.
In this work, we show that Tcf3 acts as a tumor promoter in the chemical-induced
model of mouse skin squamous cell carcinoma (SCC). Furthermore, we found that
TCF3 overexpression increases tumor growth whereas loss of function of TCF3 and
TCF4 reduces tumor growth in a xenograft model of human skin SCC. TCF3 can bind
to the transactivator β-catenin, a mediator of canonical Wnt signaling, or to the
transcriptional repressor Groucho/TLE. Using different deletion mutants of Tcf3, we
further demonstrate that Tcf3’s tumorigenic role is independent of its binding to βcatenin, suggesting that Tcf3 functions primarily as a co-repressor to promote tumor
growth. We also found that Tcf3 enhances cell migration and overrides RAS-induced
senescence independently of its role as a 
-catenin cofactor. Our findings establish a
causal role for Tcf3 as a transcriptional repressor in skin tumorigenesis and elucidate
the mechanisms underlying its tumorigenic capacity.
Contributors: Ku, Amy T; Shaver, Timothy M; Rao, Ajay S; Nguyen, Hoang
167
THE REGULATORY ROLE OF NKT CELLS IN IMMUNE RESPONSE TO VACCINES
BASED ON LIVE ATTENUATED BACTERIAL VECTORS
Suhrab Kurbanov
Advisor: Leonid Metelitsa, M.D./Ph.D.-Department of Pediatrics
Recently we reported a novel cancer vaccine, which exploits live attenuated
Salmonella strain (MVP728) to express and deliver the tumor-associated antigens
(TAA) into the cytosol of antigen presenting cells (APCs), resulting in a strong antigenspecific immune response generation. Conjugating synthetic NKT ligands with the
Salmonella-based cancer vaccine, MVP728-Survivin, further augmented the therapeutic
efficacy of this vaccine in mice with A20 murine lymphoma models. While this shows
that NKT cell activation with synthetic ligands augment Salmonella-based cancer
vaccine-efficacy, the role of the NKT cells in the initiation of the immune response to
Salmonella-based cancer vaccines remains unclear. In this study to examine the
natural role of the NKT cells in the vaccine-mediated immune response we engineered
Salmonella to express and translocate a model antigen SIINFEKL, MVP728-SIINFEKL,
and compared the immunogenicity of this vaccine in wild type, Jα18-/- (lack only type-I
NKT cells) and CD1d-/- (lack all NKT cells) mice. After 3 rounds of oral administration of
the MVP728-SIINFEKL vaccine NKT deficient mice showed 4-fold higher SIINFEKLspecific CD8+ T cell response than wild type mice (p<0.005). Moreover NKT deficient
mice showed better and longer vaccine persistance in the spleen and mesenteric lymph
nodes compared to WT mice (p<0.01). In addition comparison of the SIINFEKL-specific
CD8+ T cell generation in response to attenuated Listeria-Ovalbumin strains showed
similar results. While these findings demonstrate that in the absence of exogenous
stimulation with synthetic ligands NKT cells decrease Salmonella-based cancer
vaccine-mediated response, how previously reported NKT cell activation with synthetic
ligands augment the efficacy of the Salmonella-based cancer vaccines is unknown. To
better understand the vaccine-adjuvant role of the NKT cells in the presence of
synthetic ligands, we assessed the MVP728-SIINFEKL vaccine persistence and
immunogenicity by providing mice with synthetic NKT cell agonist, α-galactosylceramide
(αGC), at different time points in regards to vaccine challenge. We found that αGC
mediates its vaccine-augmenting role mainly through leading NKT cells into
nonresponsive anergy, thus preventing them from controlling vaccine persistence. Our
data shows that αGC administration as early as 5 days before vaccine challenge
rescues the vaccine persistence and leads to comparable SIINFEKL-specific CD8+ T
cell response to NKT deficient mice. Overall in contrast to established vaccine-adjuvant
role of NKT cells here we report that NKT cells decrease the efficacy of the cancer
vaccines based on bacterial vectors.
Contributors: Kurbanov, Suhrab; Xu, Xin; Guo, Linjie; Gao, Xiuhua; Tian, Gengwen; Metelitsa S
Leonid
168
METABOLIC REGULATION BY THE MECP2 TRANSCRIPTIONAL REPRESSOR
COMPLEX POINTS TO NEW THERAPEUTIC TARGETS IN RETT SYNDROME
Stephanie Marie Kyle
Advisor: Monica Justice, Ph.D.-Department of Molecular & Human Genetics
David Nelson, Ph.D.-Department of Molecular & Human Genetics
Rett syndrome (RTT) is a progressive neurodevelopmental disorder of females
primarily caused by mutations in the X-linked gene encoding methyl-CpG-binding
protein 2 (MECP2). To identify pathways in disease pathology for therapeutic
intervention, we carried out a dominant random mutagenesis suppressor screen in
Mecp2 null mice. One suppressor identifies a stop codon mutation in a rate-limiting
enzyme in cholesterol biosynthesis, which ameliorates RTT-like symptoms and
increases longevity in Mecp2 null mice by altering cholesterol homeostasis. Although
RTT has been classically labeled a neurological disorder, these studies suggest that a
metabolic component contributes to pathology. Here we show that Mecp2 deletion
induces hyperlipidemia, fatty liver, and metabolic syndrome in mice. These metabolic
phenotypes are strikingly similar to that in mice with a liver-specific knockout of histone
deacetylase 3 (Hdac3), a potent regulator of lipogenesis and cholesterol biosynthesis.
Consistently, we show that MeCP2 and HDAC3 work in complex to suppress
expression of the cholesterol enzyme identified in our screen, as well as other genes of
the cholesterol and de novo lipogenesis pathways. Our data suggest a novel metabolic
component in RTT, arising from loss of interaction between MeCP2 and HDAC3.
Concurrently, liver-specific deletion of Mecp2 is deleterious enough to cause fatty liver
through aberrant lipogenic gene transcription. Our ongoing studies point to additional
metabolic pathways that are prime targets in the pursuit of preventing morbidities
associated with Rett syndrome.
Contributors: Kyle, Stephanie; Justice, Monica
169
INTEGRATIVE ANALAYSIS OF GENETIC MODIFIERS OF HUNTINGTON’S
DISEASE
Andrew Marc Laitman
Program
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D.-Department of Pediatrics
Huntington’s disease (HD) is a progressive neurodegenerative disorder caused
by CAG trinucleotide repeat expansion within the Huntingtin gene. Despite the known
genetic etiology of HD, no disease modifying treatments exist. Longer repeat
expansion is inversely correlated with age of onset of neurological symptoms.
However, repeat expansion can only explain 67% of the variance of neurological onset,
which suggests that disease onset can be modified by other factors, including
environmental and genetic. Thus, the discovery of genetic modifiers could lead us to
pathways that can be targeted for drug treatment. Initial unbiased RNAi screens in a
Drosophila model of HD have identified 138 putative modifiers of HD. However, these
unbiased screens have only sampled a small percentage of human homologs in
Drosophila. We hypothesize that candidate modifier genes are more likely to be
connected in Protein-Protein interaction networks because they participate in the same
biological pathway.
To prioritize such modifiers, we used Network-Based Smoothing (NBS) on
human gene interaction networks such as STRING and HumanNet to identify new
genes that are highly connected to known modifiers. We assessed the predictive
power of this approach with leave-one-out cross-validation which tested these networks
ability to recover known modifiers. Based on receiver operating characteristic (ROC)
curves, this approach can identify modifiers at a much higher rate than random, purely
unbiased screens. However, HumanNet and STRING networks are not disease or
tissue-specific.
We then constructed a HD-specific network from RNA-seq data obtained from
the striatum of transgenic mice with progressively increasing CAG repeats. Edges
between genes were constructed using a Gaussian Markov Random Field (GMRF).
NBS on these networks were also able to recover modifier labels with ROC scores
comparable to those of HumanNet and STRING.
Further work will seek to combine HumanNet and STRING with the HDspecific GMRF to identify modules enriched for modifier genes, which could lead to
novel pathways involved in the pathogenesis of HD.
This research was funded by a training fellowship from the Keck Center of the
Gulf Coast Consortia, on the Training Program in Biomedical Informatics, National
Library of Medicine (NLM) T15LM007093.
Contributors: Laitman, Andrew; Al-Ramahi, Ismael; Botas, Juan; Liu, Zhandong
170
INFERRING READOUT OF DISTRIBUTED POPULATION CODES WITHOUT
MASSIVELY PARALLEL RECORDINGS
Janakiraman Kaushik Lakshminarasimhan
Information about task-relevant variables is often distributed among neurons
across multiple cortical areas. Neuronal responses are rarely independent of each
other, but are correlated to some degree due to common input as well as recurrent
message-passing. Consequently, determining how these neurons collectively drive
behavioural changes requires not only examining how individual neurons are correlated
with behaviour, but also estimating the correlated variability among neurons. Precisely
estimating the structure of correlated variability requires massively parallel recordings,
which remains very difficult with current technology. Fortunately, it has recently been
shown that the expansion in neural representation from sensory periphery will lead to a
predictable pattern of correlations that ultimately limits the information content in brain
areas downstream. We examined the implications of these so-called informationlimiting correlations for the readout of distributed population codes in a simple
discrimination task. Surprisingly we found that both the behavioural precision, as well as
the correlation of individual neurons with behavioural choice (choice correlation) were
determined largely by the relative magnitudes of neuronal weights in the different brain
areas and not on their specific pattern. We also found that, in the presence of
information-limiting correlations, the choice correlations of neurons within an area
should all scale by the same factor following inactivation of other potentially taskrelevant brain areas. Together, our results lead to a novel framework for inferring how
different brain areas contribute to behavioural response. Specifically, we show that the
contribution of a brain area can be inferred simply by observing how the magnitude of
choice correlations of individual neurons within the area and the behavioural precision
are affected by inactivating other areas, thus obviating the need for large-scale
recordings.
Contributors: Lakshminarasimhan, Janakiraman Kaushik; Angelaki, Dora; Pitkow, Xaq
171
A FIBRINOGEN-DERIVED MOLECULE GENERATED BY DIVERSE PROTEINASES
THAT PRIMES FUNGISTATIC IMMUNITY
Cameron Taylor Landers
Advisor: David Corry, M.D.-Department of Medicine
Adrian Casillas, M.D.-Department of Medicine
Background: Fibrinogen proteolysis within the airway lumen yields a novel ligand
that drives the expression of allergic airway disease and antifungal immunity by acting
on Toll-like Receptor 4 (TLR4). Inhibition of airway proteinases or the absence of TLR4
attenuates these processes both in vitro and in vivo. However, it remains unclear which
proteolytic product of fibrinogen acts on TLR4 and what types of proteinases are able to
induce this antifungal effect.
Methods: Fibrinogen was incubated with thrombin, the proteinase from
Aspergillus melleus (PAM), or a proteolytically active extract derived from the dust mite
Dermatophagoides farinae (DF) to generate three types of fibrinogen cleavage products
(FCPs). FCPs were subsequently analyzed by SDS PAGE to determine the size of the
fibrinogen fragments produced. Fungistasis was assayed in vitro by incubating FCPs
with murine splenocytes or bone marrow-derived macrophages (BMDMs) for 24 hours,
followed by the addition of fungal spores from Aspergillus niger. After 18 hours the
number of fungal mycelia were quantified and compared to controls.
Results: Thrombin, PAM, and DF FCP preparations all contained fibrinogen
fragments in the range of 50-250 kDa. Known fibrinogen degradation products
(domains D and E (<100 kDa)) showed no fungistatic activity as defined by a significant
reduction in fungal growth in treated as compared to sham controls. In contrast, all FCP
preparations primed murine splenocytes to significantly inhibit fungal growth in vitro
(p<0.05). Studies were also performed using murine bone marrow-derived
macrophages that yielded similar results.
Conclusions: In the presence of diverse proteinases, human fibrinogen degrades
to produce similar cytokine-like molecules that potently induce TLR4-dependent antifungal immunity from macrophages and splenocytes. Given the unique molecular size
of these fragments, we propose the existence of a novel fibrinogen-derived TLR4 ligand
with potent immunostimulatory activity.
Contributors: Landers, Cameron; Tung, Hui-Ying; Ottoboni, Fabrizio; Corry, David
172
APCDD1 STIMULATES OLIGODENDROCYTE DIFFERENTIATION AFTER WHITE
MATTER INJURY
Dylan James Laug
Wnt signaling plays an essential role in developmental and regenerative
myelination of the CNS, therefore it is critical to understand how the factors associated
with the various regulatory layers of this complex pathway contribute to these
processes. Recently, Apcdd1 was identified as a negative regulator of proximal Wnt
signaling, however its role in oligodendrocyte (OL) differentiation and reymelination in
the CNS remain undefined. Analysis of Apcdd1 expression revealed dynamic
expression during OL development, where its expression is upregulated during
differentiation. Functional studies using ex vivo and in vitro OL systems revealed that
Apcdd1 promotes OL differentiation, suppresses Wnt signaling, and associates with βcatenin. Application of these findings to white matter injury (WMI) models revealed that
Apcdd1 similarly promotes OL differentiation after gliotoxic injury in vivo and acute
hypoxia ex vivo. Examination of Apcdd1 expression in white matter lesions from
neonatal WMI and adult multiple sclerosis revealed its expression in subsets of
oligodendrocyte (OL) precursors. These studies describe, for the first time, the role of
Apcdd1 in OLs after WMI and reveal that negative regulators of the proximal Wnt
pathway can influence regenerative myelination, suggesting a new therapeutic strategy
for modulating Wnt signaling and stimulating repair after WMI.
Contributors: Laug, Dylan; Lee, Hyun Kyoung; Zhu, Wenyi; Patel, Jay; Ung, Kevin; Arenkiel,
Benjamin; Fancy, Stephen; Mohila, Carrie; Deneen, Benjamin
173
UMBILICAL VESSEL FORMATION AND REMODELING IN THE MOUSE EMBRYO
Henry H Le
Advisor: Mary Dickinson, Ph.D.-Department of Molecular Physiology & Biophysics
The allantois is the precursor of the umbilical vein (UV) and umbilical artery (UA),
which connects the embryo to the placenta. Circulation to the placenta is critical for
proper nutrition and waste exchange, as well as embryo survival and normal
development. The allantois, along with the yolk sac and dorsal aorta, undergo
angiogenesis and vascular remodeling that can be used to study vasculature
development and vascular defects. Despite the emphasis on understanding diseases
that arise from abnormal placenta function such as frequent infertility, preeclampsia,
intrauterine growth restriction (IUGR), and placental abruption, the initial placental
formation and embryo-placenta circulation is under investigated. We want to answer the
question of how the allantois remodels from a primitive plexus to a defined UV and UA
that connects to the chorion. At present, it is not known when blood flows through the
allantois and the venous return forms. Our previous works showed that hemodynamic
force is required for vascular remodeling in the embryonic yolk sac. We suspect that
hemodynamic force also induces vessel remodeling in the allantois via shear stress
and/or molecular signaling. We will use advance imaging techniques, such as,
lightsheet microscopy, confocal imaging and optical coherence tomography (OCT) to
quantify blood flow and examine the morphology of the remodeling allantois. Our
preliminary data utilized confocal and lightsheet microscopy to exam vascular
morphology of Pecam1 immunostained Tg(ϵ-globin-GFP) allantois, from 8.0 dpc (0somite) stage to 9.5 dpc (28-somites) stage. The results reveal a stepwise process of
UA/UV formation and remodeling. First, the UA forms as early as circulation begins, but
the UV forms at later stage. Interestingly, the UA diameter is initially ~40um when it first
forms, but shortly thereafter, the UA is only ~15um by 15-somites stage, but then later
expands to ~110um by the 28-somite stage. Finally, the distribution of erythroblasts
within the allantois suggests that the plexus become perfused until the UV forms and
becomes connected with the sinus venosus. These data suggest that the formation of
the UV is a key event to triggering outward remodeling because of the need for
continuous flow. In the future, we use live embryo culture and lightsheet microscopy to
obtain high spatial resolution time-lapse images from different transgenic reporter mice
to provide insight into how blood flow affects allantois-chorion development. Data from
these studies will provide significant insights into how the initial circulation between the
embryo and the placenta is established.
Contributors: Le, Henry; Hsu, Chih-Wei, PhD; Piazza, Victor; and Dickinson, Mary E., PhD
174
HIPPO SIGNALING DELETION REVERSES SYSTOLIC HEART FAILURE
John Preston Leach
Mammalian organs vary widely in regenerative capacity. All organs are prone to
failure, but poorly regenerative organs like the heart are particularly vulnerable to tissue
loss and organ failure. However, newborn mice possess an astonishing ability to
regenerate heart tissue. This ability is limited and is completely lost by seven days after
birth, thus, developmental pathways could be the key to unlocking regenerative
potential.
The organ-intrinsic, cell-cycle regulating Hippo-pathway activates during this
seven day window after birth. Therefore we postulate that Hippo signaling inhibits
regeneration in the adult heart. Indeed, we have previously shown that the Hippo
pathway controls cardiomyocyte proliferation during development to restrain heart size,
and knockout of Hippo pathway proteins Salvador and Lats1/2 leads to an increase in
cardiomyocyte renewal and is cardioprotective against ischemic damage in the adult
heart.
Furthermore, heart failure is the result of a vicious cycle deriving from the
physiologic response to low oxygen perfusion; this places stress on residual
cardiomyocytes, leading to the further loss of cardiomyocytes and decreased cardiac
function. Therefore, we investigated whether Hippo pathway deletion in failing hearts,
provided therapeutic benefits.
Hippo pathway inactivation resulted in functional recovery with extensive
repopulation of the left ventricle with contractile muscle. Sequential labeling with
nucleoside analogs revealed rare stem cell–like growth in Hippo-deficient
cardiomyocytes. An increase in capillary density provides evidence for improved
vascularization at the interface between muscle and scar tissue. Finally, RNA-seq
reveals involvement of circadian rhythm in improved cardiac function. Thus far we have
determined Hippo pathway deletion in the failing heart results in functional recovery and
has the potential for therapeutic approaches to treat systolic heart failure.
Contributors: Leach, John P.; Heallen, Todd; Zhang, Min; Hill, Matthew C.; Rahmani, Mahdis;
Willerson, James T.; Martin, James F.
175
THYMIC DEVELOPMENT OF INSULIN-REACTIVE T CELLS
Thomas Lee
Advisor: Matthew Bettini, Ph.D.-Department of Pediatrics
Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease that involves
the destruction of insulin (Ins) producing β cells found within the pancreatic islets of
Langerhans. While multiple studies have characterized the pathogenicity and
diabetogenic potential of β-cell specific T cells, we have limited mechanistic insight into
autoimmune T cell development and their escape from thymic selection. Utilizing the
retrogenic mouse technology, we show that ectopic expression of InsB9-23 or insulin
agonist mimetope (R22E) in the thymus influences the onset of diabetes. Our results
demonstrate that increasing InsB9-23 expression in the thymus is insufficient to induce
insulin reactive CD4+ T cell deletion, and upon escape from thymic selection, these
insulin reactive T cells infiltrate the pancreas of NOD mice. Interestingly, these
pancreatic-residing autoreactive CD4+ T cells are no longer able to induce
spontaneous diabetes development. In contrast, when R22E mimetope is expressed,
thymocyte development is halted at the double positive stage and INS reactive T cells
undergo negative selection. As a result, thymic expression of R22E completely prevents
autoreactive thymocytes selection and diabetes development. Collectively, these results
suggest that antigen dose or peptide:MHC complex stability of self-antigen presentation
in the thymus can lead to multiple fates of insulin reactive CD4+T cell development and
autoimmune outcome.
Contributors: Thomas Lee, Natalie Tully, Pinaki Banerjee, Maria Bettini, and Matthew L. Bettini
176
SCLEROSTIN ANTIBODY (SCL-AB) INCREASED BONE MASS AND STRENGTH IN
A MOUSE MODEL OF OSTEOGENESIS IMPERFECTA CASUSED BY WNT1
MUTATION
Yi-Chien Lee
Background: Osteogenesis Imperfecta (OI) is a brittle bone disease
characterized by low bone mass and multiple bone fractures. Recently, our laboratory
and others have identified WNT1 mutations in patients with OI. We have also
established and described a mouse model of OI caused by WNT1 mutations (swaying
mouse model). Current treatment options for OI mostly focus on bisphosphonate
therapy; however, because of its questionable efficacy in some OI patients and
concerns about long-term administration, it is necessary to explore new treatment
strategy. Sclerostin is a potent inhibitor of WNT signaling, and several studies have
shown that the inhibition of sclerostin greatly enhances bone formation. Therefore, we
hypothesized that Scl-Ab treatment could be beneficial for treating WNT1 related OI
and tested this hypothesis using our swaying mouse model.
Methods: In this study, we followed two treatment regimens: from week 3 to
week 8 (the early treatment group) and from week 9 to week 14 (the late treatment
group). Swaying mice and wild-type littermates were subcutaneously administered
vehicle or 25 mg/kg Scl-Ab twice a week for 6 weeks (n=4-10 per group). Bone mass
was assessed by
CT, bending,
-point
bone s treand
ngthmatrix
by threcomposition
e
by Raman spectroscopy.
Results: First, the Scl-Ab treated swaying mice in the early treatment group
exhibited reduced fracture rate from 90% (vehicle treated swaying mice) to 12.5%
fracture rate (Scl-Ab treated swaying mice).
CT
-Ab treated
a na lys is s howe d th
swaying mice exhibited significantly increased bone volume per tissue volume in both
the femur and lumbar spine (p-value < 0.01) in both early and late treatment groups.
Three point bending analysis at the femur confirmed that the Scl-Ab treated swaying
mice show increased bone strength parameters including maximum load, stiffness, and
post-yield energy. Finally, Raman analysis demonstrated that the Scl-Ab treated
swaying mice have increased bone collagen content in bone matrix.
Conclusions: These results suggest that Scl-Ab treatment significantly rescued
the low bone mass phenotype and increased bone strength in swaying mice. These
results support future investigations of a potential clinical benefit of Scl-Ab in the
management of OI patients with WNT1 mutations.
Contributors: Joeng, Kyu Sang; Bi, Xiaohong; Hao Ding; Ambrose, Catherine; Lee, Brendan H
177
THE ROLE OF RNF197 IN THE GROWTH OF BREAST CANCER
Yu-Ju Lee
Advisor: Weei-Chin Lin, M.D./Ph.D.-Department of Medicine
RNF197 is a cell growth regulator with a C3HC4 RING-finger domain: a binding
motif of ubiquitin-conjugating enzymes. Since the activity of most E3 ubiquitin ligases is
specified by the RING-finger domain, we hypothesize that RNF197 can function as an
E3 ubiquitin ligase.
Previous study has shown that RNF197 over-expression inhibits colony
formation of colon carcinoma, ovarian carcinoma, and glioblastoma cell lines. Another
study also found that over-expression of RNF197 inhibits cell proliferation and
enhances metformin-induced G1 arrest in endometrial cancer cell lines. However, the
molecular mechanism underlying how RNF197 inhibits cell growth remains unclear. To
understand the relationship between RNF197 and cancer, we compared the mRNA
level of RNF197 between normal and matched tumor tissues. Our results showed that
in some cancers, including breast carcinoma, the level of RNF197 is lower in tumor
tissues. We then performed survival analysis and found that lower RNF197 expression
level is strongly correlated with shorter survival of breast cancer patients.
Since the growth-inhibiting effect of RNF197 in breast cancer is still
uncharacterized, I hypothesize that RNF197 inhibits the growth of breast cancer
through its E3 ligase activity. To perform cell proliferation assay, we generated stable
RNF197-knockdown and RNF197-overexpressing breast cancer cell lines. Our results
show that knock-down of RNF197 enhances cell growth; on the other hand, overexpression of RNF197 decreases cell proliferation rate. To investigate how RNF197
inhibits cell proliferation, we performed cell cycle analysis and Western blot using these
stable cell lines to examine several growth-related signaling pathways. To study the E3
ligase activity of RNF197, we will identify its substrate and perform in vitro and in vivo
ubiquitination assays. The overarching goal of this study is to identify potential novel
therapeutic targets or pathways that shed light on breast cancer treatment.
Contributors: Lee, Yu-Ju; Lin, Weei-Chin
178
THE ROLE OF KU IN TELOMERE LENGTH MAINTENANCE IN SACCHAROMYCES
CEREVISIAE
Laramie Denise Lemon
In Saccharomyces cerevisiae, telomerase is comprised of the RNA component,
TLC1, the reverse transcriptase component, Est2, and Est1 and Est3, which are
required for telomerase activity in vivo, but not in vitro. Est1 recruits telomerase to the
telomere in late S phase by interacting with Cdc13, a single-stranded telomeric DNA
binding protein. The Ku heterodimer also contributes to telomere length maintenance
by associating with TLC1. In contrast to strains lacking telomerase, telomeres are short
yet stable and cells do not senesce in the absence of Ku or Ku:TLC1 interaction. Cells
lacking Ku or Ku:TLC1 binding, such as the yku80-135i mutant, fail to localize TLC1 to
the nucleus and have reduced levels of Est2 and Est1 at the telomere. We recently
showed that the requirement for Ku in telomere length maintenance could be rescued
by tethering Est1 to the telomere via Cdc13 (Williams et al, 2014). In addition, we
showed that Ku promotes the association of Est1 to the telomere even when Est2 is
telomere-associated. We also found Ku in a complex with Est1. Finally, we showed that
Ku’s DNA end binding (DEB) activity is required for Ku’s influence on telomere length
even if TLC1 is localized to the nucleus. Together, these results led us to propose that
Ku’s primary role in telomere elongation lies in its influence on Est1, and not on Est2
recruitment or TLC1 nuclear localization. Currently, we are exploring multiple
hypotheses involving Ku’s influence on Est1 recruitment to the telomere. One
hypothesis is that Ku binding to TLC1 and/or Ku’s DEB activity modulates the
interaction between Est1 and Cdc13. Using co-immunoprecipitation assays in
asynchronous cells, we unexpectedly found an increase in Est1:Cdc13 interaction in
yku80∆ or yku80-135i strains compared to wild type. This interaction is furthered
increased in G2/M arrested cells. We plan to further investigate this role of Ku on
Est1:Cdc13 interaction in yku80∆exo1Δ mutants, which lack a 3’ overhang, yku70R456E mutants, which cannot bind DNA ends, and in other strains with short telomeres.
We are also testing whether Ku, when bound to telomeres, modulates Cdc13
phosphorylation. Li et. al previously showed that Est1:Cdc13 interaction at telomeres is
promoted by Cdc13 T308 phosphorylation (2009). Additionally, telomeres exhibit the
same degree of shortness in yku70∆ cdc13-T308A and yku70∆ mutants suggesting that
Ku and Cdc13 T308 phosphorylation function in the same pathway (Tseng et. al, 2009).
Thus, Ku might influence Est1:Cdc13 interaction by promoting Cdc13 T308
phosphorylation.
Contributors: Lemon, Laramie; Bertuch, Alison
179
NEWLY SYNTHESIZED PERFORIN TRAFFICS TO THE IMMUNOLOGICAL
SYNAPSE VIA RECYCLING ENDOSOMES AND INDEPENDENTLY OF SECRETORY
LYSOSOMES
Kelsey Elizabeth Lesteberg
Advisor: George Makedonas, Ph.D.-Department of Pediatrics
Background: One mechanism by which cytotoxic T lymphocytes (CTL) kill their
targets is the exocytosis of specialized secretory lysosomes, lytic granules, which
contain the pore-forming protein perforin. In contrast to traditional dogma, which states
that a CTL may only replenish its perforin content upon proliferation, an antigen-specific
CTL rapidly upregulates perforin within 2 hours of stimulation. This new perforin may
replenish lytic granules, but it is also targeted to the immunological synapse for
immediate use independently of lytic granules. Therefore, this alternative trafficking of
perforin provides a potential mechanism to sustain CTL cytotoxicity. In this study, we
aimed to define the means by which this newly synthesized perforin traffics to the
immunological synapse, if not via secretory lysosomes.
Methodology: We utilized imaging flow cytometry technology to determine the
intracellular components involved in the transport of newly synthesized perforin in
human antigen-specific CTL. We stimulated human peripheral blood mononuclear cells
(PBMC) with synthetic peptides representing immunogenic epitopes from CMV, EBV, or
influenza viruses, and allowed them to expand for 10-14 days. The cells were restimulated with their cognate antigen for 2-6 hours and then stained with antibodies
against CD8 and CD56, a panel of rab and SNARE proteins, and anti-perforin clones
D48 and δG9; D48 recognizes both new perforin and lytic granule-associated perforin,
whereas δG9 recognizes only granule-associated perforin. The localization of perforin
with rab and SNARE proteins was assessed through the use of bright detail similarity
(BDS) analysis, which assigns a score to each cell based on the overlap of the probes
of interest.
Results: Newly synthesized perforin (D48+ δG9-) localized to recycling
endosome compartments, as identified by rab8, vti1b, and the transferrin receptor
(CD71). In addition, new perforin also colocalized with rab37 and VAMP4, which are
involved in transport to the synapse and to endosomes, respectively.
Conclusions: Our data suggest that newly synthesized perforin is incorporated
into recycling endosomes, where it may be sorted to subsequent destinations: the
immunological synapse for immediate use, or to lysosomes to refill the cells’ store of
lytic granules. These results define an alternative intracellular trafficking pathway for
perforin that functions to sustain CTL cytotoxicity.
Contributors: Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George
180
CENTRALLY DRIVEN ALTERATIONS IN ANGIOTENSIN II SIGNALING LEADS TO
AN INCREASE IN SYMPATHETIC TONE FOLLOWING STATUS EPILEPTICUS
Amber T Levine
50,000 to 60,000 Americans are newly diagnosed with SE (prolonged or
repeated seizures) each year. SE is associated with many comorbidities including the
development of chronic, drug resistant epilepsy. During and in the aftermath of a
seizure, some patients exhibit potentially fatal tachycardia, bradycardia, arrhythmias,
and alterations in heart rate variability. Our lab and others have shown detrimental
cardiac effects following SE that persist during the subsequent development of
epilepsy. Additionally, our preliminary data suggest heart rate variability, resting
tachycardia, and an increased propensity for lethal arrhythmia following SE. These
changes are corrected by systemic administration of atenolol, a β1-adrenergic receptor
(β1AR) blocker, suggesting that there is increased sympathetic nervous system (SNS)
drive following SE. However, the underlying mechanisms that contribute to alterations in
SNS tone following SE are unclear. SNS activity is regulated at many levels within the
central nervous system with the rostral medulla as the final converging pathway in
transferring signals from higher order structures to the peripheral SNS. Neurons in the
rostral medulla are critical modulators of SNS tone and most of the neurons are
glutamatergic and catecholaminergic. Stimulation of rostral medulla neurons
concurrently increases renal sympathetic nerve activity (RSNA) and therefore RSNA is
considered a surrogate marker of rostral medulla neuronal activity. Models of
sympathoexcitation (such as heart failure, spontaneously hypertensive rats, and others)
exhibit increased RSNA tone and elevated levels of angiotensin II (ANG II) in the rostral
medulla. ANG II binds to both angiotensin type 1 (AT1) and angiotensin type 2 (AT2)
receptors with equal affinity, and, in models of sympathoexcitation, there is an increase
in AT1 receptor levels and decrease in AT2 receptor levels in the rostral medulla.
Activation of AT1 receptors in the rostral medulla has an excitatory effect that causes
activation of reactive oxygen species-p38-MAPK signaling, which has been implicated
in the maintenance of chronic elevations in SNS tone. In contrast, the AT2 receptor has
an inhibitory effect on neuronal function via the phospholipase a2/arachidonic acid/12lipoxygenases pathway. Sympathoexcitatory models, which have similar cardiac
alterations to what we have seen in SE, have increased AT1 and decreased AT2
receptor expression in the rostral medulla. Similarly, studies in epilepsy models have
shown increased ANG II levels in the brainstem and alterations in SNS tone. I
hypothesize that centrally driven alterations in angiotensin II signaling leads to an
increase in sympathetic nervous system tone following status epilepticus.
Contributors: Levine, Amber; Anderson, Anne
181
ROLE AND REGULATION OF KLF4 AND G0S2 IN ACUTE MYELOID LEUKEMIA
Andrew H Lewis
Advisor: H Lacorazza, Ph.D.-Department of Pathology & Immunology
Leukemic stem cells (LSCs) share the key hematopoietic stem cell (HSC)
properties of quiescence and self-renewal, despite giving rise to malignant and benign
blood differentiation, respectively. These properties are epigenetically regulated during
development and dysregulated by oncogenic fusion proteins present in Acute Myeloid
Leukemia (AML). The Krüppel-like Factor 4 (KLF4) and G0/G1 Switch 2 (G0S2) genes
are key regulators of these properties in normal HSCs and the LSCs of closely-related
Chronic Myeloid Leukemia (CML), but recent data suggests that their roles and
regulation may vary depending on their cellular context. The importance of KLF4 and
G0S2 in AML LSCs, however, has not been investigated. As LSCs are thought to
generate relapse and refractory disease, identifying mechanisms regarding their
regulation of quiescence and self-renewal is vital to generate effective therapies that
address this key cell population. The central hypothesis behind this work is that KLF4
and G0S2 dysregulation enables aberrant self-renewal and quiescence in LSCs
following MLL-AF9 driven epigenetic changes in the initiating cell. Supporting this
hypothesis is the recent discovery that CDX2 expression, a common feature of the
majority of AML cases including MLL-AF9, is highly correlated with KLF4 and G0S2
repression. Publicly available gene expression data from multiple studies also
demonstrates KLF4 and G0S2 expression level changes depend on epigenetic
effectors, lineage, and MLL-AF9 transformation and our own unpublished data in CML
details a varied role in self –renewal when comparing CML LSCs with HSCs.
Completion of this work will evaluate two potential key regulators of self-renewal and
quiescence in AML LSCs and lay the groundwork for identifying the best therapeutic
approaches to targeting unique mechanisms responsible for their activity and regulation
in leukemic cells. Testing of the central hypothesis will be performed through an
approach in which we complete the following specific aims:
• Aim 1. Determine the importance of KLF4 and G0S2 in MLL-AF9 driven AML
and leukemic stem cell function.
• Aim 2. Determine epigenetic control of KLF4 and G0S2 in MLL-AF9-driven
AML differentiation and transformation.
Initially, molecular approaches using genetically modified mouse models of MLLAF9 AML will allow investigation of the effects of loss of the KLF4 and G0S2 genes on
leukemogenesis.
Completion of these aims will provide a comprehensive picture of the importance
of KLF4 and G0S2 in MLL-AF9 AML and leukemic stem cells (Aim 1) and delineate
how these two genes are epigenetically regulated in the context of oncogenic
transformation propagated by MLL-AF9 (Aim 2). We will then be able to use this
understanding to develop LSC-targeted therapy for AML.
Contributors: Lewis, Andrew; Park, Chun Shik; Shen, Ye; Puppi, Monica; Lacorazza, Daniel
182
THE ROLE OF CUGBP1 IN LIVER PROLIFERATION AND IN LIVER CANCER
Kyle Joseph Edward Lewis
Advisor: Nikolaj Timchenko, Ph.D.-Department of Pathology & Immunology
Richard Sifers, Ph.D.-Department of Pathology & Immunology
CUG triplet-repeat binding protein 1 (CUGBP1) is an RNA binding protein
expressed in several tissues including the liver. Our lab has previously shown that
CUGBP1 upregulates translation of C/EBPβ and HDAC1 by binding to the 5’ region of
both these mRNAs and by delivery of eIF2 translational complex to initiate translation of
these mRNAs. This elevation of C/EBPβ and HDAC1 leads to the formation of a
repressor complex that deacetylates chromatin and reduces expression of several
genes including C/EBPα which in turn leads to increased cell proliferation in the liver.
We have also shown that this binding to eIF2 is controlled by phosphorylation of
CUGBP1 at serine residue 302 (Ser302). To examine the role of phosphorylation of
CUGBP1 in liver biology and in liver cancer, a knock-in (KI) mouse model CUGBP1
S302A has been created which mimics the unphosphorylated state of CUGBP1, thus
CUGBP1 lacks translational activity. These mice have been implemented to study the
role of phosphorylated CUGBP1 in liver regeneration after surgical resection and liver
cancer mediated by diethylnitrosamine (DEN). CUGBP1 KI mice underwent 2/3 partial
hepatectomy and liver proliferation/regeneration was examined during the first 5 days
after surgery. H&E staining and BrdU incorporation showed a delayed regenerative
response in CUGBP1 KI mice. Expression of cell cycle proteins was also reduced in
livers of CUGBP1-S302A mice. In a second set of experiments, CUGBP1 KI mice were
treated with DEN and progression of liver cancer was investigated at 30, 34 and 38
weeks after DEN injection. We observed that CUGBP1 KI mice treated with DEN had a
higher incidence of cancer compared to control animals. CUGBP1 KI mice had
increased size and number of tumor nodules as well as increased serum transaminase
levels, indicative of liver damage. We also found a decrease in protein levels of the
mutant CUGBP1 and an increase in the oncogene Gankyrin. Gankyrin has been
shown to be elevated in liver cancer and triggers proteasome mediated degradation of
many tumor suppressor proteins. Our data demonstrate that CUGBP1-S302A interacts
with Gankyrin suggesting that the CUGBP1-S302A and un-ph-CUGBP1 might be
degraded by Gankyrin during development of liver cancer. Together, these data
demonstrate that the un-phosphorylated isoform of CUGBP1 is a new tumor suppressor
protein in the liver and that the development of liver cancer includes elimination of
CUGBP1 perhaps by the Gankyrin-proteasome system.
Contributors: Lewis, Kyle; Jiang, Yanjun; Iakova, Polina; McKay, Mary; Valanejad, Leila; Poling,
Holly; Wei, Christina; Timchenko, Lubov; Timchenko, Nikolai
183
TLR4-AGONIST CONJUGATED VLPs INDUCE A POTENT & NEUTRALIZING HIVSPECIFIC IMMUNE RESPONSE
Phoebe Elnora Lewis
Advisor: Qizhi Yao, M.D./Ph.D.-Department of Surgery
Shital Patel, M.D.-Department of Medicine
Despite 3 phase III clinical trials, an effective prophylactic human
Immunodeficiency Virus HIV vaccine candidate has yet to be identified. Virus-LikeParticles (VLPs) are structurally similar to native virions and our lab has shown the
capability of various chimeric VLPs in eliciting both cellular and humoral immune
responses in mice. Adjuvants provide an additional platform to further increase the
immunogenicity of our chimeric VLPs. In this study, we evaluated the potency and
longevity of HIV-specific immune responses induced by HIV GagIIIB/EnvBaL VLPs
conjugated to Conjugatable Adjuvant Lipid Vesicles (CALVs) containing a Toll-likereceptor 4 (TLR4) agonist (CALV-TLR4) in a C57B6 mouse model. We found that the
CALV-TLR4 potently enhanced HIV-1IIIB Gag, HIV-1BaL Env, and Gag/Env-VLPspecific total IgG responses up to 10-fold compared to PBS and 2 fold compared to
VLP alone, after intranasal priming followed by three intracheek boosts, and is
predominant IgG1 subtype. The VLP without the CALV-TLR4 immunization showed a
predominantly IgG2a subtype. Neutralization data demonstrates that the serum from
the group that received the VLP conjugated to CALV-TLR4 had 40-95% neutralization
activity against 4 different strains of HIV-1, including 2 primary isolates and a Clade C
strain. Serum from the VLP alone group demonstrated no neutralization activity.
Intracellular Cytokine Staining demonstrated Env- or Gag-specific potent IL-2 response
in CD8+ cells, the primary cytokine involved in immunological memory formation. Mice
immunized with VLPs conjugated to CALV-TLR4 had 3 fold higher Env-specific IL-2
producing CD8+ cells than the VLP only group. In addition, immunization with VLPs
plus CALV-TLR4 can elicit persistent long-term antibody responses as high levels of
HIV-specific IgG1 were detected 90 days after the last immunization. Our results
indicate that CALV-TLR4 conjugated with HIV-VLPs induce potent and enduring IgG
responses with a predominant IgG1 subtype and neutralizing activity.
Contributors: Lewis, P.1, 2, Poteet, E.2, Ho, S3, Fujii, G.3, Chen C.2, and Yao Q.2
1 Interdepartmental Program of Translational Biology & Molecular Medicine, Baylor College of
Medicine, Houston, Texas, 2 Department of Surgery, Baylor College of Medicine, Houston,
Texas, 3Molecular Express, Inc., Rancho Dominguez, CA.
184
DICTYOSTELIUM: FAST AND LARGE-SCALE GENETIC SCREEN
Cheng-Lin Li
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
Dictyostelium cells proliferate as solitary cells by binary fission while feeding on
bacteria. Upon starvation, up to 100,000 cells aggregate together and undergo
multicellular development to form a fruiting body. During aggregating development, two
immunoglobulin-like adhesive proteins, TgrB1 and TgrC1, play key roles in initiating
morphogenesis. Cells with defective tgrB1 or tgrC1 gene are developmentally lethal and
fail to undergo cell differentiation and morphogenesis. Despite the crucial roles of the
TgrB1-TgrC1 system during development, little is known about underlying signaling
pathways.
In Dictyostelium, majority of the genetic screens in the past 25 years were
done by insertional mutagenesis. Here, we devised genetic screens by integrating the
chemical mutagenesis and whole-genome sequencing, and developed tools to
streamline the identification of SNVs (single nucleotide variants). As proof-of-concept,
we screened for chemical-mutagenized cells with chemotaxis-defect or photo-killing
resistance. We showed that we were able to identify mutations on genes known to
mediate the chemotaxis and photo-killing. Therefore, we applied the chemical
mutagenesis to screen for suppressor of the tgrC1-defective phenotypes. In the screen,
the genomes of the 63 suppressor mutants were sequenced. There were an average of
~15 genic mutations per genome and nine candidate genes were mutated
independently three times or more. Interestingly, we found two recurrent mutations
(G275D and G307D) on the first immunoglobulin domain of the TgrB1. Ectopically
expressing the mutant alleles, tgrB1G275D or tgrB1G307D, partially rescued the
development of the tgrC1-defective cells. Therefore, the G275D and G307D mutations
on tgrB1 are the causative gain-of-function mutations.
In this study, we developed a novel genetic screen pipeline in Dictyostelium by
coupling chemical mutagenesis and whole-genome sequencing. We also demonstrated
that our method allows us to saturate the screen with a relatively small number of
mutants and to identify the causative mutations. Therefore, the new screening strategy
could help speeding up research progress in the Dictyostelium filed. More importantly,
nine candidate genes of the TgrB1-TgrC1-mediated signaling pathway were identified,
and may shed insight into the TgrB1-TgrC1-mediated signaling events.
Contributors: Li, Cheng-Lin
185
COMPLEXITY AND DIVERSITY OF FACTOR VIII GENETIC VARIATIONS IN THE
1000 GENOMES
Jiani Li
Advisor: Fuli Yu, Ph.D.-Department of Molecular & Human Genetics
Hemophilia A (HA) is an X-linked bleeding disorder caused by deleterious
mutations in the coagulation factor VIII (F8) gene. F8 mutations have so far been
documented predominantly in Europeans and Americans of European descendant.
Information on F8 variants with diverse ethnic backgrounds is limited. We analyzed
2,535 subjects of 26 ethnicities available in the 1000 Genomes Project (1000G) phase
3 dataset for F8 variants and their potential functional impacts. We identified 3,030
single nucleotide variants (SNVs), 31 short deletions and insertions (Indels) and a
497kb large deletion. Among all variants, 86.4% of variants were rare variants
(MAF<1%) and 55.6% were novel. Eighteen variants previously associated with HA
were found in our study. Most of these “HA variants” were ethnic-specific with low allele
frequency, however, one variant – p.M2257V was presented in 27% of African subjects.
The p.E132D, p.T281A, p.A303V and p.D422H “HA variants” were identified only in
males. Twelve novel rare non-synonymous variants were predicted to be deleterious.
The large deletion was discovered in 8 female subjects without affecting F8
transcription and the transcription of genes on the X chromosome. Characterizing F8 in
the 1000G highlighted the complexity of F8 variants and the importance of interrogating
genetic variants with different ethnic backgrounds. The haplotype analysis and the
orientation of duplicons that flank the large deletion suggested that the deletion was
recurrent and originated by homologous recombination.
Contributors: Jiani Li, Ivenise G. Carrero, Jing-fei Dong, Fuli Yu
186
PITX2 PREDISPOSES HEART TO CARDIAC ARRHYTHMIA BY REGULATING ION
TRANSPORT AND INTERCALATED DISC GENES.
Lele Li
Atrial fibrillation (AF) is the most commonly seen atrial arrhythmia. Genome-wide
association studies identified a locus on chromosome 4q25, variants of which are
associated with increased risk of atrial fibrillation. This locus locates close to the
pituitary homeobox2 (Pitx2) gene. Pitx2 is a transcription factor, which plays a critical
role in left-right asymmetry establishment and maintenance of the heart. Our previous
studies indicate that Pitx2+/− mice show symptoms of atrial fibrillation when given
programmed electrical stimulation. However, role of Pitx2 in heart function is still not
clear due to the functioning allele in these heterozygous mice. Therefore, we
hypothesize that Pitx2 deficiency in heart leads to atrial fibrillation.
We use a conditional knockout mouse model, Pitx2flox/flox MCK-Cre mice, in
which Pitx2 expression is specifically disrupted in mice cardiac/skeletal muscle after
birth. Surface ECG and ECG telemetry is used to record electric activity of the hearts in
Pitx2 knockout mice to determine phenotype in Pitx2flox/flox MCK-Cre mice. In
Pitx2flox/flox MCK-Cre mice, there is irregular R-R interval without a second triggering
event to induce arrhythmia, which is an indicator of sinus node dysfunction. From this
result, we know Pitx2 knockout leads to abnormal heart rhythm, which indicates Pitx2
plays a critical role in heart function.
Since Pitx2 is a transcription factor, we want to investigate its binding region on
chromosome. ChIP-sequencing and microarray assay is done to find target genes of
Pitx2 and determine changes in genes expression levels. Finally, we find genes whose
expression is more than two fold in mutant than in wildtype mice, including genes
related to ion channels, signaling pathways, transcription factors and so on. Majority of
the genes screened have an increase in RNA level in mutant mice, indicating Pitx2 may
function mainly as a repressor for the candidate genes.
To confirm the result of microarray, and evaluate molecular mechanisms of the
arrhythmia in Pitx2 CKO mice, RNA level of a number of candidate genes, which
regulates cardiac function, are analyzed using qRT-PCR. Result indicated upregulation
of RNA level of majority of candidate genes and down regulation of a small number of
genes, indicating Pitx2 may function mainly as repressor in regulation of genes
transcription in cardiac function. Luciferase reporter assay was conducted and
confirmed Pitx2 targets, indicating Pitx2’s regulation of candidate genes. TEM showed
that Pitx2 CKO mice have damaged mitochondria and disrupted intercalated disc
structure, suggesting Pitx2 regulates heart function by stabilize integrity of intercalated
disc structure.
Contributors: Tao, Ye; Zhang, Min; Bai, Yan; Zhou, Yuefang; Moon, Anne M.; Kaminski Henry
J.; Martin, James F.
187
A NOVEL UBIQUITINATION PATHWAY REGULATES USHER SYNDROME AND
MYH9 RELATED DISORDER PROTEINS IN DROSOPHILA AUDITORY ORGANS
Tongchao Li
Andrew Groves, Ph.D.-Department of Neuroscience
To identify novel genes that affect hearing, we screened a collection of mutations
in essential genes on the X chromosome for morphological defects in the fly auditory
organ, the Johnston’s Organ (JO). We isolated two mutations in the gene ubr3, which
encodes a conserved, RING type E3 ubiquitin ligase. Loss of ubr3 causes apical
detachment of the auditory sensory cells and functional defects in hearing.
Apical detachment of auditory sensory cells is a specific defect that was
previously only associated with mutations in the fly homolog of Myosin VIIA (myo7a)
gene. myo7a encodes an unconventional myosin. Mutations in this gene cause Usher
syndrome in human, a disease that causes blindness and deafness and accounts for
50% of the blind-deaf patients. We found that ubr3 and myo7a genetically interact.
Through biochemical assays, we found that Ubr3 negatively regulates the monoubiquitination of another myosin protein MyoII and that MyoII-Myo7a interact.
Interestingly, mutations in MYH9, the human homolog of myoII, cause MYH9 related
disorders. MYH9 related disorders are four syndromes that were thought to be different
diseases but later found to be caused by mutations in the same gene MYH9. The
patients exhibit a series of symptom including sensorineural deafness. Consistent with
the ubiquitination change of MyoII and increased MyoII-Myo7a binding in ubr3 mutant
cells, we also observe genetic interactions between ubr3 and myoII, myo7a and two
other Usher genes sans and Cad99C. Moreover, Ubr3 regulates the monoubiquitination of MyoII through down-regulation of the protein level of Cullin1 (SCF E3
ligase). The fact that both over-expression and down-regulation of Cullin1 cause similar
apical detachment in JO and increased mono-ubiquitination of MyoII suggests that
Cullin1/SCF is not the direct E3 ligase for MyoII.
To determine if the newly identified ubiquitination pathway has any pathological
relavence to MYH9 related disorders, we over-expressed four dominant mutations of
MyoII in JO first identified in patients. Interestingly, we observed a similar scolopidial
detachment upon over-expression of the MyoII mutant forms, but not wild type Myosin
II. In addition, all mutant MyoII form puncta in the auditory neurons, very different from
the observed diffuse wild type MyoII. Interestingly, manipulating the ubiquitination of
MyoII leads to similar puncta of wild type MyoII proteins. These data clearly suggest
that ubiquitination of MyoII, which is regulated by Ubr3 and Cul1, is relevant to the
pathogenesis of MYH9.
Contributors: Li, Tongchao; Nagarkar Jaiswal, Sonal; Eberl, Daniel; Godt, Dorothea; Groves
Andrew; Bellen Hugo
188
THE ROLE OF MULTI-ENZYME AMINOACYL TRNA SYNTHETASE COMPLEX
COMPONENT AIMP1/P43 IN DENDRITIC CELL TH1 IMMUNITY
Dan Liang
Advisor: William Decker, Ph.D.-Department of Pathology & Immunology
Of professional antigen presenting cells (APC), only the dendritic cells (DC) are
regarded as initiators of adaptive immune responses. Previously we identified a Th1promoting phenotype of DC simultaneously loaded with overlapping (homologous) MHC
class I and II determinants: tumor specific mRNA and cell lysate. This event, termed
“homologous antigenic loading”, augmented DC IL-12 production, generation of CD8+
cytolytic effectors, and IFN-γ T-cell secretion, all downstream hallmarks of Th1
immunity. We further confirmed this Th1-promoting phenotype across a variety of
antigenic systems in which DC class I and II are loaded with determinants of
homologous origin, indicating DC intrinsic mechanisms to recognize and compare
antigenic epitopes. In an effort to characterize underlying mechanisms, we looked for
secreted factors in DC culture supernatants and detected high levels of AIMp1/p43
release from DC loaded with homologous tumor lysate/mRNA, single protein antigen, or
overlapping viral peptides in comparison to singly-loaded or heterologous controls.
AIMp1 is a structural component of the multi-enzyme aminoacyl-tRNA
synthetase complex (mARS) which consists of at least 8 aminoacyl-tRNA synthetases
and 3 structural proteins. Previous studies have shown that AIMp1 is released by both
tumor and macrophages under stress or TLR stimulus, upregulates proinflammatory
gene expression in monocytes and macrophages, and induces Th1 polarization of bone
marrow derived DC (BMDC). We subsequently established that the level of AIMp1
release is closely correlated to the degree of homology between loaded class I and II
epitopes. AIMp1 deficiency abrogated the ability of homologous loaded DC vaccines to
mediate Th1 immune response against B16 melanoma tumors in vivo. In vitro studies
confirmed that AIMp1 deficiency reduced the expression of costimulatory markers
(CD86, CD40), MHC molecules and Th1 cytokines (IL12) expression from BMDCs.
Further, AIMp1 plays opposite roles in effector T cell type 1 and 2 polarization. The
production of IFNγ and IL4 is regulated differently both in vitro and in vivo in the
absence of AIMp1. These data suggest AIMp1 is critical for Th1 immune responses
induced by homologous loading of DC.
We also observed that AIMp1KO animals are more susceptible towards
influenza virus infection (H3N2 influenza virus). We will further investigate the role of
dendritic cells in this scenario. Significant differences in protein expression profile
between WT and AIMp1KO BMDCs are indicated by reverse phase protein array
(RPPA), with the help of which we could elucidate the molecular and cellular
mechanisms of AIMp1 mediated crosstalk between DC and T cells. Finally, we seek to
apply AIMp1 as a novel vaccination adjuvant and will test its potential in anti-tumor
immunity and infectious diseases models. Most critically, these studies will underscore
the importance of homologous antigenic signals in DC polarization.
Contributors: Liang, Dan, Halpert, Matthew M, Konduri, Vanaja, Irene Yunyu Chen, Doyeun
Kim, Sunghoon Kim, Levitt, Jonathan M, Decker, William K
189
EFFECTS OF MILD WHOLE-BODY BLAST WAVE TRAUMA TO THE VESTIBULAR
RECEPTOR ORGANS, NUCLEI, AND VOR IN MICE
Steven Donald Lien
Advisor: J. Dickman, Ph.D.-Department of Neuroscience
The increased use of close range explosives has led to higher incidence of
exposure to blast-related head trauma. Exposure to primary blast waves is a significant
cause of morbidity and mortality. Active service members who have experienced blast
waves report high rates of vestibular dysfunction, such as vertigo, oscillopsia,
imbalance, and dizziness. Accumulating evidence suggests that exposure to blast wave
trauma produces damage to both the peripheral and central vestibular system; similar
to previous findings that blast-induced damage of auditory receptors. Here we
examined the vestibular receptor, central nuclei, and vestibulo-ocular reflex (VOR)
behavior in mice after exposure to mild whole body blast wave trauma. Mice were
implanted with a head stability post, and the VOR was then measured using a
horizontal rotation stimulus (0.5 - 2 Hz; 20 deg/s). Next, each animal was exposed to a
single air blast wave overpressure of 55, 75, or 95 kpa while anesthetized and
suspended inside a rigid tube. Following blast exposure, animals were allowed to
survive for periods of 1, 7, 14, or 28 days. At each survival time point, the VOR was
again measured after which the animal was euthanized and perfused for histological
analyses. Vestibular receptors and central nuclei were histologically prepared and the
following measures quantified: stereocilia hair bundle counts, density of hair cells (both
type I and type II) in the central and peripheral zones, density and size of otoconia in
central and peripheral zones, axon density and diameter beneath the stroma. Ongoing
measurements also include number and length of axonal initial segment, terminal
innervation patterns of calyx, dimorph, and bouton afferents. The medial and lateral
central vestibular nuclei were measured for the density of neurons at the caudal,
central, and rostral regions of each of these nuclei. The VOR gain and phase of the eye
movement responses were compared for the normal (pre-blast) and the blast exposed
animals. To date, we have observed a reduction in horizontal VOR gains and stereocilia
loss in the highest, 95, kPa blast wave exposed animals. These results suggest that
blast-wave exposure can lead to peripheral vestibular damage (possibly central deficits
as well) and provides some insight into causes of vestibular dysfunction in blast-trauma
victims.
Contributors: Lien, Steven; Dickman, J David
190
RECURRENT SOMATIC MUTATIONS IN RAS-ERK PATHWAY CHARACTERIZE
LANGERHANS CELL HISTIOCYTOSIS
Karen Phaik Har Lim
Advisor: Carl Allen, M.D./Ph.D.-Department of Pediatrics
Donald Parsons, M.D./Ph.D.-Department of Pediatrics
Purpose: Langerhans Cell Histiocytosis (LCH) is a myeloproliferative disorder
characterized by lesions composed of pathologic CD207+ dendritic cells (DCs) with an
inflammatory infiltrate. BRAF-V600E remains the only recurrent mutation described in
LCH.
Methods: In order to characterize the extent of somatic mutations in LCH, whole
exome sequencing (WES) followed by AmpliSeq validation was performed on a series
of matched LCH and normal tissue. Lesions from other histiocytic disorders, juvenille
xanthogranuloma (JXG), Erdhim-Chester disease ECD), and Rosai-Dorfman Disease
(RDD) were also evaluated.
Results: LCH lesions were characterized by a low mutation frequency of ~ 2
somatic mutations per sample. BRAF-V600E mutations were identified in 20 of 41
cases (48%), and included in all cases previously found to harbor BRAF mutations by
qPCR. Among the 21 LCH patients with wild-type BRAF, 7 of 21 (33%) cases were
found to have novel in-frame deletions in exons 2 and 3 of MAP2K1 (c.158_172del,
c.171_185del, c.301_306del x 2), which encodes MEK1. Additional mutations in genes
with potential influence on the RAS-ERK and related signal transduction in LCH
samples include the tyrosine kinase receptors ERBB3, ARAF, PICK1 and PIK3R2. In
JXG, germline NF1 and somatic ERBB4 were identified.
Conclusions: We have confirmed recurrent mutation in BRAF-V600E and
identified novel mutations in the RAS-ERK pathway, supporting a model of ERK hyperactivation as the key driver of LCH pathogenesis. The functional impact and clinical
consequences of different combinations of MAP2K1, BRAF, and other RAS-ERK
pathway variants will require further investigation and may inform future diagnostic and
therapeutic
strategies
for
LCH
as
well
as
other
ERK-associated
neoplasias/malignancies.
Contributors: Chakraborty, Rikhia1; Hampton, Olive2; Shen, Xiaoyun2; Shih, Albert1,
Simko,Stephen1, Abhkandar, Harshal1; Lim, Karen Phaik Har1; Hicks, John1; McClain,
Kenneth L1; Wheeler, David1; Allen, Carl E1; Parsons, D.Will1
1Texas Children's Cancer Center, Texas Children's Hospital, Houston, TX, United States;,
2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX,
United States;
191
THE EFFECT OF DELETING BMI1 IN MESENCHYMAL STEM CELLS TO HSC
MAINTENANCE DURING AGING
Angelique Lin
Being the apex of the hematopoietic system, hematopoietic stem cells (HSCs)
give rise to and maintain all the other blood cell lineages in the blood. In the process of
advanced aging, HSCs undergo dramatic intercellular changes, results in the decline of
HSC function and biased differentiation capacity. In addition to internal cellular
changes, the changes of extrinsic factors have an influence on diminishing HSC
function as well. HSCs reside in a specific microenvironment in the bone marrow,
consisting of mesenchymal stem cells (MSCs), endothelial cells, and other types of
cells, where HSCs are maintained and regulated. Regulatory signals within the niche
tightly regulate the quiescence, proliferation, and differentiation of HSCs, preventing
HSCs from exhaustion. The HSC niche undergoes dramatic changes upon aging,
including the reduction of bone formation due to decreased number of osteoblasts,
accumulation of adipocytes in the bone marrow, and alteration of the extracellular
matrix. These reduce the capacity of HSC niche to support HSCs, but the precise
mechanisms are largely unknown.
To study the effect of aging-associated changes in the HSC niche, we
conditionally deleted Bmi1in MSC using MSC-specific promoter Prx1. Bmi1 is a
polycomb group protein that maintains gene silencing through chromatin modifications.
It has an essential role in regulating stem cell self-renewal. However, is not clear in
which cell type Bmi1 functions to the maintenance of HSCs. To study the role of Bmi1
in MSC aging and its effect to HSC function, we generated Prx1-Cre; Bmi1fl/fl mice and
examined the impact of specifically deleting Bmi1 in MSCs to HSC function upon aging.
Prx1-Cre; Bmi1fl/fl mice showed an increase of adipogenesis in trabecular bone
comparing with the wild type. The bone marrow cellularity was significantly decreased
with large variation in the HSC frequency in the Prx1-Cre; Bmi1fl/fl mice. To study the
effect of deleting Bmi1 in MSCs on HSC reconstitution capacity, we performed HSC
transplantation, introducing genotypically normal HSCs isolated from 3-month-old or 6month-old Prx1-Cre; Bmi1fl/fl mice into young (2-month-old) wild type recipients. We
found that HSCs isolated from a Prx1-Cre; Bmi1fl/fl niche showed the aged phenotype
of myeloid-biased reconstitution. But these HSCs also showed a significantly higher
overall reconstitution. These results suggest that deleting Bmi1 in the MSC has an
effect on HSC function, but more tests are required to examine how MSC regulates
HSC aging.
Contributors: Nakada, Daisuke; Kitano, Ayumi; Takeichi, Makiko; Saitoh, Yusuke; Chapple,
Richard Harrison; Hoegenauer, Kevin
192
MICROBE-HOST CHEMICAL COMMUNICATION REGULATES HOST METABOLIC
ADAPTATION TO ENVIRONMENTAL VARIATIONS
Chih-chun Janet Lin
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Gut microbiota exists virtually in all coelomate animals. Besides, microbe-derived
metabolites can directly influence specific cellular pathways in the host. These microbehost metabolic axes play pivotal roles in maintaining the homeostasis of host
physiology, and in modulating host susceptibility to diseases. Although the composition
of the gut microbiota is essentially stable throughout adulthood, microbial metabolism is
highly dynamic and flexible in response to environmental variations. Therefore,
environmental factors not only directly exert their effects on host physiology but also
indirectly through tuning microbe-host metabolic communication. However, the
molecular interactions between environment, microbe and host remain largely
unknown. In this project, we showed that environmental methionine tunes bacterial
methyl metabolism, which in turn modulates mitochondrial dynamics and lipid
metabolism in Caenorhabditis elegans through NR5A nuclear hormone receptor NHR25. We discovered that methionine deficiency decreases the production of bacterial
metabolites
that
are
essential
for
phosphatidylcholine
synthesis in C.
elegans. Reduction of diundecanoyl and dilauroyl phosphatidylcholines suppressed the
activation of their receptor NHR-25, and led to increased lipid accumulation by
promoting mitochondrial fragmentation. Together, our work reveals a molecular
mechanism that connects host-microbe interactions with environmental variations. The
components in this pathway have well-conserved homologous counterparts in human.
Therefore, these results could advance our knowledge on the evolutionarily conserved
chemical dialogues between microbiota and human under environmental variations.
Contributors: Wang, Meng
193
THERAPEUTIC TARGETING OF INI1 DEFICIENCY IN PEDIATRIC ATRT: A PRECLINICAL STUDY UTILIZING PATIENT-DERIVED ORTHOTOPIC XENOGRAFT
MODELS (PDOX)
Holly Brianne Lindsay
Advisor: Xiaonan Li, M.D./Ph.D.-Department of Pediatrics
INTRODUCTION: Atypical teratoid rhabdoid tumors (ATRTs) are molecularlydefined by deletions or mutations in tumor suppressor INI1. INI1 deficiency causes
dysfunction of chromatin-remodeling complexes which normally oppose EZH2, a
epigenetic gene silencing protein. In the setting of INI1 deficiency, EZH2 is overexpressed, leading to ATRT oncogenesis. Using novel patient-derived orthotopic
xenograft (PDOX) models, we investigated the pre-clinical utility of therapeutic EZH2
inhibition in ATRT using the drug DZNep.
METHODS: Two PDOX models of ATRT were established by directly implanting
tumor cells into mouse brains in the matched location with patient tumor. For in vitro
testing, ATRT cells were exposed to DZNep at doses from 78 to 5000 nM for 2 weeks;
cell viability was measured every 1- 3 days. For in vivo testing, 40 mice were divided
into 4 treatment groups: control, DZNep (5 mg/kg i.p. daily x 14 days), standard of care
(cranial radiation 2 Gy daily x 5 days, cisplatin 5 mg/kg i.p. on days 8 and 11), and
combination therapy. Survival times were compared using log-rank analysis.
RESULTS: DZNep showed dose- and time-dependent in vitro inhibition of tumor
cell viability in both ATRT models tested. One of these models (IC-L1115ATRT) was
then subjected to in vivo studies. ATRT tumors were present at the time of xenograft
death in all 4 treatment groups including DZNep- and combination therapy-treated
animals. Survival time did not differ between the control- and DZNep-treated groups or
the standard of care- and combination therapy-treated groups (P>0.05).
CONCLUSIONS: While the EZH2 inhibitor DZNep is effective in vitro in killing
ATRT tumor cells, it does not appear to have effects in vivo when tested in PDOX
models of pediatric ATRT acting alone or in combination with combined chemo- and
radio-therapies. Extensive further pre-clinical evidence should be obtained before an
EZH2 inhibitor is brought into pediatric clinical trials.
Contributors: Lindsay, Holly; Kogiso, Mari; Qi, Lin; Murray, Jeffrey; Perlaky, Laszlo; Su, Jack;
Baxter, Patricia; Adesina, Adekunle; Parsons, D. Williams; Chintagumpala, Murali; Li, Xiao-Nan
194
COMPLEMENT C3-INDUCED MODULATION OF AMYLOID PATHOLOGY THROUGH
ASTROCYTE-MICROGLIA INTERACTION IN MOUSE MODELS OF ALZHEIMER`S
DISEASE
Alexandra Litvinchuk
Advisor: Hui Zheng, Ph.D.-Department of Molecular & Human Genetics
Alzheimer`s disease (AD) is the most common form of dementia, affecting
millions of people worldwide. A common feature of AD is the activation of the immune
response in the brain. Activation of the complement pathway has been implicated in this
process.
Recent studies in our lab showed that NF-kB pathway can activate complement
C3 component in astrocytes but not neurons. Moreover, elevated astroglial C3 through
its receptor C3aR in neurons can modulate intraneuronal calcium levels, and
dysregulation of calcium homeostasis has been implicated in the pathogenesis of AD.
Importantly, astroglial NF-kB and C3 can be induced by Aβ and are upregulated in AD
brains.
In the current work we report that astroglial upregulation of C3 also regulates Aβ
dynamics and pathology through microglial C3aR.
In primary microglia culture experiments we show that acute C3 treatment
enhances while chronic C3 exposure attenuates microglial phagocytosis and this effect
can be blocked by C3aR antagonist treatment or by genetic deletion of
C3aR.Furthermore, in our in vivo work we demonstrate that C3 upregulation through
NF-kB pathway activation increases amyloid pathology in APP transgenic mice while
C3aR antagonist treatment can reduce plaque load and microgliosis.
Together, this data suggests that complement C3 plays a detrimental role in AD
pathogenesis and blocking of C3aR with its antagonist can be used as a potential
therapeutic target.
Contributors: Lian Hong, Chiang Angie C-A, Aithmitti Nadia, Jankowski Joanna, Zheng Hui
195
GLIAL LIPID DROPLETS AND ROS INDUCED BY MITOCHONDRIAL DEFECTS
PROMOTE NEURODEGENERATION
Lucy Liu
Reactive oxygen species (ROS) and mitochondrial defects are implicated in
many neurodegenerative diseases. My colleagues and I found that a key consequence
of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets
(LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol
Regulatory Element Binding Protein (SREBP) activity in neurons, leading to LD
accumulation in glia prior to or at the onset of neurodegeneration. The accumulated
lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and
lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly
delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD
accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting
that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved
phenomenon, and represents an early, transient indicator and promoter of
neurodegenerative disease. In these mutant animals, lipid synthesis originates in the
neurons and is transferred to the glia where LDs are formed. Lipid transfer between
neuron and glia has not been previously documented and the mechanism of lipid
transport is ill-defined. I found that monocarboxylate (lactate) transporters are critical for
the transport of lipids from the neuron to the glia irrespective of ROS levels. The
pharmacological or genetic perturbation of monocarboxylate transporters and/or lactate
levels reduces cell death in vitro and ameliorates neurodegeneration in mutant animals
with ROS induced LD accumulation.
Contributors: Liu L, Zhang K, Sandoval H, Yamamoto S, Jaiswal M, Sanz E, Li Z, Hui J,
Graham BH, Quintana A, Bellen HJ
196
UNDERSTANDING THE LINK BETWEEN BONE HOMEOSTASIS AND BREAST
CANCER BONE METASTASIS
Hin Ching Lo
Background: Bone metastasis is the most common form of disseminated
disease in breast cancer, affecting 20-40% of patients. Early stage bone metastasis
presents an attractive therapeutic target, but very little is known about it. Recent work in
our lab revealed that early stage micrometastasis is associated with a
microenvironment niche with features of osteogenesis. Moreover, direct cell-cell
interaction with osteogenic cells increases cancer cell proliferation via the mTOR
pathway. Based on this finding, we hypothesize that molecular and cellular changes
that lead to increased osteogenesis in the bone will also promote early bone metastasis
progression.
Experimental design and methods: To perturb bone homeostasis, I used 2
pharmacological agents: Teriparatide (increases osteogenic activity) and Zoledronic
acid (decreases osteogenic activity by suppressing general bone turnover). Drug or
vehicle treatment began in mice 2 weeks prior to the inoculation of cancer cells. Intrailiac artery (IIA) injection was then performed to deliver breast cancer cells into the hind
limb bones of mice, and the growth of early bone metastatic lesions was monitored
weekly using in vivo bioluminescence imaging. Structural and cellular changes to the
bone were quantified by microCT scanning and immunohistochemistry. Experiments
were conducted in mice with or without supplemented estradiol pellets to mimic the preand post-menopausal settings.
Results: In mice with estradiol supplementation, zoledronic acid treatment
appears to slow down early bone metastatic development. In the treatment group
without estradiol, tumor growth between different mice is highly variable, and no
significant difference is found between the different drug treatment groups.
Conclusion: Zoledronic acid may have an effect in suppressing early stage bone
metastatic progression.
Contributors: Lo, Hin Ching Flora; Yu, Cuijuan Melanie; Wang, Hai; Zhang, Xiang
197
SPDEF ENFORCES QUIESCENCE OF CANCER INITIATING CELLS BY SHIFTING
THE TRANSCRIPTIONAL TARGETS OF ACTIVATED Β-CATENIN
Yuan-Hung Lo
Advisor: Noah Shroyer, Ph.D.-Department of Pediatrics
Background: Colorectal carcinogenesis is driven by a series of genetic and
epigenetic changes that results in the oncogenic transformation of normal colonic
mucosa. Although constitutive activation of canonical Wnt signaling resulting in high βcatenin transcriptional activity is believed to drive most human colorectal cancers
(CRCs), current molecular targeted therapies have limited efficacy. We have previously
reported that SAM Pointed Domain Ets transcription Factor (SPDEF) is a colonic tumor
suppressor that negatively regulates canonical Wnt/β-catenin signaling pathway. Under
homeostatic condition, SPDEF plays an important role in cell cycle exit and regulates
goblet cell terminal differentiation in the adult intestine. However, the molecular
mechanism behind SPDEF-mediated tumor repression is still largely unknown. Here we
aim to elucidate the molecular mechanism that SPDEF-mediated repression of
canonical Wnt/β-catenin activity in CRCs.
Methods: To achieve our goal, we analyzed the effects of SPDEF expression in
β-catenin-driven intestinal tumors in vivo using an inducible mouse model
(Lgr5CreERT2; β-cateninexon3; Rosa26rtta-ires-EGFP; TRE-Spdef) and human
colorectal cancer xenografts. Moreover, wildtype or truncated SPDEF mutants were
used for β-catenin transcriptional activity assay, co-immunoprecipitation, and chromatin
immunoprecipitation in human colon cancer cell lines.
Results: We found that SPDEF is sufficient to inhibit β-catenin-driven intestinal
tumorigenesis and shrink established tumors in vivo. Consistent with these finding,
SPDEF inhibits both ligand- and oncogene-mediated β-catenin transcriptional activity in
vitro. The inhibitory effect of SPDEF on β-catenin transcriptional activity is mediated by
protein-protein interaction, which appears distinct from SPDEF’s function as a DNAbinding transcription factor. Moreover, our data suggested a novel mechanism that
SPDEF selectively displaces β-catenin from the promoter/enhancer regions of cell cycle
genes without affecting the other intestinal stem cell signature genes by disrupting
binding between β-catenin and specific TCF/LEF partners. Finally, we directly
demonstrated that SPDEF promotes cell cycle exit, but does not enhance terminal
differentiation of cancer cells, resulting in a quiescent state of β-catenin-driven cancer
initiating cells. Taken together, here we unveil a novel mechanism by which SPDEF
directs cancer initiating cells to switch between active and quiescent states, by shifting
the transcriptional targets of activated β-catenin.
Contributors: Lo, Yuan-Hung; Noah, Taeko; Chen, Min-Shan; Zou, Winnie; Shroyer, Noah
198
MANGANESE ENHANCED MRI (MEMRI): A NOVEL, NON-INVASIVE APPROACH
TO ASSESS SKELETAL MUSCLE PATHOLOGY IN MDX MICE
James Anthony Loehr
Advisor: George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Duchenne muscular dystrophy is an X-linked progressive degenerative disease
resulting from a mutation in the gene that encodes dystrophin, leading to decreased
muscle mechanical stability and force production. Evidence indicates that increased
sarcolemmal Ca2+ influx is an early indicator of disease pathology and occurs prior to
alterations in muscle histology or inflammation. A variety of therapeutic approaches for
DMD are being tested in clinical trials, however, there currently are no reliable in vivo
methods to track the molecular progression of the disease or assess the benefit of
different treatments. Because Mn2+ ions can pass through Ca2+ channels, Mn2+ has
long been used to indirectly monitor sarcolemmal Ca2+ influx. Interestingly, Mn2+ also
is paramagnetic and a potent magnetic resonance imaging (MRI) contrast agent. While
MRI has been used to study dystrophic muscle, a major limitation is the inability to
differentiate between disease progression and inflammation, edema, or fat. However,
manganese enhanced MRI (MEMRI) may mitigate these limitations by utilizing Mn2+
ability to enter muscle through Ca2+ channels in mdx mice. Therefore, we hypothesized
MEMRI would be a viable, non-invasive method to monitor dystrophic muscle in the
same subject over time. In vitro, Mn2+ quench demonstrated an increased basal Ca2+
level in skeletal muscle of mdx mice and Mn2+ administration had no effect on muscle
function. In vivo, MEMRI revealed increased Mn2+ contrast in mdx skeletal muscle
compared to wild-type that dissipated 2 days later. Furthermore, Mn2+ infusion had no
effect on in vivo muscle function immediately after or 2 days post-MEMRI. These data
are in agreement with the in vitro Mn2+ quench and muscle function results. Previously
we have shown that eliminating Nox2 ROS production in mdx mice decreases
sarcolemmal Ca2+ influx in vitro and reduces muscle pathology; here we demonstrate
MEMRI can be used to differentiate between these different pathological states. Our
data indicate that MEMRI is a viable, non-invasive technique to monitor dystrophic
muscle over time and evaluate the effectiveness of potential therapies for Duchenne
muscular dystrophy.
Contributors: Stinnett, Gary R; Pautler, Robia G; Rodney, George G.
199
ANKYRIN-G: A NOVEL MECHANISTIC LINK BETWEEN EPILEPSY AND BIPOLAR
DISORDER
Angel Y Lopez
Advisor: Edward Cooper, M.D./Ph.D.-Department of Neurology
The co-morbidity of mood disorders and epilepsy is well established
epidemiologically but poorly understood. Intronic single nucleotide polymorphisms
(SNPs) upstream of the alternative-first-exons of ANK3, exon-1b, have shown
association with bipolar disorder in multiple genome-wide association studies.
Furthermore, individuals with these SNPs have reduced levels of exon-1b-contianing
transcripts in many brain regions. However, the mechanism(s) by which ANK3
contributes to this disorder is still unknown. Here we show that parvalbumin (PV)interneurons exclusively express isoforms of AnkG encoded by exon-1b transcripts, and
that reduced expression of these isoforms results in reduced AnkG and NaV channel
densities at the axon initial segment (AIS) of PV-interneurons. We also observed
spontaneous seizures in exon-1b-knockout mice associated with altered PV-interneuron
firing properties. Thus, loss of AnkG from the AISs of PV-interneurons leads to network
hyperexcitability, which may underlie the genetic association of ANK3 and mood
disorder and provide a mechanistic link to epilepsy.
Contributors: Wang, Xinjun; Xu2, Mingxuan2; Maheshwari3, Atu3l; Sun4, Qian-Quan4;
Noebels5, Jeffrey5; Cooper6, Edward6
200
HIGH-THROUGHPUT ENGINEERING AND FUNCTIONAL ANNOTATION OF
CANCER FUSION GENES
Hengyu Lu
Next generation sequencing (NGS) technologies are rapidly being incorporated
into the clinic to facilitate decisions on cancer patient care. Recognizing this, large-scale
efforts by The Cancer Genome Atlas (TCGA) and others are generating a compendium
of genomic aberrations found across major cancer types with the goal of identifying new
therapeutic targets. The challenge now is to find ways to identify functional “driver”
aberrations, as targeting driver events or their activated pathways offers the greatest
hope of improving patient outcomes. Oncogenic transcript fusions resulting from
chromosomal rearrangements represent an important class of such events. The
successful targeting of fusion oncoproteins such as BCR-ABL1 and EML4-ALK with
imatinib and crizotinib, respectively, provide strong rationale for comprehensive testing
of cancer fusion genes. Unfortunately, the functional interrogation of fusion genes is
complicated by the large quantity identified, inability to accurately predict those with
driver activity, and significant technical roadblocks preventing fusion gene construction
for biological assays. To circumvent these bottlenecks, we developed novel
technologies permitting (1) high-throughput fusion gene construction using a novel
multi-fragment DNA recombineering strategy with our platform of >35,000 human open
reading frame gene clones, and (2) rapid lentiviral delivery of fusion genes to
generalized and context-specific cell models to identify those with in vitro and in vivo
driver activity and responsiveness to available therapeutics. As proof-of-concept, we
used this approach to engineer known fusion oncogenes (BCR-ABL1, EML4-ALK, and
ETV6-NTRK3) and validated their transforming ability using our in vitro and in vivo
driver screening systems, demonstrating our ability to rapidly deliver fusion genes with
functional activity. In a pilot screen of fusion genes identified in pan-negative melanoma
(i.e., devoid of signature mutations in BRAF and NRAS) by TCGA, we identified multiple
uncharacterized BRAF and RAF1 fusion events among others that strongly activate
MAPK signaling and exhibit potent transforming activity. Moreover, cells carrying the
BRAF and RAF1 gene fusions revealed a marked sensitivity to RAF and MEK
inhibitors, a finding that may indicate use of these agents for patients whose tumors
harbor these events. We are now scaling these efforts for the comprehensive analysis
of uncharacterized gene fusions, ultimately allowing functionalization of thousands of
fusion events across diverse cancer types. These systems will reveal the highest
priority fusion gene targets to enroll in deep mechanistic biology studies, drug discovery
and development programs ultimately leading to personalized treatment strategies.
Contributors: Lu, Hengyu; Pantazi, Angeliki; Dogruluk, Turgut; Wu, Chia-Chin; Neill, Nicholas;
Kucherlapati, Raju; Scott, Kenneth.
201
ATXN1-CIC CO-REPRESSOR COMPLEX IS CRITICAL FOR PROPER CORTICAL
DEVELOPMENT
Hsiang-Chih Lu
Expansions of glutamine-encoding CAG repeats in ATAXIN-1 (ATXN1) cause
the neurodegenerative disorder spinocerebellar ataxia type 1 (SCA1). Genetic studies
have provided insight into SCA1 pathogenesis by demonstrating that SCA1 is mostly
caused by a gain-of-function mechanism due to enhanced function of ATXN1. Little is
known, however, about the specific function of ATXN1. Thus, our lab sought out protein
interactors of ATXN1 to understand its function better. We discovered that the most
salient of the interactors (that also modify the SCA1 phenotype) are ATXN1’s
functionally redundant paralog Ataxin-1-like (ATXN1L) and the transcriptional repressor
Capicua (CIC). These three proteins, all highly expressed in the brain, form a corepressor complex in vivo, and depend on one another for stability. Moreover, loss-offunction mutations in ATXN1 or CIC have been implicated in developmental disorders
such as autism spectrum disorder and intellectual disability, suggesting that this protein
complex is important for the development of the nervous system. Atxn1-/-; Atxn1l-/(double knockout, DKO) mice and Cic-/- mice die perinatally with defects in multiple
organs, demonstrating that these proteins have critical roles during development. We
therefore propose that ATXN1-CIC complex is important for the proper development
and function of the nervous system.
To test this hypothesis and to understand the function of this complex in the
brain we must bypass the perinatal lethality. Accordingly, we used the Cre-lox
technology to delete the genes encoding this complex conditionally in the nervous
system. We generated conditional knockout of either Atxn1-Atxn1l or Cic in the
developing forebrain using an Emx1-Cre. Loss of either Atxn1-Atxn1l or Cic causes
multiple behavioral abnormalities including increased motor activity, reduced anxiety,
and impaired memory. Histological studies showed that while the layering architecture
of the cortical neurons is largely preserved in these animals, the thickness of superficial
cortical layers (layer 2-4) is reduced. Moreover, there is a reduction in the size of the
barrel cortex, suggesting that ATXN1-CIC complex is also important for regional
patterning of the cortex. These results show that ATXN1-CIC complex is indispensable
for normal brain development and its absence results in multiple behavioral deficits. We
are currently investigating the electrophysiological changes in the cortex as well as
dissecting the molecular changes that lead to the histological defects in the mice.
Contributors: Lu, Hsiang-Chih; Rousseaux, Maxime; Kim, Ji-Yoen; Liu, Xiuyun; Zoghbi, Huda
202
FUNCTIONAL ROLES OF MIR205 IN MAMMARY GLAND DEVELOPMENT AND
STEM CELL MAINTENANCE
Yang (Daisy) Lu
While miRNAs play an important role in normal development and cancer, little is
known about the functional role of miRNAs in mammary gland development. The
analysis of miRNA expression in the mouse mammary epithelial cells has revealed high
expression of miR-205 in basal stem cell-enriched population. Consistent with this
observation, miR205 over-expression in the mouse mammary epithelial cell (MEC) line
COMMA-DβGeo leads to an expansion of the Sca-1+ progenitor cell population,
decreased cell size, increased cellular proliferation and colony-forming potential. Based
upon these results, we hypothesize that miR205 is required in mammary gland
development and stem cell maintenance. To test our hypothesis, we obtained a miR205
conditional knock-out mouse model from Dr. McManus lab in UCSF. The LacZ
fragment in the miR205 conditional allele allowed us to study miR205 expression
pattern by X-gal staining. To study the functional results of miR205 loss, we utilized
adenovirus-cre to induce miR205 deletion ex vivo and performed several in vitro and in
vivo assays to evaluate the effect of losing miR205 on mammary stem cell population.
We also collected WT and miR205-/- basal/stem cell-enriched population and
performed RNA-sequencing to identify potential targets of miR205. Finally, we
performed ChIP-qPCR to evaluate the regulation of p63, the well-known stem cell
regulator, on miR205 transcription. Our results showed that miR205 was predominantly
expressed in the basal/stem cell-enriched population. Loss of miR205 severely
impaired the stem cells repopulating potential and also resulted in several cellular and
stromal changes. Primary RNA-seq result analysis indicated that miR205 may affect
mammary stem cell population through canonical/non-canonical Wnt pathways and
hippo pathway. Thus, our finding suggests a pivotal role of miR205 in maintaining the
mammary gland stem cell potential. Unraveling miR205’s function in normal
development will further help us to study its role in promoting tumorigenesis, possibly
through regulating the function of cancer stem cells.
Contributors: Lu, Yang; Rosen Jeffrey
203
COLONIZATION WITH BIFIDOBACTERIUM DENTIUM ALTERS BEHAVIOR AND
SYNAPTIC PLASTICITY-RELATED GENE EXPRESSION IN THE CENTRAL
NERVOUS SYSTEM OF AN ADULT GERM-FREE MOUSE MODEL
Berkley Luk
Background: Accumulating data indicates that certain members of the gut
microbiota communicate with the host central nervous system (CNS). Bifidobacterium
species are known to produce many neuroactive metabolites, and previous studies
have demonstrated that bifidobacteria can alter CNS gene expression, neurotransmitter
function, and behavior in adult rodents. In humans, bifidobacteria are detectable within
the first week after birth and are the predominant genus of the infant intestinal
microbiota. Given that Bifidobacterium spp. colonize the human system during a critical
period of neural circuit development and organization, the Bifidobacterium-gut-brain
interaction may have pervasive and lasting effects on brain function and behavior.
However, we currently lack detailed knowledge regarding which CNS genes are altered
by the presence of these species, and how modulation of these genes may affect
neurodevelopment. We hypothesized that colonization with a representative
Bifidobacterium species (Bifidobacterium dentium) would alter CNS expression of
synaptic plasticity-related genes and behavioral phenotypes. Method & Results: Germfree mice were utilized to examine the effects of B. dentium mono-association. Adult
male and female germ-free mice were colonized with B. dentium by oral gavage. Agematched Specific Pathogen Free (SPF) mice that had a complex murine gut
microbiome were also tested for comparison. We found that B. dentium colonization
resulted in alterations to intestinal morphology, increased mucus production, and
increased luminal concentrations of serotonin in the ileum. Due to its simple,
conserved, and well-defined circuit, the cerebellum was used as a model to study how
the microbiota alters gene expression and circuit development. Cerebellar gene
expression was quantified via qRT-PCR. Significant expression differences were
observed in 10 of 30 synaptic plasticity-related genes tested. Mono-association with B.
dentium resulted in a gene expression profile that resembled that of SPF mice for many
of the genes tested, suggesting a rescue effect. Based on the qRT-PCR results, 5
genes of interest were selected to examine via RNA In-Situ Hybridization to further
quantify and localize expression differences in whole brain sections. Finally, three
behavioral testing paradigms were utilized to assess motor and non-motor behavioral
differences between the groups. We observed increased stereotypic digging behavior in
the B. dentium-colonized mice, as assessed by the marble burying test. Conclusion:
Together these data demonstrate that mono-association of germ-free mice with B.
dentium alters intestinal morphology and neurochemical concentrations, as well as CNS
gene expression which may correspond with the observed behavioral changes.
Contributors: Luk, Berkley; Versalovic, James
204
SUBUNIT ARRANGEMENT OF CHAPERONIN TRIC IN ITS APO STATE
Boxue Ma
In eukaryotic cells, group II chaperonin TCP-1 ring complex (TRiC) assists
protein folding in the presence of ATP. TRiC is a hetero-oligomeric complex containing
two rings sitting back to back. Each ring consists of eight homologous subunits, known
as CCT1-CCT8. The eight subunits have varies affinity to ATP and substrate,
suggesting the unique heterogeneity plays an important role in the interaction between
TRiC and its substrate.
TRiC undergoes large conformational change upon ATP binding and hydrolysis,
from asymmetric apo state to closed state with pseudo 8-fold symmetry. Previous study
has identified each CCT subunit in the closed state, thus determined the ordering of the
eight subunits. However, earlier structural studies failed to identify the subunits in the
apo state due to its intrinsic flexibility, which limited the resolution.
Because there is a 2-fold symmetry between the two rings, there are only two
possible models for the apo state, and both had been proposed with biochemical
evidence. The apo state TRiC has an asymmetric feature where one of the subunit is
protruding outward. One of the apo state model assigned the protruding subunit as
CCT1 and the other model assigned it CCT7.
To eliminate the controversy, we studied the structures of two recombinant
TRiCs with GFP tag fused onto subunit CCT6 (CCT6-GFP-TRiC) or CCT1 (CCT1-GFPTRiC) by cryo electron microscopy. At median resolution, single particle reconstruction
of each apo state GFP-TRiC allows the recognition of the subunit that attached the
GFP tag. The 10Å structure of CCT6-GFP-TRiC and the 12Å structure of CCT1-GFPTRiC achieved in this study both agree with the first model above.
The GFP labeled TRiC allowed for the first time to recognize CCT subunits in the
apo state structure of TRiC unambiguously. It can also be used in future structural
studies of the TRiC-substrate complexes where the GFP tag will help identify which
CCT subunits are associated in the interaction between TRiC and each substrate.
Contributors: Gestaut, Dan; Roh, Soung-Hun; Frydman, Judith; Chiu, Wah
205
HEPATIC GLYCOGEN ACCUMULATION AND LIVER DYSFUNCTION IN ASL
DEFICIENCY
Simran Arun Madan
Argininosuccinate lyase (ASL) is a urea cycle enzyme responsible for conversion
of argininosuccinate to arginine and fumarate. ASL deficiency (ASLD) is an autosomal
recessive disorder. Neonatal symptoms of ASLD include hyperammonemia, vomiting,
lethargy and seizures. Despite early intervention, some patients develop long-term
complications including liver dysfunction, neurocognitive deficits, developmental delays
and hypertension. Liver pathology in patients with ASLD may include swollen
hepatocytes, glycogen accumulation, fibrosis and cirrhosis, and liver transplantation
may eventually be required in some patients. The ASLneo/neo hypomorphic mouse
serves as a good model for evaluating liver dysfunction in ASLD because we have
discovered that these mice, like the human patients, have hepatomegaly, elevated liver
enzymes, and hepatic glycogen accumulation. To further evaluate this glycogen
accumulation, we evaluated the activity of hepatic enzymes involved in glycogen
metabolism and discovered that ASL deficient mice have decreased activity of hepatic
glycogen phosphorylase, an enzyme responsible for breakdown of glycogen. Thus, we
hypothesize that this decreased activity of glycogen phosphorylase may explain the
hepatic glycogen accumulation and may contribute to liver dysfunction observed in mice
and humans with ASLD. Preliminary studies investigating the cause for decreased
hepatic glycogen phosphorylase activity revealed similar hepatic RNA expression levels
of glycogen phosphorylase between ASL deficient and wild type mice but reduced
protein levels in ASL deficient mice. We are now investigating post-translational
modifications on glycogen phosphorylase. In particular, we have shown that ASL is
required for nitric oxide production and that loss of ASL leads to a global decrease in
protein nitrosylation in several tissues, including the liver. Interestingly, hepatic
glycogen phosphorylase is one protein that is differentially nitrosylated in the liver of
ASL deficient vs. wild type mice. To address whether loss of nitrosylation of glycogen
phosphorylase contributes to the hepatic glycogen accumulation in ASLD, we are
testing if supplementation with a nitric oxide donor increases the activity of glycogen
phosphorylase in vitro and in vivo and whether this results in improvement in the
hepatic glycogen accumulation or liver dysfunction phenotype in ASL deficient mice.
Studies evaluating the mechanism underlying hepatic glycogen accumulation and liver
dysfunction in this mouse model of ASLD may potentially lead to the discovery of new
therapeutic strategies for liver dysfunction in human patients with ASLD.
Contributors: Madan, Simran*; Burrage, Lindsay*; Paschalis, Doulias; Cela, Racel; Jiang, Ming
Ming; Chen, Yuqing; Bertin, Terry; Ischiropoulos, Harry; Finegold, Milton; Lee, Brendan.
*Contributed equally to this work
206
THE ROLE OF PSTAT3 IN THE ANTIGEN-PRESENTING CELLS ASSOCIATED
WITH SMOKE-INDUCED LUNG DISEASE
Matthew Cameron Madison
Advisor: Farrah Kheradmand, M.D.-Department of Medicine
Anita Sabichi, M.D.-Department of Medicine
Background and Translational Relevance: Cigarette smoke is a well-defined
factor contributing to the incidence of pulmonary diseases worldwide. Smoke-induced
inflammatory lung diseases are among the leading causes of death in the U.S., and
they serve as primary risk factors for the development of other chronic diseases, such
as lung cancer. Given the prevalence of smoke-associated disease and the limited
treatment options currently available, a strong urgency exists to better understand the
complex molecular mechanisms underlying smoke-induced lung inflammation and its
ability to promote carcinogenesis.
Objective: Inflammatory, lung antigen-presenting cells are recruited to the lung
upon exposure to cigarette smoke and orchestrate pathogenic Th1 and Th17
responses which promote tissue destruction and disease persistence. Consequently,
we seek to better understand the molecular mechanisms by which these lung APCs are
manipulated in smoke-induced microenvironments by studying a protein that is
important to both immune cell function and lung inflammation, signal transducer and
activator of transcription 3 (STAT3). STAT3 is critical in inflammatory diseases as it is a
downstream target of multiple inflammatory cytokines, including interleukin 6 (IL-6).
However, its role in the function of APCs is not well understood. Cytokines associated
with smoke-induced lung inflammation (i.e. IL-6) signal through STAT3-associated
pathways and are known to alter immune cell function. Therefore, we hypothesize that
this protein is a key molecular factor harnessed to alter APC phenotype and aid in
driving lung disease.
Design: Using SH2 domain inhibitors of STAT3 along with genetic ablation
strategies (shRNA), we will determine the functional significance of STAT3 in lung
antigen-presenting cells and assess how the protein is employed by smoke-initiated
cytokine environments to alter APC identity and drive pathogenic T lymphocyte
responses.
Results: Preliminary data reveals that treatment with our STAT3 inhibitor during
smoke exposure reduces phosphorylation of STAT3 and mitigates the production of key
soluble factors by APCs (IL-6, MMP9 and MMP12), which are known to drive the tissue
destruction associated with smoke-induced emphysema. Further, we have
demonstrated that treatment of inflammatory CD11c+ APCs with the inhibitor ex vivo
reduces expression and secretion of IL-6.
Contributors: Madison, Matthew; McMurray, John; Kheradmand, Farrah
207
ESTABLISH HIGH RESOLUTION NK CELL PHENOTYPING AND IDENTIFY
DIFFERENCES BETWEEN HEALTHY CHILDREN AND ADULTS
Sanjana Mahapatra
Background: Natural killer (NK) cells comprise 5-20% of peripheral lymphocytes
and recognize virally infected cells or tumor cells without prior antigen sensitization.
They possess a wide array of germline-encoded activating and inhibitory receptors. The
balance of these receptors in addition to their adhesion ligands and developmental
stage determine the two main effector functions of NK cells - cytotoxicity and cytokine
release.
Rationale: NK cell percentages in the peripheral blood are known to vary with
age. The percentage of NK cells is high at birth, then goes down from 5 to 9 months of
age and then rises until 16 years of age after which it goes back down. While NK cells
originate and primarily develop in the bone marrow, further maturation in the peripheral
blood is not well understood. Therefore, in order to understand the maturation process,
we need to distinguish the expression of NK cell receptors and corresponding function
in the peripheral blood of healthy children and adults.
Methods: We have used multi-dimensional flow cytometry to determine the
phenotype and function of NK cells in the peripheral blood of 20 healthy adults (>21
years) and 25 healthy children (5-21 years). Our five extensive multicolor flow cytometry
panels each encompass NK cell activation state, inhibition state, adhesion properties,
developmental stage and cytokine profile.
Results: Using our approach, we have established high resolution NK cell
phenotype and defined normative ranges in 20 healthy adults and 25 healthy pediatric
individuals. With the Spanning Progression Analysis of Density Normalized Events
(SPADE) analysis, we have also defined several unique NK subsets in both children
and adults.
Conclusion/ Relevance: The normative ranges in both children and adults display
great inter-individual variation for most subsets. Thus, the proposed work seeks to
reveal novel NK cell phenotypic characteristics across various ages. This has broad
clinical relevance since the findings from the study can be used to identify NK cell
phenotypic abnormalities seen in patients, to facilitate the diagnosis and treatment of
immunological disorders.
Contributors: Shearer, William; Orange, Jordan
208
MESH-1, A METAZOAN HOMOLOG OF BACTERIAL STRESS RESPONSE
MEDIATOR, IS A LYSOSOMAL REGULATOR AGAINST MULTIPLE STRESSES
Keng Hou Mak
Advisor: Jue Wang, Ph.D.-Department of Molecular & Human Genetics
Lysosomes play an important role in maintaining cellular homeostasis against
stress. As a center for degradation, the organelle resolves stress induced by a variety of
sources, such as xenobiotics, damaged organelles and misfolded proteins.
Understanding how lysosomal functions are regulated in response to stress is thus
crucial and has implications to diseases associated with lysosomal dysfunctions.
Using C. elegans as a model, my thesis project focuses on mesh-1, a gene
evolutionarily conserved among metazoans that is poorly characterized, but the
bacterial homolog of which is a master regulator of stress response. My study finds that
mesh-1 regulates tolerance to multiple stresses, including hydrogen peroxide, cold
stress, and an endoplasmic reticulum stress inducer tunicamycin. Expressed in
intestine and epidermis, the MESH-1 protein localizes to lysosomes and other
endolysosomal compartments. Supporting its role in regulating lysosomal functions,
mesh-1-mediated tunicamycin tolerance requires genes involved in lysosomal
degradation and lumen acidity. In addition, preliminary data indicate an increase in
lysosomal acidity in mesh-1 mutants. Since mesh-1 homologs in bacteria regulate
stress response through metabolizing highly phosphorylated nucleotide signals, current
work examines the role of mesh-1 in stress signaling. Altogether the data support a
model that mesh-1 is a lysosomal regulator important for stress tolerance. The study
may link lysosomal regulation to the highly phosphorylated nucleotide signals, the
existence and physiological functions of which are controversial in metazoans. It may
also suggest why mesh-1 family is conserved in metazoan genomes.
Contributors: Mak, Keng Hou; Wang, Meng; Jue Wang
209
A HIGH-THROUGHPUT FLUORESCENCE MICROSCOPY SCREEN TO IDENTIFY
PROTEINS ASSOCIATED WITH RNA FOCI IN MYOTONIC DYSTROPHY
Kathleen Seger Manning
Advisor: Thomas Cooper, M.D.-Department of Pathology & Immunology
Myotonic dystrophy type I (DM1) is caused by an unstable CTG repeat
expansion in the 3’ UTR of the muscle kinase gene DMPK. The expanded CTG repeat
is transcribed into CUG expanded repeat (CUGexp) RNA transcripts, which are retained
in the nucleus and aggregate into distinct foci. CUGexp RNA foci sequester
Muscleblind-like (MBNL) family splicing factors and alter the activity of other RNA
binding proteins, leading to misregulated post-transcriptional gene regulation for
multiple genes. These CUGexp RNA foci are a characteristic feature of DM1, yet the
full composition of CUGexp RNA foci is unknown. We have designed a high-throughput
immunofluorescence-based assay to objectively screen for proteins associated with
CUGexp RNA foci. Given that RNA foci in DM1 are known to associate with the core
nuclear matrix, we immunized mice with purified DM1 fibroblast nuclear matrix extracts
to generate a monoclonal antibody (mAb) library enriched for foci-associated proteins.
This mAb library was robotically screened in high-throughput fluorescence microscopy
to identify mAbs that co-localize in characteristic nuclear foci in DM1 cells. Although no
mAbs co-localized strongly with DM1 RNA foci, we identified mAbs with unique staining
patterns that could be of interest in future DM1 studies. This project also provides a
framework for future studies of aggregate composition in other repeat expansion
disorders.
Contributors: Manning, Kathleen; Cooper, Thomas
210
VAPB, A PROTEIN IMPLICATED IN ALS, IS CLEAVED, AND THE SECRETED
MOIETY AFFECTS FGFR SIGNALING
Dongxue Mao
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease
that affects both upper and lower motor neurons, leading to muscle atrophy and
paralysis. VapB is one of the ~20 disease causing genes for ALS. Several mutations in
VapB (P56S, T46I, V234I) have been identified in ALS patients and VapB mRNA level
is decreased in the spinal cord of sporadic ALS patients. Therefore, loss of VapB may
contribute to ALS disease.
VapB is implicated in lipid transport, muscle mitochondrial dynamics and ER
stress. The N-terminal MSP domain of VapB is cleaved and secreted. Ectopically
expressed VapBP56S aggregates in the ER, leads to the elevation of ER stress and
affects MSP secretion in Drosophila and C. elegans. MSP is observed in most tissues
including spinal cord, cortex, cerebellum and muscle in mice. MSP cleavage is
decreased in both muscle and spinal cord of SOD1G93A mice, suggesting that
decreased MSP cleavage may contribute to ALS disease progression. Secreted MSP is
also detected in human blood serum and cerebrospinal fluid. MSP prevents the
degeneration of the motor neurons that are co-cultured with astrocytes derived from
SOD1G93A mice, suggesting that MSP may activate signaling pathways that are
beneficial to motor neuron survival. Addition of recombinant MSP in HEK293 cells
induces tyrosine phosphorylation of RTKs (receptor tyrosine kinases) and loss of VapB
in Drosophila reduces AKT phosphorylation, suggesting that MSP controls cellular
signaling through modulating RTK phosphorylation. Partial loss of function mutations of
VapB (P56S) exhibit an age-dependent climbing defect and reduced life span that are
severely exacerbated by the loss of FGFR (fibroblast growth factor receptor, Heartless),
a RTK.
In summary, the neuronal or muscular degeneration correlates with loss of
function of VapB, or defect in MSP secretion, and addition of MSP exerts a beneficial
effect on neuronal survival. We are currently exploring the mechanisms by which MSP
functions as a hormone by modulating RTKs and assess the role of this pathway in
ALS.
Contributors: Mao, Dongxue; Lin, Guang; Tepe, Burak; Bellen, Hugo
211
REASSEMBLY OF EXCITABLE DOMAINS AFTER CENTRAL NERVOUS SYSTEM
REGENERATION
Miguel Alejandro Marin
Myelinated axons are divided into distinct excitable domains, including the axon
initial segment (AIS) and nodes of Ranvier. The AIS serves as both a physical barrier
between the axonal and somato-dendritic compartments of the neuron and as the site
of action potential (AP) initiation. Nodes of Ranvier are responsible for the rapid and
efficient propagation of APs along the axon. Disruption of the AIS or nodes of Ranvier
by injury or disease has a dramatic impact on neuronal function. Thus, any therapeutic
strategy aimed at nervous system repair or regeneration must include the
reestablishment or maintenance of these excitable domains. Myelinated axons of the
adult mammalian central nervous system are incapable of self-regeneration after injury.
However, intravitreal administration of proinflammatory glucans as well as deletion of
the PTEN gene in retinal ganglion cells (RGC) after injury to the optic nerve allows for
substantial long distance growth of axons. When these treatments are combined with
elevation of cAMP, some regenerating axons can grow past the optic chiasm and
reinnervate the dorsal lateral geniculate, suprachiasmatic nucleus, and other central
target areas. Using this model, we performed a detailed analysis of the dismantling and
reorganization of the AIS and nodes of Ranvier. In the absence of any treatment,
immunofluorescence reveals a dismantling of the excitable domains within 48 hours of
the crush. Loss of nodes of Ranvier is detectable by 6 hours after crush in the
immediate vicinity of the crush site, and across the entire optic nerve within a week of
the crush. Analysis of the PTENf/f+Zymosan+cAMP regeneration model by both
immunofluorescence and electron microscopy (EM) demonstrates the reestablishment
of excitable domains and remyelination of regenerating axons in the optic nerve.
Analysis by immunofluorescence revealed the reestablishment of RGC-AIS in the retina
6 weeks after crush. We also observed growth of axons and reestablishment of nodes
of Ranvier proximal, distal and within the crush site of the optic nerve 6-12 weeks after
crush. Furthermore, EM analysis of regions close to the chiasm shows remyelination of
regenerating axons 12 weeks after lesion. Thus, regenerating axons are capable of
reestablishing excitable domains and undergoing remyelination, supporting the
possibility of efficient signal conduction.
Contributors: Marin, Miguel; de Lima, Silmara; Gilbert, Hui-Ya; Martinez, Ana; Benowitz, Larry;
Rasband Matthew
212
IDENTIFICATION OF NOVEL DRIVERS OF MAMMARY TUMORIGENESIS USING
AN RCAS-TVA MOUSE MODEL
Jarrod Don Martinez
Advisor: Jianming Xu, Ph.D.-Department of Molecular & Cellular Biology
The tumor suppressors Pten and P53 are commonly lost in malignant breast
cancers. Specifically Pten is lost in 25-30% of triple negative breast cancers (TNBCs).
While P53 is lost in approximately 80% of TNBCs. Furthermore, it has been reported
that Pten and P53 are inactivated together in approximately 10-20% of TNBCe. The
effects of combined inactivation of both Pten and P53 are poorly understood. To date
few studies have examined the effects of P53 and PTEN dual deletions. One important
insight of these studies is that there appears to be a selective pressure to deactivate
P53 when Pten is lost. Our model system will use the RCAS-CRE/TVA system from
which we will be able in infect mammary epithelial cells, subsequently causing the
deletion of both P53 and PTEN alleles and the cleavage of the RFP to GFP reporter.
Sequential genomic profiling of the cancers pathological progression from initiation to
distant metastases will allow us to obtain a comprehensive view of the genomic
changes that occur throughout the course of the disease. Despite the heterogeneity of
mutations an genomic alterations that accompany cancer progression, we hypothesize
that there are certain genomic changes that must occur sequentially during the
progression of the disease that allows for BC cells to initiate progress, and metastasize
to distant organs. To test this hypothesis we will use the following aims: Aim 1: To
characterize and profile the malignant progression of the resulting Pten and P53 null
tumors from our novel TVA+ R/G+ PTENf/f P53f/f mouse model. Aim 2: To determine
the genomic changes at DNA/RNA levels that occur after the deletion of Pten and P53
in various stages of tumorigenesis and metastasis of the transformed mammary
epithelial cells. The understanding of the genomic changes and molecular mechanisms
regulating the various stages of tumorigenesis and metastasis is essential in the
development of high risk management and new therapies for patients.
Contributors: Martinez, Jarrod; Xu, Yixiang; Qin, Li Ph.D.; Li, Yi Ph.D.; Xu, Jianming Ph.D.
213
EXTRACELLULAR MECHANISMS OF TFEB-MEDIATED CLEARANCE OF PTAU IN
TAUOPATHIES
Heidi Martini-Stoica
Joshua Shulman, M.D./Ph.D.-Department of Neurology
Presently, tau-targeting therapies to treat Alzheimer’s disease (AD) are limited by
efficacy and safety. To date, the majority of therapeutic trials have focused on Aβ
because of the strong genetic evidence linking mutations in APP and presenilins to
familial AD. However, tau-based neurofibrillary tangle (NFT) pathology correlates more
closely with cognitive decline than Aβ plaques. Therefore, targeting AD’s NFT pathology
may prove to be a complimentary, alternative strategy to Aβ directed AD therapies,
which up to this point have been generally unsuccessful. Thus, the long-term goal of
this work is to gain a better understanding of the cellular mechanisms of NFT clearance
in order to develop more effective therapies. The overall objective is to dissect the
multiple roles of transcription factor EB (TFEB), a critical regulator of lysosomal
biogenesis, in clearing pathological forms of tau. The central hypothesis is that
astroglial TFEB expression enhances uptake and clearance of aberrant extracellular
tau, preventing the neuronal spreading of tau pathology, in addition to TFEB’s cellautonomous effect in enhancing the autophagy-lysosomal pathway (ALP). This
hypothesis has been formulated based on recent work demonstrating the therapeutic
potential of TFEB in ameliorating tau pathology in a mouse model of tauopathy. In
addition, TFEB is known to upregulate endocytosis genes and enhance
macropinocytosis of protein aggregates. With studies demonstrating the existence of
extracellular tau and cell-to-cell tau spreading, targeting extracellular tau will likely prove
crucial to halting disease progression. Thus, through TFEB-mediated enhancement of
uptake mechanisms and the ALP, astrocytes are prime candidates to take up and clear
pathologic extracellular tau, reducing overall tau pathology and preventing further
seeding of aggregation and spreading. The central hypothesis will be tested by
pursuing the following specific aims: 1) Determine the mechanistic effect of TFEB on
tau handling by astrocytes in vitro through measuring TFEB-mediated astrocyte uptake
and clearance of synthetic tau aggregates and soluble tau. 2) Determine the effect of
astroglial TFEB on tau pathology in vivo via astrocyte-specific TFEB overexpression in
tauopathy mouse models. Changes in overall tau pathology and cognitive behavior will
be assessed, in addition to the impact on the propagation of NFT-like neuronal tau
pathology. The successful completion of these aims will define the extracellular
mechanisms of TFEB’s action and provide valuable insight into the poorly defined role
of astrocytes in tauopathies. Clarification of the extracellular mechanisms of TFEBmediated pTau clearance is expected to identify novel therapeutic targets to guide the
development of new therapeutic strategies in the treatment of AD. Contributors: MartiniStoica, Heidi; Cole, Allysa; Swartzlander, Daniel; Mitchell, Patrick; Zheng, Hui
214
MYD88/CD40-BASED INDUCIBLE CO-STIMULATION TO IMPROVE CAR T-CELL
THERAPY
Melinda Mata
BACKGROUND: Adoptive immunotherapy with genetically modified T cells holds
promise in improving outcomes for cancer patients. While a broad array of genetic
modification strategies are being explored, few allow for the specific manipulation of
adoptively transferred T cells in vivo. One successful example includes the introduction
of an inducible 'suicide gene' to enable selective T-cell killing in the event of toxicities.
Given the limited antitumor activity of adoptively transferred T cells for solid tumors in
early clinical studies, we reasoned that introducing an inducible co-stimulatory molecule
into T cells would allow for the selective activation of adoptively transferred T cells in
vivo resulting in enhanced antitumor activity. Due to the role of MyD88 and CD40
signaling pathways to fine tune T-cell activation, and the recent success of using
inducible (i) MyD88 and CD40 molecules to activate antigen-presenting cells, the goal
of this project was to explore if T cells can be activated with iMyD88 and/or iCD40
molecules.
METHODS/RESULTS: We constructed a panel of retroviral vectors encoding
mOrange as a marker gene and an inducible co-stimulatory molecule (iCO-STIM)
consisting of a myristoylation tag, two FKBP dimerizer domains, and i) MyD88, ii) CD40,
or iii) MyD88 + CD40. T cells expressing iMyD88, iCD40, or iMyD88.CD40 were
generated by retroviral transduction, and transduction was confirmed by FACS analysis
and Western blot. T cells expressing iCO-STIMs were activated with the CD3
monoclonal antibody OKT3 in the presence or absence of the chemical inducer of
dimerization (CID), AP20187. iMyD88.CD40 T cells secreted the highest amount of IL2
in the presence of OKT3 + CID in comparison to iMyD88 or iCD40 T cells. To evaluate
if activating iMyD88.CD40 in CAR T cells also enhances cytokine production, we
generated T cells that expressed HER2-CARs and iMyD88.CD40 (HER2-CAR/iCOSTIM T cells). CID enhanced IL2 production of HER2-CAR/iCOSTIM T cells that were
either stimulated with recombinant HER2 protein or HER2+ cell lines. Enhanced IL2
production was observed with 1st, 2nd, and 3rd generation HER2-CARs.
CONCLUSION: We have generated CAR T cells with an inducible co-stimulatory
molecule based on MyD88 and CD40. Preliminary functional analysis of CAR/iCO-STIM
T cells is encouraging, warranting further active exploration of this approach to improve
current T-cell therapy approaches for cancer.
Contributors: Gerken, Claudia; Spencer, David; Gottschalk, Stephen
215
A NEXT-GENERATION TRANSPOSON MUTAGENESIS TOOLSET FOR FORWARD
GENETIC SCREENING
Nick Matinyan
Advisor: Koen Venken, Ph.D.-Department of Biochemistry & Molecular Biology
Forward genetic screens are powerful approaches in the geneticist’s tool box for
conducting large scale, unbiased genomic analyses. Such screens have been carried
out using one of three mutagenesis methods: radiation, chemical mutagenesis, or
transposition. While chemicals are the most efficient of the three, obtained lesions are
difficult to map, isolate and validate. Transposons offer a genetically encoded
alternative that can be readily controlled to create a diverse array of mutations.
Moreover, resulting insertion sites are easily identified using standard molecular
methods. However, transposon insertions are not random due to insertion site
preference, therefore limiting genome coverage. Additionally, while somatic transposon
mutagenesis has been successfully employed in cancer modeling using mice and
zebrafish, transposon-based models have not been applied to other diseases or
phenotypes. Neither have such approaches been demonstrated in any other species.
Identifying candidate genes underlying disease and mutant phenotypes is critical for
understanding disease initiation, progression, and, potentially, the development of more
effective therapeutics, but systematic identification of candidate genes across different
species is currently impossible. Hence, a new generation of high-throughput genetic
tools able to screen genomes across species in an unbiased manner is highly desired.
Here, we propose a novel transposon-based mutagenesis toolset as the foundation of a
highly dynamic forward genetic screening technology. We propose, three
complimentary approaches as part of this toolset that each seek to maximize
transposon mutagenesis for the discovery of candidate genes in disease and mutant
phenotypes. Through this work, we aim to significantly improve current transposon
mutagenesis technology using Drosophila melanogaster as a model system, ultimately
extending efficient transposon-based high-throughput genetic screening towards any
phenotype in any species.
Contributors: Matinyan, Nick; Sarrion-Peridognes, Alejandro; Venken, Koen
216
MUS81 AND CONVERGING FORKS LIMIT MUTAGENESIS AT BROKEN
REPLICATION FORKS
Ryan Mayle
Advisor: Grzegorz Ira, Ph.D.-Department of Molecular & Human Genetics
DNA breaks are toxic to cells and must be repaired for cells to survive. Most
spontaneous DNA double strand breaks result from replication fork breakage. Breakinduced replication (BIR), an error prone repair mechanism characterized by high rates
of mutations and template switch mediated rearrangements that requires the
Pol32/POLD3 subunit of eukaryotic DNA Polδ, was proposed to repair broken forks.
Whether BIR contributes to broken fork repair, and if so how genome destabilization is
avoided, was unknown. To study the mechanism and fidelity of fork breakage repair, we
employed an inducible Flp-nickase mediated broken fork repair assay in
Saccharomyces cerevisiae.
We show that, while BIR is not required for broken fork repair, initial synthesis is
error-prone and mediated by Pol32. However, mutagenic synthesis is limited to within a
few kilobases from the break by the endonuclease Mus81 and a converging fork.
Therefore, we conclude that BIR is not the primary mechanism of repair at broken forks.
Instead, we propose that repair is mediated by Mus81, likely via cleavage of the D-loop
to form a Y-fork structure and prevent further BIR, and replication can be completed by
converging forks. We find that Mus81 suppresses template switches between both
homologous sequences and diverged human Alu repetitive elements, highlighting its
importance for stability of highly repetitive genomes. We predict that lack of a timely
converging fork or Mus81 may propel genome instability observed in cancer, and
possibly promote tumor progression.
Contributors: Mayle, Ryan; Campbell, Ian; Beck, Christine; Yu, Yang; Wilson, Marenda; Shaw,
Chad; Bjergbaek, Lotte; Lupski, James; Ira, Grzegorz
217
DYSREGULATION OF PURINERGIC SIGNALING IS ASSOCIATED WITH POOR
RECURRENCE-FREE SURVIVAL IN PATIENTS WITH HEPATOCELLULAR
CARCINOMA
Janielle P Maynard
Advisor: Sundararajah Thevananther, Ph.D.-Department of Pediatrics
John Goss, M.D.-Department of Surgery
Background: Hepatocellular carcinoma (HCC) is the 2nd most lethal cancer
worldwide. Recent studies suggest that extracellular ATP-mediated activation of P2
purinergic receptors induce hepatocyte proliferation in vitro and P2 purinergic receptors
are overexpressed in certain cancer tissues. However, the pathophysiologic relevance
of purinergic signaling in HCC remains unknown. Hypothesis: Dysregulation of
purinergic signaling facilitates aberrant cell proliferation underlying hepatocellular
carcinogenesis.
Methods: Human livers (HCC, n=42; normal, n= 6), mouse livers (Mst1/2-/(hippo), WT - 1, 3, & 6+ months) and human derived HCC cell lines (Huh7, Hep3B,
SNU387, PLC/PRF/5) were analyzed by qRT-PCR for all 15 P2 purinergic receptor
isoforms. Immunohistochemistry was performed on select patient liver tissues. An
independent HCC patient cohort (n=188) was used for gene expression profiling. HCC
cells, maintained in serum free media for 24h, were treated with ATP(S, ATP or ADP +/SP600125 (c-Jun N-terminal Kinase inhibitor) or AF-353 (P2X3 antagonist). Western
blotting, qRT-PCR and BrdU incorporation analysis were done.
Results: Multiple P2 purinergic receptor isoforms were elevated ≥2-fold in
patient liver tumors compared to uninvolved areas in up to 60% of patients. High P2X3
or low P2Y13 receptor expression was associated with poor recurrence-free survival.
Dysregulation of P2 purinergic receptor expression was observed in HCC cell lines.
Extracellular nucleotide treatment alone was sufficient to induce cell cycle progression
in Huh7 cells, evidenced by increased BrdU incorporation and increased cyclin D3, E,
and A mRNA and protein expression. JNK inhibition attenuated nucleotide-induced
cyclin protein expression. Extracellular ATP-mediated activation of P2X3 promoted
proliferation in HCC cells. Interestingly, ATP(S treatment induced a distinct
downregulation of cyclin D1 expression in Huh7 cells, which was associated with poor
prognosis in HCC patients. Mst1/2-/- mouse tumors exhibit dysregulated expression of
multiple P2 purinergic receptor isoforms as compared to WT while nucleotide treatment
of Huh7 cells induced hippo target genes implicated in HCC pathogenesis.
Conclusions: Our analysis of HCC patients, Mst1/2-/- livers and HCC cells in
vitro identifies a novel role for dysregulation of P2 purinergic signaling in the induction of
a hyper-proliferative HCC phenotype and identifies P2X3 purinergic receptors as
potential new targets for therapy.
Contributors: Maynard, Janielle; Johnson, Randy; Lee, Ju-Seog; Sohn, Bo Hwa; LopezTerrada, Dolores; Finegold, Milton; Goss, John; Thevananther, Sundararajah
218
INVESTIGATION OF HOST GENETICS FACTORS THAT INFLUENCE THE HUMAN
NASOPHARYNGEAL MICROBIAL COMMUNITY
Tyler Jordan McCue
Since the completion of the Human Microbiome Project there have been
numerous studies investigating the links between the resident microbiota and host
health. While there have been great advances in understanding how microbial
communities can influence human health, there has been far less work into
investigating how host genes can influence what bacteria inhabit various body sites. To
investigate the link between host genetics and the resident microbial community of the
nasopharyngeal cavity, nasal swabs were collected from over 200 individuals for 16S
RNA gene sequencing analysis. Individuals of this cohort have previously had DNA
collected and analyzed using the Affymetrix 6.0 Human SNP Array. To expand the
SNP coverage to 37 million sites across the genome SNP imputation was performed
using VAAST and PLINK analysis tools. Identification of the microbial communities
present in the study subjects was analyzed using QIIME 1.7 in order to determine the
presence or absence of specific taxa. Then using a multi-omics approach, the human
genetic data and resident microbial data were combined to identify potential human
genes that may be influencing what bacteria reside in the nasopharyngeal cavity.
Preliminary data from this study has shown potential links between presence of the
Bacillus genus and SNPs involved in glucose homeostasis. The findings of this study
will help lead to the identification of host markers that influence microbial communities
that in turn can alter an individuals risk of various disease states.
Contributors: McCue, Tyler; Below, Jennifer; Bryson, Rebecca; Ajami, Nadim J.; Hanis, Craig
L.; Brown, Eric L.; Petrosino, Joseph F.
219
THE ROLE OF NR4A ORPHAN NUCLEAR RECEPTORS IN NPM1C+ ACUTE
MYELOID LEUKEMIA
Catherine Teresa McNamara
Acute myeloid leukemia (AML) develops from mutations at the stem or
progenitor level during hematopoiesis, which leads to the generation of malignant
leukemia initiating cells (LICs). LIC progeny are incapable of normal myeloid
differentiation, resulting in immature blast accumulation. AML is very heterogeneous
due to diversity in the LIC cell of origin and the large number of recurrent mutations,
making it difficult to develop targeted treatments against this disease. Genomic
sequencing performed by The Cancer Genome Atlas recently identified a set of
recurrently mutated genes in AML. One of the most frequent is the nucleophosmin
(NPM1) gene, which is associated with a normal karyotype and is mutated at its Cterminus (NPM1c+) in 27% of AML patients. NPM1 functions as a homodimer to
regulate DNA repair, transcription and apoptosis. Heterozygous NPM1c+ mutants
dimerize with wildtype NPM1 (and other proteins such as p19Arf) causing its abnormal
retention in the cytoplasm and disruption of its normal function. The molecular
mechanisms underlying NPM1c+ AML development remain poorly understood, and
targeted strategies for therapy are lacking. Our lab discovered that NR4A disruption in
mice causes AML and that NR4A expression is silenced in leukemic blasts from AML
patients with distinct cytogenetic backgrounds. Furthermore, NR4A rescue establishes
a core tumor suppressive gene signature and decreases human AML cell viability,
suggesting that the NR4As are tumor suppressors and novel therapeutic targets.
Although NR4A expression is silenced in NPM1c+ AML, their functional role as tumor
suppressors in this subset of AMLs has not been addressed. Our preliminary data show
that NR4A1 can interact with NPM1 in mouse myeloid progenitor cells, but the role of
this interaction is unknown. Our goals are to: 1) Examine the cellular consequences of
the NPM1c+ interaction on the NR4As’ tumor suppressive capabilities, 2) Examine the
molecular consequences of the NPM1c+ interaction on NR4A localization and
expression of the NR4A dependent gene signature, 3) Use small molecule activators of
the NR4As to inhibit NPM1c+ cell viability and AML development in NPM1c+ mouse
xenograft models.
Contributors: McNamara, Catherine; Conneely, Orla
220
CLINICALLY OBSERVED VARIANTS OF KPC-2 β-LACTAMASE TRADE-OFF
INCREASED CEFTAZIDIME HYDROLYZING ABILITY WITH DECREASED
STABILITY
Shrenik Chetan Mehta
The rise in Carbapenem-resistant enterobacteriaceae (CRE) infections has
resulted in increased mortality rates and healthcare costs. The production of βlactamase enzymes such as Klebsiella pneumoniae carbapenemase-2 (KPC-2) is the
primary mechanism of resistance in CREs. KPC-2, a class A β-lactamase can
hydrolyze penicillins, cephalosporins and carbapenems but not cefamycins and
ceftazidime. Thus, KPC β-lactamase is a threat to public health as it confers multi-drug
resistance. A number of KPC-2 variants have been recently identified all over the
world. We investigated the evolution of KPC-2 function among nine clinically isolated
variants by examining the effects of the substitutions on the antibiotic resistance profile
and antibiotic hydrolyzing ability. We also investigated the subsequent effect of the
mutations on protein stability. We show that the amino acid substitutions associated
with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime
hydrolysis accompanied by an in vivo increase in ceftazidime resistance. The increase
in catalytic efficiency for ceftazidime hydrolysis and resistance to ceftazidime is gradual,
with double mutants displaying additively higher resistance as compared to single
mutants. The largest increase in ceftazidime hydrolysis and resistance was due to the
P104R, V240G and H274Y substitutions as single and double mutations. In silico
docking studies suggests that the increased catalytic efficiency of ceftazidime
hydrolysis may be due to formation of new hydrogen bonding interactions between
R104 and the oxyimino carboxyl of ceftazidime and Y274 with the amine functionality of
the aminothiazole ring of ceftazidime. Finally, the gain of catalytic function for
ceftazidime hydrolysis, correlated well with the loss of enzyme stability. Thus, as per
‘stability-function tradeoff’ phenomenon the gain of catalytic function in the KPC-2
variants is accompanied by a concomitant loss in enzyme stability.
Contributors: Mehta, Shrenik; Rice, Kacie; Palzkill, Timothy
221
DENDRITIC CELL RESPONSES TO PNEUMOCOCCAL VACCINES ARE IMPAIRED
IN HIV-INFECTED SUBJECTS ON ANTIRETROVIRAL THERAPY
Daniel Mendoza
Background. HIV+ subjects have high burden of invasive pneumococcal disease
(IPD). The pneumococcal vaccines, the 23-valent polysaccharide vaccine (PPV) and
the 13-valent polysaccharide conjugate vaccine (PCV), have reduced efficacy in HIV+
patients. PPV contains pure pneumococcal capsular polysaccharides (PS), and PCV
has PS conjugated to the protein carrier CRM197 to enhance immunogenicity. Dendritic
cells (DC) play essential roles in vaccine responses by producing cytokines. We
hypothesize that DC produce cytokines in response to pneumococcal vaccines and that
HIV impairs this DC function. Methods. We performed a clustering analysis of 41
cytokines produced in the supernatant of peripheral blood mononuclear cells (PBMC)
exposed to pneumococcal vaccines for 6 h ex vivo using a Luminex machine. PBMC
were from healthy unvaccinated adults. We exposed PBMC to PCV and PPV for 6 h to
measure production of the identified cytokines in DC using flow cytometry. We used
PBMC of HIV+ patients off antiretroviral therapy (ART), HIV+ patients on >6 months of
ART and healthy controls. We evaluated whether pre-vaccine DC production of
cytokines ex vivo predicted post-vaccine serum opsonophagocytic killing activity (OPA),
which is an in vitro assay that correlates with protection against IPD. Results. PPV and
PCV induced similar cytokine profiles consistent with an antibacterial innate response.
Among the cytokines produced were the chemokine IL-8, and IL-6, which induces
inflammation and B cell differentiation. DC produced IL-8 in response to pneumococcal
vaccines (median percent of DC producing IL-8: media: 5.1%, PPV 10%, PCV 23%,
p<0.001). These DC responses targeted the PS component in both vaccines and not
the protein carrier: median percent of DC producing IL-8: media: 11.1%, CRM197:
9.3%, PS: 21.2%, p=0.01; and of DC producing IL-6: media: 1.6%, CRM197: 2.2%, PS:
5.4%, p=0.02. DC production of cytokines against PCV was reduced in HIV+ subjects
(percent of DC producing IL-6: HIV+ subjects off ART: 0%, HIV+ subjects on ART:
0.7%; uninfected subjects: 17.5%, p<0.0001; percent of DC producing IL-8: HIV+
subjects off ART: 6%, HIV+ subjects on ART: 10%, uninfected subjects: 43%,
p<0.0001. Pre-vaccine DC production of IL-8 in response to PCV predicted OPA
against PS (serotype 6B) measured 1 month post-vaccination (r=0.48, p=0.001).
Conclusion. DC production of cytokines against pneumococcal vaccines is impaired in
HIV+ patients and this defect is not reversed by ART. This could explain why HIV+
patients have reduced vaccine responses. DC production of cytokines in response to
pneumococcal vaccines predicted post-vaccine OPA, highlighting the presence of a
mechanism that could be exploited to improve vaccine efficacy and reduce IPD among
HIV+ subjects.
Contributors: Mendoza, Daniel; De La Rosa, Indhira; Xu, Yi; Rodriguez-Barradas, Maria; Lewis,
Dorothy; Keitel, Wendy; Chen, Min; Corry, David.
222
MECP2 DEFICIENCY PARTIALLY DISABLES EXCITATORY GLUTAMATERGIC
NEURONS,LEADING TO SEVERE NEUROLOGICAL DYSFUNCTIONS
Xiangling Meng
Background: Rett Syndrome (RTT) is a postnatal neurological disorder caused
by loss of function mutations in the gene encoding methyl-CpG-binding protein 2
(MeCP2). Deleting Mecp2 only from brain tissue at embryonic day 12 leads to
phenotypes identical to those of the null mutation, indicating that loss of MeCP2 from
the CNS is responsible for the RTT phenotypes. Neuronal subtype-specific Mecp2
deletion replicates select aspects of the null phenotype. For instance, deletion of Mecp2
only from inhibitory GABAergic neurons recapitulates many RTT phenotypes including
the stereotypies and altered social interaction, but spares anxiety-like behaviors and
tremor. The role that excitatory glutamatergic neurons play in the pathogenesis of RTT
has not been explored in detail.
Method: We conditionally deleted or expressed Mecp2 only in glutamatergic
neurons in the mouse brain using a Vglut2-Cre line, and characterized the mice by a
comprehensive battery of behavioral assays as well as neurophysiological methods.
Results: The glutamatergic conditional knockout mice (CKO) became obese, and
developed impaired acoustic startle and motor deficits. Interestingly, unlike the
GABAergic CKO, the glutamatergic CKO showed anxiety-like behaviors as early as 5
weeks of age, and developed severe tremor. Furthermore, they died early with half of
them dead by 10 weeks of age. These phenotypes are identical to the disease
progression pattern of the Mecp2 null mutation. Patch-clamp recording from the layer V
pyramidal neurons revealed reduced spontaneous activity in the CKO mice, which
replicated the circuit deficit of null mutation. Expressing MeCP2 only in glutamatergic
neurons from an otherwise Mecp2 null background rescued most of the phenotypes
displayed in CKO mice.
Conclusion: These data demonstrate that dysfunction of MeCP2 in glutamatergic
neurons contributes to numerous neuropsychiatric phenotypes. Especially, it drives the
onset of anxiety-like behaviors, tremor, and obesity in RTT, indicating an excitatory
neuron-dependent mechanism underlying these phenotypes of Rett syndrome.
Contributors: Wang, Wei; Lu, Hui; Zoghbi, Huda.
223
THE STRINGENT STRESS RESPONSE IS REQUIRED FOR MUTAGENIC REPAIR
OF DNA BREAKS
Phillip Jay Minnick
Mutagenesis mechanisms controlled by stress responses increase mutation
rates during stress, potentially accelerating adaptation to stressors such as nutrient
limitation, antibiotics, antifungal drugs and hypoxia, in bacteria, yeast and human
cancer cells. Inhibiting these mechanisms could provide new ways to combat cancer
and infectious disease. One stress-induced mutagenesis mechanism in Escherichia
coli is mutagenic repair of DNA double-strand breaks (mutagenic break repair or MBR).
Under stress, or if the general (RpoS) stress response is activated artificially, repair of
double-strand breaks (DSBs) switches from use of the high fidelity DNA Pol III to use of
error-prone DNA Pol IV causing base-substitution and indel (“point”) mutations, or use
of Pol I causing copy-number variations. Mutagenic repair of DSBs causes new
mutations non-randomly both in time (under stress), and in genomic space (near
DSBs). Three stress responses promote MBR in E. coli: the envelope-protein stress
response promotes spontaneous DSB formation; the DNA-damage response
upregulates Pol IV (promoting point mutagenesis); and the general stress response
licenses use of Pols IV, II and I in repair. Here we demonstrate that a fourth stress
response must be activated for MBR to occur: the stringent (starvation) response. We
show that stringent-response-defective ∆relA or ∆dksA cells are defective for both point
mutagenesis and gene amplification. Moreover, the stringent response promotes MBR
by a mechanism other than activation of the other three stress responses. First, DksA is
still required for MBR if DSBs are given artificially with I-SceI enzyme. Thus, the
stringent response role in MBR is not in formation of DSBs, the role of the envelopeprotein-stress response. Second, the RelA/DksA role is not via activating the DNA
damage-response because stringent-response-defective relA or dksA cells are still
MBR defective if Pol IV is upregulated, the primary role of the damage response in
MBR. Third, mutations that allow general-stress-response-independent MBR do not
compensate for relA/dksA mutations in MBR, demonstrating a role of the stringent
response in MBR other than it’s known role of promoting general-stress-response
activation. We conclude that the stringent response constitutes a fourth, independent,
stress response input into MBR. The stringent response presumably regulates the
expression of a currently unknown protein(s), critical to MBR. Our data imply that cells
regulate mutagenesis very carefully, requiring four independent stress-response inputs
to unleash their mutagenesis “program”.
Contributors: Frisch, Ryan; L. Gibson, Janet; Terwilliger, Austen; McCue, Tyler; Darrow,
Michele; Herman, Christophe; Rosenberg, Susan
224
THE EFFECT OF HEPATITIS B VIRUS REPLICATION ON THE CULLIN 4A-RING E3
LIGASE COMPLEX
Marissa Mie-Kehaulani Minor
Advisor: Betty Slagle, Ph.D.-Department of Molecular Virology & Microbiology
Chronic infection with hepatitis B virus (HBV) affects over 400 million people
worldwide and is a significant risk factor for severe liver disease, including
hepatocellular carcinoma (HCC). HBV likely contributes to cancer by a complex
mechanism. Our research focus is on the cellular cullin 4A RING E3 Ubiquitin Ligase
(CRL4) pathway that functions to regulate protein levels by recruiting substrate proteins
for degradation. Several viruses encode proteins that bind to CRL4 to modify important
cellular functions including cell cycle, innate immunity, and apoptosis. Deregulation of
CRL4 is associated with cancer. HBV encodes the regulatory HBx protein that binds to
DDB1, a major adaptor protein of CRL4. DDB1 acts, in part, by binding and recruiting
cellular DDB1 Cullin Accessory Factor (DCAF) proteins to CRL4 that then selectively
recruit and ubiquitinate substrate proteins for degradation. The HBx protein is a viral
DCAF, and its binding to DDB1 is required for virus infection. However, the outcome of
the HBx-DDB1 interaction on CRL4 function remains unknown. We hypothesize that
binding of HBx to DDB1 alters the binding of cellular DCAFs within CRL4 and that this
benefits virus replication. Immunodeficient mice transplanted with human hepatocytes
(human liver chimeric mice) were infected with HBV, and viremia was monitored in
mouse serum by real-time PCR.
Uninfected chimeric mice, repopulated with
hepatocytes from the same human donor, were used as negative controls. Mice were
sacrificed at peak viremia and livers were analyzed by immunoprecipitation (IP) with
anti-CUL4A antibody followed by mass spectrometry (MS) analysis to identify cellular
proteins in the CRL4 complex. Four DCAFs were identified in uninfected livers
(DCAF8, DCAF11, CRBN, and PAFAH1B1), and the same four DCAFs were identified
in HBV-infected liver. In HBV-infected livers, DCAF11 was markedly reduced in the
complex compared to uninfected controls in two independent experiments. This
suggests that HBx expression during HBV replication may have displaced DCAF11
from CRL4. These results are being validated by IP followed by western blot in plasmid
transfected cells. To investigate the impact of HBx on DDB1-related function(s), cell
lines were created encoding doxycycline-inducible HBx that express either wildtype HBx
or point mutant versions of HBx that do not bind DDB1. Studies are in progress to
examine the impact of HBx expression on the cellular damaged DNA repair response.
Contributors: Minor, Marissa M.; Hollinger, F. Blaine; McNees, Adrienne L.; Jung, Sung Yun;
Slagle, Betty L.
225
A GPI-ANCHORED ANTI-HIV ENV SINGLE CHAIN FRAGMENT VARIABLE REGION
NEUTRALIZES HIV-1 AT BOTH EARLY AND LATE STEPS OF THE VIRAL LIFE
CYCLE
Anisha Misra
Advisor: Jason Kimata, Ph.D.-Department of Molecular Virology & Microbiology
Infection of memory CD4+ T cells by HIV-1 drives their progressive depletion and
the impairment of cellular immunity. Although combination antiretroviral therapy
dramatically improves survival of HIV-infected individuals, toxic side effects can occur
with use, and it fails to fully restore immune function or cure infection. Thus, the
development of alternative treatment strategies to eliminate the viral reservoir are of
high interest. T cell immunotherapy has been proposed as a way to restore immune
function and eradicate HIV in infected patients. However, protecting memory CD4+ T
cells from further infection will be necessary to maintain immunity. In previous studies,
we demonstrated that a single-chain variable fragment (scFv) from an anti-HIV-1 Env,
monoclonal antibody, X5, could function as potent entry inhibitor when attached to the
surface of target cells by a glycosyl-phosphitidylinositol (GPI) anchor. Surprisingly, even
if a virus escaped inhibition at entry, its replication was efficiently controlled. We
hypothesized that in addition to functioning as a fusion inhibitor, GPI-X5 scFv interacts
with Env in the infected cell, thereby interfering with viral infectivity. To test this
hypothesis, we cotransfected HIV proviruses with plasmids expressing GPI-X5 scFv or
a control GPI-scFv and analyzed the level of virus production, infectivity, and viral
protein expression. We found that the GPI-anchored X5 scFv restricted the production
of virions from cells. Furthermore, infectivity of the virions that were released was
decreased by 7-10-fold compared to control GPI-scFv. An analysis of Env expression
in cell lysates and virions demonstrated that processing of Env gp160 precursors in
cells was almost completely blocked in GPI-X5 scFv expressing cells, resulting in a
reduction in the amount of Env gp120 associated with virions. These data indicate that
GPI-X5 scFv has the ability to inhibit Env processing and function, thereby restricting
production and infectivity of HIV. Together, with our previous findings, we conclude that
GPI-X5 scFv is a unique anti-HIV molecule, as it derives its potent inhibitory activity
against HIV by interfering with both early (receptor binding) and late (Env processing
and incorporation into virions) stages of the viral life cycle.
Contributors: Gleeson, Emile; Couturier, Jacob P2; Lewis, Dorothy2; Zhou, Paul1; and Kimata,
Jason T
1 Institut Pasteur of Shanghai-Chinese Academy of Sciences; 2 The University of Texas
Medical School at Houston
226
THE STRUCTURE AND FUNCTION OF MICROTUBULE ASSOCIATED PROTEIN
TAU IN ALZHEIMER’S DISEASE AND OTHER TAUOPATHIES
Patrick Gerald Mitchell
Microtubule associated protein tau (MAPT, or tau) is implicated as an associated
factor in the pathology of Alzheimer’s Disease (AD), as well as several other
neurodegenerative disorders, collectively referred to as tauopathies. The disease
associated form of tau aggregates in paired-helical filaments (PHF), which interfere with
tau’s ability to bind and stabilize microtubules, and cause higher-order intracellular tau
aggregate formation. These aggregates, or neurofibrillary tangles (NFT’s), are a
common feature of neurological diseases characterized by neuronal dysfunction and
eventual cell death. While it is well supported that pathologically modified tau and tau
aggregates can play a role in neurodegeneration, the specific neurotoxic species, and
the mechanisms underlying their pathology, remain unclear.
Although tau has been the subject of increasing interest in the Alzheimer’s
Disease research community, a comprehensive structural study of tau aggregates has
yet to be performed. Recent technological advances in cryo-electron microscopy (cryoEM) allow for the elucidation of structural features of tau fibrils at a resolution that has
not previously been attainable using this technique. A detailed structural study can
provide insight into the important differences between wild-type tau and aggregationprone tau mutants.
In addition to high-resolution features of the tau fibril, the role of tau aggregation
within the neuron is also an important consideration in our understanding of tau
pathology. Eukaryotic cells have been conventionally considered to exceed the
maximum specimen thickness for investigation by cryo-EM. However, as sample
preparation techniques and imaging technologies have improved, it has been
demonstrated that cryo-EM is a useful technique for imaging of some eukaryotic cells,
especially in thin cellular projections, such as neurites. As studies of larger specimens
have become more common in the EM community, a hybrid technique known as
correlative light and electron microscopy (CLEM) has emerged as a useful bridge
between light microscopy, and the molecular resolution of EM. This technique will be
useful to visualize the structural characteristics of tauopathies, as well as the
mechanistic action of potential treatments. Treatments to be tested include upregulation
of autophagy using Transcription Factor EB to clear Tau aggregates within the cell,
exposure of aggregates to TRiC chaperonin to block elongation of the tau fibril, and
utilization of astrocytes to take up and degrade tau aggregate species aberrantly
deposited in the extracellular matrix.
Contributors: Mitchell, Patrick; Martini-Stoica, Heidi; Dai, Wei; Xu,Yin; Li, Hongmei; Zheng, Hui;
Chiu, Wah
227
STRUCTURAL POLYMORPHISM IN INFLUENZA VIRUS NS1 AND ITS POSSIBLE
EFFECT ON BINDING TO CELLULAR LIGANDS
Sayantan Mitra
Advisor: B Prasad, Ph.D.-Department of Biochemistry & Molecular Biology
Influenza viruses cause acute highly contagious respiratory disease and affect
500 million people annually worldwide. Flu virus is unpredictable and can undergo
antigenic drift or antigenic shift to infect new host species including humans, and this
might lead to pandemic. Recent emergence of influenza virus strains resistant to the
available antiviral drugs makes it necessary to identify new drug targets. One of the
promising targets is the non-structural protein 1 (NS1) of influenza virus. NS1 is a
potent antagonist of various cellular antiviral interferon response pathways and is
critical for virus replication, spread, and pathogenesis. This multifunctional protein is
capable of interacting with various cellular ligands and consists of a RNA binding
domain (RBD) and an effector domain (ED) separated by a flexible linker. NS1 exhibits
strain-dependent structural polymorphism. NS1 of H5N1 strains with a 5 amino acid
deletion adopts two conformations - an “open” and a ‘closed’ conformations, whereas in
the NS1 of non-H5N1 strains it exhibits a “semi-open” conformation. It is hypothesized
that such a conformational polymorphism within a particular strain may also exist to
facilitate context-dependent interactions with various cellular ligands during the virus
replication. Here we show that deletion of 5 amino acid residues together with E to G
mutation at position 71 in the linker region can reversibly alter the conformation from
‘closed’ to ‘open’ conformation. Further biochemical and crystallographic studies are in
progress to examine how the strain-dependent structural polymorphism of NS1 affects
its interactions with various cellular ligands such as F2F3 domain of the cleavage and
polyadenylation specificity factor (CPSF30) to modify 3’ end processing of cellular premRNA including IFN-β pre-mRNA CPSF30 and PI3K to suppress the cellular apoptosis
to ensure efficient virus replication.
Contributors: Sayantan Mitra, B V Venkatar Prasad
228
FUNCTIONS OF EPITHELIAL, STROMAL, AND SYSTEMIC PTCH1 IN MAMMARY
GLAND DEVELOPMENT
Teresa Monkkonen
Advisor: Michael Lewis, Ph.D.-Department of Molecular & Cellular Biology
The hedgehog signaling network regulates organogenesis, stem cells, and
cancers including breast. Breast lesions show increased activity of hedgehog signaling.
We have decided to study the normal roles of Ptch1 with respect to normal
development to understand normal functions of this inhibitor of signaling. Mice
homozygous for a hypomorphic Ptch1 allele showed stunted, dysplastic ducts, while
mice heterozygous for a null allele showed hyperproliferative and hyperplastic
mammary glands wwith filled-in ducts and terminal end buds (TEBs). Transplantation
experiments using these mutants demonstrated a requirement for epithelial, stromal,
and systemic Ptch1 in regulating morphogenesis and ductal elongation, but these
functions have not been defined. We hypothesize that Ptch1 mediated inhibition of
canonical hedgehog signaling has discrete epithelial, stromal, and systemic functions to
regulate mammary gland morphogenesis, histology, and proliferation.
Outgrowths from transplanted adenovirus-Cre treated, conditional null Ptch1fl/fl
mammary epithelial cells yielded hyperbranched, hyperproliferative mammary ducts
with intact histology. Next, we evaluated stromal loss of Ptch1 via Fsp-Cre-mediated
deletion in fibroblasts and immune cells, which produced divergent phenotypes. Glands
from mice heterozygous for the null allele were hyperbranched and hyperproliferative.
Homozygous null Fsp-Cre, Ptch1fl/fl animals showed severely stunted ductal
outgrowths and loss of proliferation in mature ducts and terminal end buds (TEBs).
Both mutants displayed aberrant microlumen formation and ducts filled with cells of
luminal cell fate. Fsp-Cre, Ptch1fl/fl animals had altered hormone receptor positivity at 8
weeks, implicating altered cell fate or a perturbed hormone signaling axis. Whole gland
transplantation to evaluate systemic effects rescued the stunted ducts, but filled-in
ducts and microlumens were still present, indicating this is a local requirement for
Ptch1. To determine whether the Fsp-Cre, Ptch1fl/+ and Ptch1fl/fl phenotypes may be
Smoothened mediated, we crossed Fsp-Cre animals to SmoM2 animals conditionally
expressing a conditionally activated allele. We found a very slight defect in outgrowth,
however, TEB and ductal histology phenotypes were recapitulated, suggesting that
these phenotypes were due to Smoothened-mediated mechanisms.
In summary, epithelial Ptch1 suppresses branching and proliferation, but is
dispensable for normal ductal histology. In contrast, Ptch1 promotes branching and
proliferation via a systemic function in Fsp positive cells. Signaling downstream of Ptch1
in the local mammary stroma regulates histomorphology of the mammary epithelium.
Our data also indicate that Ptch1 functions via Smoothened to regulate terminal end
bud morphology. These data are consistent with clinical observations in human breast
cancer, and underscore the importance of tissue-tissue crosstalk in the mammary
gland.
Contributors: Landua, John; Visbal, Adriana; and Lewis, Michael
229
OVEREXPRESSION OF A PRO-PROLIFERATIVE, TRANSCRIPTIONALLY ACTIVE
YAP IN ADULT CARDIOMYOCYTES CAUSES SUDDEN CARDIAC DEATH
Tanner Oliver Monroe
George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Developmental biologists have long sought mechanisms that govern organ size.
About 12 years ago, separate groups identified the Hippo pathway as responsible for
limiting Drosophila organ size by restraining cell proliferation during development. Our
lab predicted that this same pathway would be evolutionarily conserved in mammals.
Through a series of genetic and histological approaches, we discovered that, indeed,
the Hippo pathway turns on in the postnatal mouse to limit the final cardiomyocyte (CM)
number. The Hippo pathway is a kinase cascade, largely regulated by contact inhibition,
which ultimately phosphorylates and inactivates the transcriptional co-activator, Yap.
When Yap is active, it promotes the transcription of a variety of pro-proliferative genes.
Directly upstream of Yap is the kinase, Lats, which phosphorylates serine residues at
the HXRXXS peptide consensus motif. We created two lines of transgenic mice with
inducible overexpression of a phosphorylation deficient, and thus, constitutively active
form of Yap1α, in which all 5 phosphorylatable serine residues are mutated to alanine.
The transgene design is as follows: CAG-loxp-eGFP-STOP-loxp-2xflagYap5SA-lacZ,
which we term, “JoJo-Yap5SA.”
Adult CMs are highly resistant to cell division, and postnatal growth of the heart
is due to CM hypertrophy. Therefore, when CMs die, either due to ischemic attack or
trauma, they are forever lost, and replaced by fibrotic scar. Therefore, we are interested
in therapeutically turning CM proliferation back on in the adult. Since the Hippo pathway
halts CM proliferation during development, we hypothesized that inactivating it in the
adult mouse would promote CM cell cycle re-entry. Our lab showed that not only did
that happen, but the induced CM proliferation was sufficient to restore cardiac function
and limit scar formation after damage.
Given our previous data, we predicted that overexpression of our JoJo-Yap5SA
transgene would cause dramatic CM cell cycle re-entry; this in-turn would facilitate
dramatic myocardial regeneration after ischemia. To test that hypothesis, we crossed
the JoJo-Yap5SA mice with the cardiomyocyte-specific, and tamoxifen-inducible Cre,
αMyHC-MerCreMer. Surprisingly, we found that activation of the gene in the adult
mouse causes sudden cardiac death. We are currently investigating the mechanism by
which these mice are dying.
Contributors: Monroe, Tanner O.; Leach, John; Rodney, George; Martin, James
230
REGULATION OF HISTAMINE PRODUCTION BY LACTOBACILLUS REUTERI BY
OTHER BACTERIAL METABOLITES
Christina Narie Morra
Amino acid metabolism by the gut microbiome is critical for human health. The
decarboxylation of L-histidine to histamine by Lactobacillus reuteri reduces the
production of the pro-inflammatory molecule, TNF. This work examines histamine and
histidine metabolism by L. reuteri, as well as the effects of microbial histidine
metabolism on the host. Supernatants from bacterial cultures with 13C615N3 Lhistidine were processed and NMR was used to identify novel bacterial metabolites. We
have determined that the addition of carnosine (β-alanyl-L-histidine) in the media
significantly increases histamine production. We have also determined that media
concentrations of acetate and folate have effects on viability, histamine production and
TNF production by THP-1 cells treated with bacterial supernatants. Currently we are
working to determine the mechanism by which carnosine, folate, and acetate affect
histamine production. Our plans include the oral administration of 13C615N3 L-histidine
to mice and co-culture of L. reuteri with human ileal enteroids. These studies will
determine the effects of L. reuteri and histidine/histamine metabolism on the human
intestinal epithelium. Together the data will facilitate understanding of bacterial
metabolites produced by this probiotic and their effects on the mammalian intestine.
Contributors: Morra, Christina; Roeth, Daniel; Kalkum, Markus; Versalovic, James
231
THE ROLE OF PERINATAL EXPOSURES IN MODULATION OF
NEURODEVELOPMENT USING THE SHANK3e4-9/+ MUTANT MOUSE MODEL
Jaclyn Bravo Murry
Advisor: Ignatia Van Den Veyver, M.D.-Department of Obstetrics & Gynecology
Autism spectrum disorder (ASD) manifests with repetitive behaviors, social and
communication impairments. Early environmental exposures concurrent with genetic
factors are implicated in ASD. Genes like SHANK3, affecting synaptic function, are
causative in a subset of ASD patients and Shank3 null mice exhibit ASD-like behavior.
Perturbed serotonergic signaling has been reported in ASD and FLX, a Selective
Serotonin Reuptake Inhibitor, is an increasingly common early life exposure. We tested
our hypothesis that perinatal exposures (FLX) in conjunction with ASD-associated risk
alleles (Shank3) modulate neurodevelopment, increasing penetrance of offspring
behavior disturbances. Shank3ex4-9/+ (HET) dams were mated with HET males and
given FLX (15mg/kg) in drinking water (+FLX n=36) or drinking water only (-FLX n=37)
from day 0 of pregnancy (dpc0) until postnatal day 7 (pnd7). Adult male knockout and
wild-type (KO n=10, WT n=20) +FLX and (KO n=18, WT n=14) -FLX offspring were
tested for anxiety, repetitive, and social behaviors. Two-way ANOVA was used to test
gene-environment (GXE) interactions, with Bonferroni posthoc test. Student’s t-test was
used to test social preferences. Litter sizes and pregnancy outcomes were similar in
HET (+FLX) and HET (-FLX) dams. In all, four behavioral assays had significant
differences in social and anxiety behaviors. In the elevated plus maze, mutant mice had
greater distance traveled in the open arm than WT males (Significant genotype effect,
P<0.01), but WT (+FLX) males trended towards less time in the open arm. In the open
field assay, vertical locomotion was lower in KO (+FLX) males vs. KO (-FLX) males (P
<0.05) (Significant GXE interaction, P <0.05; and significant genotype effect, P<0.05).
KO (+FLX) males had a borderline reduction in vertical locomotion vs. WT (+FLX)
males (P=0.06). Additionally, mutant males had lower center/total distance traveled
ratios vs. WT males (significant genotype effect, P=0.02), with KO (+FLX) animals
exhibiting a trend towards further ratio reduction. For sociability, KO (+FLX) males
lacked a social preference in the 3-chamber test by chamber time or sniffing time (P
=0.11; P =0.48). In novel mouse sniffing time, WT (+FLX) trended towards greater
sociability, while KO (+FLX) males trended towards reduced sociability (borderline GXE
interaction, P=0.06). Also, WT (+FLX) males had significantly increased active social
contact vs. passive social contact (P <0.01) in the direct social interaction test. No
significant interactions were observed in repetitive behavior assays. These data suggest
chronic FLX exposure alters social behavior and anxiety-like behaviors differently in
Shank3-mutant and WT mice. Ongoing experiments will examine the molecular and
structural correlates of chronic and developmental FLX exposure in these mice.
Contributors: Murry, Jaclyn; He, Fang; Engler, David; Balasa, Alfred; Van den Veyver, Ignatia
232
BREAST CANCER CELLS UTILIZE BALLOON LIKE STRUCTURES TO FACILITATE
INTERCELLULAR CROSSTALK WITH OSTEOBLASTS
Aaron Matthew Muscarella
The bone is the organ most frequently affected by breast cancer metastasis, and
can serve as a reservoir of cells for further disease spread in addition to having its own
pathology. While the mechanism of osteolytic bone metastases has been established,
little is known about the early stage of bone metastasis in which disseminated tumor
cells (DTCs) arrive in the bone, enter the bone marrow, and begin dividing again. The
Zhang lab recently published that luminal breast cancer cells which grow from single
cells to multicellular lesions are almost always in direct contact with osteoblast cells.
This relationship, termed the “osteogenic niche” allows the cancer cells to grow and is
mediated by heterotypic adherens junctions between the two cell types, activating
mTOR signaling in the cancer cells. In order to further understand how cells make the
initial contact with the niche cells, we set out to image the interaction process in real
time.
We utilized cryo-electron microscopy (cryoEM) and tomography (cryoET) as well
as florescence light microscopy (FLM) to image metastatic breast cancer cells MCF7
both in the presence of osteoblasts and without. This was done in sequence on the
same cells, allowing us to match high resolution electron microscopy data with the
bigger-picture aspects of whole-cell florescence.
Using these techniques, we were able to observe a unique, undescribed
membrane-extension phenotype in which the cancer cells project long, thin (20-40nm
diameter) tubes of membrane across long distances (2-3 cell lengths, 40-60 µm). These
tubes branch out and end with larger (0.5 -1 µm) spheroid blubs which we have termed
“Balloon-Like Structures” or BLS. We have found while MCF7 exhibit these extensions
occasionally in normal culture conditions, they are greatly enriched in the presence of
osteoblast cells. Curiously, under cryoET these structure do not appear to contain actin
or microtubules, and this was supported through the use of inhibitors. We observed that
BLS have a propensity to grow towards and on top of osteoblasts, even if the main
cancer main cell body is not nearby. Additionally there is a high local concentration of
ribosomes where the two cell types touch, indicating a possible signaling component.
Using deconvolution fluorescence microscopy we have observed that gapjunction permeable dye is transferred from the cancer cells to the osteoblasts via BLS,
but not vice versa, at this stage of contact. When exposed to osteoblast cells, MCF7
exhibit long, cytoskeleton-free extensions across many cell lengths. These extensions
end in small blubs of membrane about one micron across. We are working to
characterize the functionality of these structures both in vivo and in vitro.
Contributors: Muscarella, Aaron; Dai, Wei; Mitchell, Patrick; Chiu, Wah; Zhang, Shawn;
233
ABO INCOMPATIBLE PEDIATRIC LIVER TRANSPLANT: TITER BASED
MANAGEMENT
Krupa Ramaprasad Mysore
Advisor: Robert Shulman, M.D.-Department of Pediatrics
Xian Li, M.D./Ph.D.Purpose: Children with end stage liver disease have high morbidity and mortality
awaiting transplant as a result of organ shortage. ABO incompatible liver transplants
(ABO-ILT) is an alternative for select patients. The clinical outcomes in children
undergoing ABO-ILT are largely undefined and treatment protocols are not
standardized. We present our experience with 7 ABO-ILT with a standardized protocol.
Methods: Charts of children who underwent ABO-ILT between 9/2013 and 8/2014 were
retrospectively reviewed. Children with high Isohemagglutinins (Ih) titers (≥1:32)
received additional immune suppression (IS) including Plasmapheresis, Rituximab,
IVIG, and Mycophenolate. Children with Ih titers <1:32 received standard steroids and
Tacrolimus
Results: Seven children (3 F and 4 M) with median age of 8 months (5 mo - 9 yrs)
underwent ABO-ILT out of total forty pediatric liver transplants. All ABO-ILT recipients
had PELD score of 30 points or higher, reflecting high medical acuity. Indications for
transplant were: Biliary atresia (4/7; 57%), acute liver failure (1/7; 14%), Cryptogenic
cirrhosis (1/7; 14%) and metabolic liver disease (1/7; 14%). Ih titers were monitored in
5/7 patients pre-transplant and 7/7 patients post-transplant. 2/7 (28%) patients
underwent alternate IS due to high pre-op Ih. Post-operative complications included
biliary stricture in 2/7 (28%, one of whom had high pre-op Ih), viral infection in 2/7 (28%,
neither received enhanced IS). All 7 ABO-ILTs had no acute cellular rejection in the
first 6 months post-transplant and have normal ALT, GGT and function at subsequent
follow up. No patients had elevation in relevant Ih in the first 6 months.
Conclusion: ABO-ILT is safe and feasible in children with appropriate modifications of
IS in those with high Ih and standard IS in patients with low Ih. There was 100% graft
and patient survival. In our cohort, none of the patients made immune response to their
mismatched ABO antigen, indicating tolerance. There is a great need for a prospective
study to better assess outcomes in children undergoing ABO-ILT and further determine
optimal immunosuppression regimens
Contributors: Mysore, Krupa; Himes, Ryan; Goss, John; Teruya, Jun; Desai, Moreshwar;
Srivaths, Poyyapakkam; Zaruca,Kimberly; Nicholas, Sarah
234
BROAD THERAPEUTIC SUPPRESSION IN TRIPLE NEGATIVE BREAST CANCER
BY TARGETING COORDINATE TYROSINE KINASE DYSREGULATION
Amritha Nair
Triple-negative breast cancer (TNBC) is a collection of genetically
heterogeneous diseases with distinct clinical behaviors and molecular features. Herein,
we present evidence of a novel tumor suppressor network that is frequently
compromised in TNBC, and a credentialed strategy to target this pathway for TNBC
therapeutic intervention. Our group has previously identified via an unbiased genetic
screen that the tyrosine phosphatase PTPN12 is a bonafide tumor suppressor in TNBC.
PTPN12 function is compromised in more than 60% of human TNBCs and notably, the
tumorigenic and metastatic potential of PTPN12-deficient TNBCs is severely impaired
by restoring PTPN12. In this study using integrative biochemical, genetic, and
pharmacologic approaches, we demonstrate that PTPN12 serves as a common
negative regulator of mitogenic signaling from a subset of proto-oncogenic receptor
tyrosine kinases [RTKs] including MET, PDGFRBand others in TNBC. The loss of
PTPN12 phosphatase commonly seen in TNBC, results in coordinated activation of
these RTKs. Importantly, combined pharmacological inhibition of these PTPN12regulated TKs leads to synergistic inhibition of TNBC cell survival and robust tumor
regression across a panel of 14 in vivo patient-derived xenograft (“PDX”) models of
human TNBC, with drug response closely correlating with PTPN12 dysfunction in the
PDXs. Broadly these studies suggests that loss of a phosphatase like PTPN12 can
result in coordinate RTK dysregulation in a subset of breast cancers and that targeting
this dependency pharmacologically might be a viable therapeutic strategy clinically
Contributors: Nair, Amritha1; Sun, Tingting2; Tyagi, Siddhartha2; Dominguez-Vidana, Rocio3;
Dobrolecki, Lacey.E. 4 ; Petrovic, Ivana 4 ; Schmitt, Earlene 3; Osborne, C.Kent 4 ; Schiff,
Rachel 4 ; Shaw, Chad.A. 1; Lewis, Michael.T. 4; Westbrook,Thomas.F. 1,2,3,4.
1Department of Molecular and Human Genetics, Baylor College of Medicine, TX. 2The Verna
and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of
Medicine, Houston, TX. 3Interdepartmental Graduate Program in Cell and Molecular Biology,
Baylor College of Medicine, Houston, TX, 4Lester & Sue Smith Breast Center, Baylor College
of Medicine, Houston, TX.
235
ACTIVIN DEFICIENCY REDUCES ADIPOSITY AND CAUSES ABERRANT
DEVELOPMENT OF WHITE ADIPOSE TISSUE
Maria Namwanje
Advisor: Chester Brown, M.D./Ph.D.-Department of Molecular & Human Genetics
Obesity and its associated metabolic disorders are increasing worldwide. Several
members of the TGF-β family play important roles in body composition and energy
metabolism. In this study we utilized mouse models with adipose-selective conditional
knockout of Inhba and/or global Inhbb knockout to better understand the direct roles of
activins in adipose tissues and metabolism. Activin A and B proteins exhibit temporal
expression differences in visceral and subcutaneous white adipose tissue. Loss of
either Inhba or Inhbb has a modest suppressive effect on diet-induced obesity.
However, activin double mutants show severe weight loss and markedly reduced
adiposity. In contrast to the subcutaneous white adipose depot predominance of
browning induced by cold stress, combined activin A and activin B deficiency induces
the robust appearance of multiloculated, UCP-1 positive cells, predominantly within
visceral white adipose. Our results demonstrate that activins A and B contribute to early
white adipose tissue expansion and adipocyte cell fate in a depot selective manner,
thus highlighting activin signaling in early adipose tissue development
Contributors: Namwanje, Maria; Huang, Lihua; and Brown Chester W.
236
THE MYCOBIOME OF THE HUMAN MICROBIOME PROJECT HEALTHY COHORT
Andrea Kathleen Nash
The human gut microbiome has been repeatedly characterized in both healthy
and diseased states, but this characterization has mostly emphasized the bacterial
compartment. The fungal microbiome (known as the mycobiome) is now beginning to
gain recognition as a fundamental part of our microbiome and has been implicated in
several diseases, including inflammatory bowel disease, irritable bowel syndrome, and
colorectal cancer. Previously, much of the characterization of the human mycobiome
has focused on subjects with a particular disease of interest, yet there is a need to
explore and understand the healthy human mycobiome. Using stool samples from the
Human Microbiome Project (HMP), we have discovered that there is low fungal diversity
in the gut, mostly dominated by yeast genera such as Saccharomyces, Malassezia, and
Candida. These three genera are present in 90%, 77% and 77% of samples,
respectively, as well as at least one time point in 100%, 97%, and 100% of volunteers,
respectively. These data suggests that Saccharomyces, Malassezia, and Candida
might be part of our core gut mycobiome. Clinical metadata revealed lower alpha
diversity in samples donated from males compared to those donated by females.
Diversity of fungal communities also tends to decrease with increasing blood pressure.
Furthermore, the relative abundance of the Debaryomycetaceae family, including
species such as Candida albicans, Candida topicalis, and Candida parapsilosis,
significantly decreases with increasing resting pulse rate, indicating that these taxa may
be associated with a healthier heart. The HMP samples present a unique opportunity to
define the mycobiome in healthy individuals, knowledge that is lacking in the field. This
information on the healthy mycobiome could aid in research identifying diseasecontributing fungi or imbalances of fungal-bacterial relationships.
Contributors: Nash, Andrea; Auchtung, Thomas; Wong, Matthew; Smith, Daniel; Ross,
Matthew; Metcalf, Ginger; Muzny, Donna; Gibbs, Richard; Ajami, Nadim; Petrosino, Joseph
237
MECHANISMS UNDERLYING THE TUMOR SUPPRESSIVE ROLE OF PTPN12 IN
TRIPLE-NEGATIVE BREAST CANCER
Nicholas J Neill
Triple-negative breast cancer (TNBC) is a common and aggressive subtype of
breast cancer that is newly diagnosed in approximately 220,000 women annually.
TNBC is refractory to current targeted therapies and despite recent efforts to
characterize TNBC genomes and epigenomes, a major barrier to developing TNBC
therapies is the paucity in our understanding of the molecular drivers of TNBC.
Identifying the signaling networks whose dysregulation drives TNBC would have
enormous impact on our understanding of the disease and how we treat afflicted
patients.
Using a forward genetic screen, we recently identified the tyrosine phosphatase
PTPN12 as a tumor suppressor in TNBC (Sun, Cell 2012). Our preliminary data
indicate PTPN12 is compromised in many epithelial cancers, including more than 70%
of TNBCs, making it the second most frequently inactivated tumor suppressor in TNBC
(behind p53). Loss of endogenous PTPN12 leads to transformation of human
mammary epithelial cells (HMECs) and mammary carcinogenesis in mouse models.
Moreover, restoring PTPN12 function dramatically impairs tumor progression and
metastasis in TNBCs. These and other studies suggest PTPN12 functions as a
suppressor of human TNBC.
Our objectives are to elucidate the mechanisms by which PTPN12 suppresses
human TNBC and discover new vulnerabilities of PTPN12-deficient TNBCs that can be
used as therapeutic entrypoints. To define such vulnerabilities, we performed unbiased
genetic screens in PTPN12-overexpressing and PTPN12-deficient TNBC models to
identify genes that regulate tumor survival and proliferation in a PTPN12-dependent
manner. By combining this genetic-interaction profiling with quantitative proteomics
data, we have identified several cellular processes that are dysregulated in PTPN12deficient cancers and selectively required to support the growth of these tumors. We
are currently exploring these pathways as novel therapeutic entrypoints that can be
exploited to treat patients with TNBC.
Contributors: Neill, Nicholas; Sun, Tingting; Westbrook, Thomas
238
GI DYSBIOSIS AND ITS CONTRIBUTION TO CEREBRAL SMALL VESSEL
DISEASE
James Willard Nelson
Advisor: Robert Bryan, Ph.D.-Department of Anesthesiology
In my study, I will develop the novel idea that a pathological change in the gut
bacteria, dysbiosis, is an underlying cause of cerebral small vessel disease (CSVD).
CSVD consists of pathological alterations in cerebral small vessels leading to damage
of deep structures in the brain. CSVD is responsible for ~45% of all the dementias,
second only to Alzheimer’s disease. I propose that decreases in bacterial populations
that produce the short chain fatty acid (SCFA) butyrate in the gut are responsible for
CSVD. Butyrate produced by commensal bacteria residing in the gut are known to
maintain integrity of both the intestinal barrier and the blood-brain barrier (BBB). It
follows that a decrease in butyrate-producing bacteria allows bacteria and endotoxin in
the gut to enter the systemic circulation through a compromised gut barrier and
ultimately to the brain through a compromised blood brain barrier (BBB). The end result
of this deficit in butyrate is cerebral inflammation. Interestingly, disruption of the BBB
and brain inflammation are considered initiating events in the CSVD disease process. I
propose to test the hypothesis that decreases in butyrate-producing commensal
bacteria in the gut initiates CSVD. I propose to use the spontaneously hypertensive
stroke prone rat (SHRSP), an animal model that closely mimics human CSVD as the
rats ages to 20-30 weeks.
Contributors: Nelson, James; Bryan, Robert
239
THE PHYSIOLOGICAL ROLE OF A-TO-I EDITING IN RESPONSE TO
ENVIRONMENTAL STIMULI
Isaiah Adam Alexander Neve
Living organisms constantly respond to environmental stresses with reactions
ranging from drastic systemic alterations to molecular scale changes. One possible
method of managing environmental insults comes in the form Adenosine-to-Inosine
RNA editing (RNAe) performed by the highly conserved ADAR (adenosine deaminases
acting upon RNA) enzymes. These enzymes can facilitate the generation of alternative
isoforms of essential proteins or regulatory RNA; granting the organism flexibility while
maintaining genetic fidelity (Savva et al., 2012). Recently our lab has elucidated a novel
gene-environment interaction pathway in which diet significantly alters the reproductive
strategy of C. elegans (Sowa, Mutlu, Xia, & Wang, 2015). Cultivated strains of C.
elegans are generally fed a B-strain derived E. coli known as OP50. However, when
worms are fed HB101, an alternative K-12 derived E. coli strain, they display a faster
reproductive lifespan (RLS), an increased germline proliferation rate, and reduced
germline maintenance. Furthermore, we have now determined that the RNAe
machinery in C. elegans is required for some of these gene-environment interactions.
adr-2 knockout worms fed HB101 show long OP50-like RLS, and altered late life
fertility. Additionally, the changes induced by knocking out the RNAe machinery may be
suppressed by perturbation of the endogenous C. elegans RNA interference pathway.
These preliminary studies suggest that the HB101 dietary signal may induce specific
RNAe events which modify reproductive strategy in an RNA interference (RNAi)
dependent manner. This study will provide significant insights into the importance of the
environment on RNAe-RNAi interactions and their subsequent effects on major
phenotypic characteristics.
Contributors: Neve, Isaiah; Sowa, Jessica; Wang, Meng
240
GLOBAL METABOLOMICS ANALYSIS OF A MAMMALIAN HOST INFECTED WITH
BACILLUS ANTHRACIS
Chinh Thi Quynh Nguyen
Whereas DNA provides the information to design life, and proteins the materials
to construct it, the metabolome can be viewed as the physiology that powers it. As
such, metabolomics, the field charged with the study of the dynamic small molecule
fluctuations in response to changing biology, is now being used to study the basis of
disease. A comprehensive metabolomic analysis of a systemic bacterial infection was
conducted using Bacillus anthracis, the etiological agent of anthrax disease, as the
model pathogen. An organ and blood analysis identified approximately 400 metabolites,
including several key classes of lipids involved in inflammation, as being suppressed by
B. anthracis. Metabolite changes were detected as early as 1 day post-infection, well
before the onset of disease or the spread of bacteria to organs, which testifies to the
sensitivity of this methodology. Functional studies using pharmacologic inhibition of host
phospholipases support the role of these key enzymes and lipid mediators in host
survival during anthrax disease. Finally, the results are integrated to provide a
comprehensive picture of how B. anthracis alters host physiology. Collectively, this
study provides a blueprint for using metabolomics as a platform to identify and study
novel host-pathogen interactions that shape the outcome of an infection.
Contributors: Shetty, Vivekananda; Maresso, Anthony
241
MTOR-ASSOCIATED KV1.1 CHANNEL DYSREGULATION IN THE NEURONAL
SUBSET-SPECIFIC PTEN KNOCKOUT MOUSE MODEL OF CORTICAL DYSPLASIA
Lena Nguyen
Background: Hyperactivation of the mechanistic target of rapamycin (mTOR)
pathway has been demonstrated in human cortical dysplasia (CD), which is a prevalent
cause of intractable pediatric epilepsy. Previous studies in our lab have shown that
inhibition of mTOR attenuates epilepsy in the neuron subset-specific Pten knockout
(NS-Pten KO) mouse model of CD. However, the underlying mechanisms remain
elusive. Given that emerging research has suggested a role for mTOR in the regulation
of voltage-gated ion channels, which are critical modulators of neuronal excitability, we
investigated whether mTOR-associated dysregulation of voltage-gated ion channel
expression occurs in NS-Pten KO mice.
Methods and results: Western blotting analyses on whole hippocampal
homogenates from 2, 4, 6, and 8 week-old NS-Pten KO and wildtype (WT) mice
revealed progressively aberrant Kv1.1 protein levels with age in NS-Pten KO mice. By
postnatal weeks 6-8, Kv1.1 protein levels were significantly increased in NS-Pten KO
compared to age-matched WT mice (p<0.001, n=6-15 mice/group). No changes in
Kv1.2 and Kv1.4 protein levels were observed between age-matched NS-Pten KO and
WT mice at any time points. Further analyses with real-time quantitative PCR revealed
no changes in Kv1.1 mRNA expression between the two genotypes (n=7-8 mice/group).
Treatment with the mTOR inhibitor rapamycin (10mg/kg i.p. 5 days/week) at an age
before Kv1.1 protein levels were dysregulated (during postnatal weeks 4 and 5) in NSPten KO mice prevented the aberrant increase in Kv1.1 levels (p<0.01, n=7-16
mice/group). Treatment with rapamycin at an age after Kv1.1 protein levels were
dysregulated (during postnatal weeks 9 and 10) in NS-Pten KO mice restored the
aberrant increase in Kv1.1 levels to WT levels (p<0.05, n=6-14 mice/group). Rapamycin
treatment had no effects on Kv1.2 and Kv1.4 protein levels.
Conclusion: Our results demonstrate the presence of altered Kv1.1 protein levels
in association with mTOR hyperactivation in NS-Pten KO mice, and provide support for
mTOR-associated ion channel dysregulation as a candidate mechanism underlying
epilepsy in this model. Future studies will further explore the potential mechanisms by
which mTOR regulate Kv1.1 protein levels and investigate the functional relevance of
altered Kv1.1 protein levels in this model.
Contributors: Nguyen, Lena; Anderson, Anne E.
242
NOVEL STRATEGY FOR DECODING THE NONCODING RNA REGULATOME OF
TRANSLATION IN BREAST CANCER AND STEM CELLS
Tuan Minh Nguyen
FGF and WNT signaling, crucial pathways in mammary gland development and
stem cell self-renewal, are frequently deregulated in cancer progression. Previously, we
demonstrated that simultaneous overactivation of FGF and WNT signaling using an
inducible system of receptor tyrosine kinase activation accelerates mammary
tumorigenesis. The dramatic reduction in tumor latency is accompanied by enhanced
recruitment of polysomes to known WNT-target genes, implicating that disruption of
translational control in stem cell self-renewal may contribute to malignancy.
Furthermore, recent studies provide evidence for the roles of noncoding RNAs in stem
cell self-renewal and translational regulation. Interestingly, the PI3K/AKT/mTOR
pathway, a key downstream pathway of FGF signaling, can modulate functions of
noncoding RNAs (i.e. microRNAs and long noncoding RNAs), a number of which have
been shown to regulate translation of WNT signaling components. We hypothesize that
noncoding RNAs participate in FGF-WNT-induced translational reprogramming that
results in deregulation of stem cell self-renewal and subsequent cancer progression
and poor prognosis. We have optimized ribosome profiling (Ribo-Seq) in vitro as well as
ex vivo on freshly isolated mammary epithelial cells (MECs) to quantify translational
efficiencies of individual transcripts across the transcriptome. The technique is currently
being applied on normal and premalignant MECs to interrogate translatomic alterations
during FGF-WNT-driven tumorigenesis. In addition, we are developing a method,
termed RNA-RNA interactome capturing (RIC), to capture intermolecular RNA-RNA
interactions exclusively in the cytoplasm to identify noncoding RNA-mRNA partners, in
which the noncoding RNAs function as regulators of translation of the mRNA partners.
The combination of ribosome profiling and RIC will systematically decode the
noncoding RNA regulatome of translation during FGF-WNT driven tumorigenesis and
provide novel insights into the cytoplasmic functions of long noncoding RNAs, whose
roles in gene regulation remain poorly understood.
Contributors: Nguyen, Tuan; Kabotyanski, Elena; Roarty, Kevin; Zhang, Xiang; Neilson, Joel;
Rosen, Jeffrey
243
LINK BETWEEN SEQUENCE, STRUCTURE, AND FUNCTION IN NON-CODING RNA
Ilya B Novikov
Functional non-coding RNAs regulate gene expression, translation, and maintain
genome stability. They are important in subcellular biology and known to cause human
disease. It is important to understand functional determinants in these understudied
molecules. Using Evolutionary Trace, a sequence analysis method that leverages
evolutionary history of a molecule, we identified evolutionary important nucleotides in a
set of 2450 functional RNAs. We discovered that these nucleotides universally shared
five remarkable properties: (1) they clustered non-randomly in the tertiary and
secondary structure; (2) they overlapped significantly with known catalytic nucleotides,
protein binding sites, antibiotic binding sites, post-transcriptionally modified nucleotides,
and inactivating mutations; (3) the clustering quality of these nucleotides predicted the
quality of overlap with known functional nucleotides; and finally sequence selection,
optimized for (4) clustering of evolutionary important nucleotides and (5) smoothing of
evolutionary importance over the structure improved the quality of functional site
recovery. The results show that sequence, structure, and function are strongly linked in
functional RNA, and that we can use evolutionary information to discover functional
sites in novel RNAs. This points to idea that RNA evolves under the same principals as
proteins suggesting that evolutionary trace could be effectively applied to any evolving
polymer molecule.
Contributors: Wilkins, Angela; Lichtarge, Olivier
244
CHARACTERIZATION OF THE NOVEL NPAS2 CKO MOUSE MODEL INDICATES
ROLE IN REGULATING METABOLIC HOMEOSTASIS IN RESPONSE TO A HIGH
FAT DIET
Derek Steven O’Neil
William Gibbons, M.D.-Department of Obstetrics & Gynecology
Introduction: Npas2, a circadian gene, is associated with metabolic regulation in fetal
development. Previously, we have demonstrated that a maternal high fat diet is associated with
the disruption of the circadian pathway in the fetal liver. It is not known how circadian rhythms
are established in fetal development with the lack of light/dark cues through the central clock
nor is it known how Npas2 plays a role in adult metabolic function. We have developed a novel
Npas2 conditional knock out (cKO) mouse model to investigate the role of the peripheral clock
(liver) in establishing metabolic homeostasis. Our objective is to understand the role of Npas2
in maintaining metabolic homeostasis in adult life, especially under the metabolic stress of a
high fat diet. Npas2 cKO were generated by targeting the deletion of exon 3 through the CreLoxP system of conditional gene deletion.
Methods: Mice with the Albumin Cre (AlbCre) transgene and heterozygous for the LoxP
flanked (Fl) region of the Npas2 gene (AlbCre;Fl/+) were mated to controls (Fl/Fl) to generate
Npas2 cKO (AlbCre;Fl/Fl, n=62) and control mice (AlbCre;Fl/+, n=48 and Fl/Fl, n=49) for this
study. Mice were genotyped between postnatal day 14-20 (p14-20) by PCR amplification of tail
genomic DNA then weaned at p21 onto control (CD, Harlan Teklad TD.08485, n=76) or high fat
diet (HFD, Harlan Teklad TD.88137, n=83). Mice were weighed biweekly, and after 25 weeks
post weaning, a glucose tolerance test (GTT, 2g/kg glucose by oral gavage) was performed.
Then, animals were rested two weeks before fat and lean mass was measured by quantitative
magnetic resonance (QMR). On a different cohort of mice we performed a restricted feeding
study in which 16 week old mice were limited to 4 hours of food availability each day (9AM1PM) for 16 days, mice were weighed every 2 days.
Results: As expected, both the male and female mice on a HFD gained significantly
(p<0.01) more weight than mice placed on the CD. Male cKO mice on a HFD have significantly
(p<0.05) larger mass than control mice on a HFD starting at 13 weeks after weaning. QMR
measurements of fat mass showed that male cKO mice on CD had significantly less fat mass
(p<0.05, 11.27% fat) than male control mice (Fl/Fl, 17.53% fat) on CD. The results of the GTT
study indicate that loss of hepatic Npas2 lowers the blood glucose levels after a 6 hour fast and
in response to a glucose challenge. Finally, we showed that under a restricted feeding regimen
cKO females have a significantly greater loss of weight compared to controls (*=p<0.05).
Conclusion: We successfully generated a novel mouse model to study to role of Npas2
expressed in the liver in regulating metabolic homeostasis. The initial phenotype indicates that
Npas2 in the peripheral clock (liver) has a significant role in regulating weight gain, lipid
accumulation, and in responding to glucose challenge. Additionally, Npas2 has a protective
effect against excessive weight loss when food is restricted. Our novel mouse model
demonstrates the importance of the peripheral circadian clock, independent of the central
circadian clock, to regulate metabolic homeostasis.
Contributors: O’Neil, Derek; Goodspeed, Danielle; Aagaard, Kjersti
245
ADENYLYL CYCLASE COPY NUMBER VARIANTS ARE ASSOCIATED WITH
CONGENITAL GENITOURINARY ANOMALIES
Marisol Ann O’Neill
Anomalies of the genitourinary (GU) system are among the most common birth
defects in males. GU anomalies such as hypospadias, the improper placement of the
urethral opening on the underside of the penis; cryptorchidism, the failure of testicles to
descend; and ambiguous genitalia, are severe conditions which require surgical
intervention. In order to identify the genetic causes of GU anomalies, our lab has
previously performed comparative genomic hybridization arrays to identify “hotspots” of
chromosomal imbalance in patients with GU anomalies. These hotspots contain copy
number variants (CNVs), microdeletions or microduplications, present in patients with
GU anomalies that are not common in the general population. One such region
identified was 5p15.3; two patients were identified with CNVs in this region, one of
which consisted of only the Adenylyl Cyclase 2 (ADCY2) gene. ADCY2 is a member of
the adenylyl cyclase class III family of genes that are responsible for converting ATP
into cAMP. Through the use of literature and database (DECIPHER, ISCA) searches,
we have been able to identify an association between the development of GU
anomalies and several of the ADCY genes, the most prevalent of which are ADCY2,
ADCY7, and ADCY9. We have established the prevalence of ADCY2 and ADCY9
CNVs in our cohort of patients with GU anomalies to be 1.9% and 3.0% respectively.
We have found that ADCY2, ADCY7, and ADCY9 are expressed throughout the
developing GU tract, thereby providing evidence for the involvement of ADCY 2,7, and
9 in GU development.
Contributors: O’Neill, Marisol; Lamb, Dolores J.
246
IDENTIFYING CANCER SUSCEPTIBILITY GENES IN PEDIATRIC LEUKEMIA
Ninad R Oak
Advisor: Sharon Plon, M.D./Ph.D.-Department of Pediatrics
In childhood cancers, identification of driver and cancer susceptibility mutations
can have a great impact on risk stratification, therapeutic regimens and surveillance for
subsequent malignancies. To find cancer susceptibility genes, we performed whole
exome sequencing of constitutive DNA from childhood cancer patients with family
history of cancer. This dataset consisted of nine kindreds with at least one case of
acute lymphocytic leukemia (ALL) and a second case of a childhood cancer in a first
degree relative. A total of 21 exome sequencing samples from affected individuals were
analyzed to find possibly deleterious mutations in genes that are rare with minora allele
frequency of <1% which segregate with the disease within a family. We have started
the functional studies on the handful of candidates from each family to test their
potential role in leukemia.
To further expand the discovery of genetic factors of cancer susceptibility, I
decided to explore the mutations within microRNAs in this cohort. The miRNA mutation
analysis pipeline was developed to retain variants within 200bp upstream and
downstream of a precursor miRNA. I focused on rare mutations that were shared by
affected individuals within a family. We identified a germline mutation in the primary
sequence of a miRNA, MIR1301, which was shared between a daughter-mother pair
affected by ALL and melanoma respectively. We used bioinformatics algorithms to
predict the targets of MIR1301 and next shortlisted a list of candidate targets based on
literature and their functional relevance to leukemia. We functionally tested 10 predicted
targets using luciferase reporter assays, and 6 genes, MLL, EZH2, NOL4, ZNF181,
ARL5A and TCF4 were positively validated. We further tested whether the knockdown
of endogenous MIR1301 has an effect on its target expression, but we could not affirm
this effect due to lack of a cell-line that expresses this miRNA at high levels. Next, we
designed an assay to test the effect of the miRNA mutation on its processing by cloning
pri-miRNA transcript into a mammalian expression vector followed by site-directed
mutagenesis to create candidate and positive control mutations. Upon transfection of
HEK293 cells with these vectors, we observed that miRNA processing was completely
abolished in case of the positive control mutation while the candidate mutation had no
effect on the mature MIR1301 expression. Given the important genes regulated by
MIR1301, we are further testing whether this miRNA has a potential role in
leukemogenesis.
In summary, we are using diverse approaches to identify cancer susceptibility
genes in familial cancer patients. Discovery of such genes will help us understand the
underlying genetic mechanisms of the disease as well as have an impact on clinical
surveillance of the patients.
Contributors: Oak, Ninad; Ritter, Deborah; Powell, Bradford, Wheeler, David; Plon, Sharon
247
CARDIAC SPECIFIC FKBP12 DEFICIENCY PRODUCES DILATED
CARDIOMYOPATHY WITH MARKEDLY ENHANCED SUSCEPTIBILITY TO
PERIPARTUM DEATH
Joshua Oakes
Advisor: Susan Hamilton, Ph.D.-Department of Molecular Physiology & Biophysics
Two immunophilins, FKBP12 and FKBP12.6, regulate the activity of cardiac
Ca2+ release channels (RyR2). Mice with a cardiac-specific deficiency in FKBP12
display an early onset dilated cardiomyopathy. To define the mechanisms that drive
this myopathy, we assessed changes in Ca2+ handling in isolated cardiomyocytes.
Ca2+ spark frequency and amplitude are increased by FKBP12 deficiency. We found
that this increase in SR leak is due to decreased SR stores and increased cytoplasmic
Ca2+ levels following 1 Hz pacing. In addition, we found that there was increased
extracellular Ca2+ influx into the cytoplasm during 1 Hz pacing.
Female FKBP12 deficient mice also displayed dilated cardiomyopathy but
following birth have an increased risk of dying due to heart failure. We found that 25%
of all births from FKBP12 deficient females led to the development of heart failure and
subsequent death. In summary, these results indicate that FKBP12 plays a vital role in
cardiac function at both baseline and during pregnancy, and Ca2+ handling in
cardiomyocytes.
Contributors: Oakes, Joshua; Hanna, Amy; Hamilton, Susan
248
EMERGENCE OF PENICILLIN-NONSUSCEPTIBLE PNEUMOCOCCAL SEROTYPE
35B AMONG U.S. CHILDREN
Liset C Olarte
Advisor: Sheldon Kaplan, M.D.-Department of Pediatrics
Background: Streptococcus pneumoniae serotype 35B is a nonvaccine serotype
associated with high rates of penicillin-nonsusceptibility. Its prevalence has increased
since the introduction of the 13-valent pneumococcal conjugate vaccine. We describe
the epidemiology of serotype 35B in US children.
Methods: We identified patients with serotype 35B invasive pneumococcal
disease (IPD) from 1994-2014 at 8 US children’s hospitals from our surveillance
database. Pneumococcal isolates were collected prospectively. Serotyping and
antibiotic susceptibility testing were performed at a central laboratory. Multidrug
resistance (MDR) was defined as penicillin-nonsusceptibility (MIC ≥ 0.12 μg/ml) plus
resistance to ≥2 non-β-lactam antimicrobials. Multilocus sequence typing was
performed on 2008-2014 isolates. Descriptive statistics were used.
Results: We identified 76 serotype 35B cases (1994-2014); 54% occurred in
2010-2014. Serotype 35B IPD represented 0.1% of all IPD cases in 1994-1999, 0.9% in
2000-2004, 2.2% in 2005-2009 and 5.9% in 2010-2014 (Chi square for trend p<0.001).
In 2014, the most common serotype was 35B, accounting for 14% of IPD cases. The
median age was 19 months (IQR 8.4 -72.0). Thirty-five patients (46.1%) had an
underlying condition. Bacteremia represented 43.4% of cases, meningitis 23.7%,
pneumonia 10.5% and bone/joint infections 7.9%. 87% of isolates were penicillinnonsusceptible and 14.6% had a penicillin MIC ≥2μg/ml. 17.1% of isolates were MDR.
The most common genotype was ST558 (30/53; 56.6%), 17% of them were
MDR. ST156 (associated with MDR vaccine serotypes like 9V) represented 11.3% of
the isolates; 83.3% were MDR. ST156 was first observed in 2011.
Conclusions: Serotype 35B has emerged as the most common serotype causing
IPD in 2014 in our study. High rates of penicillin-nonsusceptibility and MDR are
concerning. Genotyping suggests capsular switching has occurred between vaccine
serotypes and serotype 35B.
Contributors: Olarte, Liset; Barson, William; Romero, Jose; Tan, Tina; Hoffman, Jill; Lin,
Philana; Givner, Larry; Bradley, John; Hulten, Kristina; Mason, Edward; Kaplan, Sheldon.
249
EPIGENOMIC DECONVOLUTION YIELDS INSIGHTS INTO THE BIOLOGY OF
BREAST TUMOR CELLS WITHIN THEIR NATIVE MICROENVIRONMENT
Vitor Onuchic
Tumor phenotypes result from interactions of a diversity of cell types and yet it is
currently not possible to measure epigenomic and transcriptomic states of constituent
cell types without perturbing their interactions by physically isolating them from their
microenvironment. To gain insights into cancer-related processes within epigenetically
defined subpopulations of breast tumor cells within their native microenvironment, we
developed Epigenomic Deconvolution (EDec), a two-stage procedure for estimating
methylation and gene expression profiles of constituent cell types, as well as
proportions of constituent cell types in each input sample. We validate high accuracy of
EDec using both experimental and simulated mixtures of purified cell types in known
proportions. When applied to 1184 breast tumor methylation profiles from the TCGA
collection the method infers methylation profiles of constituent cell types that closely
match the reference methylation profiles of cell types known to constitute breast
tumors. The inferred cell type proportions are highly concordant with pathologist’s
estimates based on H&E staining. By comparing the cell type specific gene expression
profiles, we detected gene expression changes that are highly consistent with known
hallmarks of cancer. We confirmed significantly longer survival for triple negative breast
cancer patients with high immune cell infiltration within their tumors. We detected downregulation of cellular respiration pathways in triple negative tumor associated stroma in
conjunction with an up-regulation of those same pathways in the cancerous epithelial
cells, a metabolic shift consistent with the previously proposed Reverse Warburg Effect.
Lastly, by analyzing gene expression changes that are specific to epithelial cells of triple
negative tumors, we identified gene expression changes in a large number of SP1
regulated genes, including CDH1. Such changes are consistent with the downregulation of SP1 that is also identified specifically in epithelial cells of basal-like breast
cancers. The down-regulation of SP1 is consistent with a single copy deletion affecting
the SP1 gene that is present in nearly 60% of basal-like breast cancers, but is rarely
detected in other subtypes of breast cancer. Despite not being previously reported, the
down-regulation of SP1 and deregulation of its targets specifically in basal-like breast
cancers is highly consistent with the more aggressive and EMT-like phenotype of that
subtype of breast cancers. We show that these cancer cell perturbations could not be
detected without EDec because of averaging across diverse cell types within complex
tumor tissue. We therefore conclude that EDec in conjunction with newly available
reference epigenomes provides unique new insights into the biology of tumor cells
within their native microenvironment.
Contributors: Onuchic, Vitor; Hartmaier, Ryan; Boone, David; Oesterreich, Steffi; Samuels,
Michael L.; Roth, Matt E.; Lee, Adrian V.; Milosavljevic, Aleksandar
250
THE INFLUENCE OF CHOLINERGIC SIGNALING ON FEEDING IN THE AMYGDALA
Joshua Ortiz
To maintain balanced energy requirements, energy expenditure must be properly
matched with caloric intake. Deficits to either of these is associated with a variety of
diseases which may affect the immune system, nervous system, internal organs, and
may also lead various eating and/or metabolic disorders. In fact, obesity-associated
diseases constitute the leading causes of death in the United States. Importantly, food
intake is largely controlled in the brain. Although the hypothalamus has been identified
as a master regulator controlling feeding behavior, it has recently been found that other
brain regions play important roles in this process. In our lab, the diagonal band of
Broca (DBB), a cholinergic signaling center in the basal forebrain, has been identified
as an important regulator of food intake. Anatomical tracing studies from these
cholinergic neurons revealed dense cholinergic innervation to the amygdala. While
classic lesion-based studies have shown that the amygdala has an important role in
regulating food intake, little is known about the underlying circuitries which control this
process. Investigating the role of cholinergic signaling within the amygdala may provide
novel insight into feeding regulation and may help advance the development of novel
strategies to treat feeding-associated disorders. Therefore, we hypothesize that
cholinergic signaling in the amygdala influences feeding behavior. To test this
hypothesis, we are currently implementing optogenetic strategies to enhance or inhibit
cholinergic drive from the DBB to the amygdala to better understand how cholinergic
signaling mechanisms influence feeding behavior. Furthermore, we are currently
generating novel tools to profile gene expression in candidate feeding-associated cell
types to identify changes in gene expression which may be actively influenced by
cholinergic signaling.
Contributors:
251
NEUROENDOCRINE REGULATION OF LIPID HOMEOSTASIS IN C. ELEGANS
Ayse Sena Ozseker
My project aims to dissect the regulation of lipid storage and metabolism by
sensory perception using Caenorhabditis elegans as a model organism. Based on the
preliminary studies, my hypothesis is that signaling in sensory neurons modulates the
release of neuroendocrine factors, which transduce signals to systemically regulate lipid
homeostasis.
To find the neurons and neuropeptides that are involved in the regulation of lipid
storage, I have examined the lipid storage in 30 chemosensation mutants using
stimulated Raman scattering (SRS) microscopy. SRS microscopy is a quantitative
chemical imaging technology that does not require labeling lipids or fixing worms and
assesses the levels of triacylglycerides, the main lipid storage form in the worm adipose
tissue, the intestine. Our best hit from this screen are the daf-11 mutants that have
increased lipid levels in the intestine. daf-11 mutants lack a guanylyl cyclase expressed
in five different head sensory neurons all of which show defective neuronal signaling in
the mutant worms.
The intestine of C. elegans is not innervated and daf-11 mutants display normal
food intake, suggesting that secreted factors from these sensory neurons regulate
intestinal lipid storage in a cell non-autonomous manner. Consistent with this
hypothesis, EGL-21, a carboxypeptidase essential for neuropeptide processing, is
required for the lipid storage increase in daf-11 mutants. Currently, we are analyzing
which neuropeptides are differentially expressed in daf-11 mutants in comparison to
wild-type. But in the meantime, we performed an RNAi screen targeting all of the known
G-protein coupled receptors (GPCRs) in C. elegans and found that two GPCRs, srh266 and T23F1.3, are required for the increase in lipid levels. Both of these receptors
are expressed in multiple tissues including the intestine.
Additionally, inactivation of the worm homolog of FoxO transcription factor, daf16, suppresses the increased lipid storage phenotype of daf-11 mutants. daf-16 have 3
different isoforms: a, b and d/f. We found that specifically daf-16d/f is required for the
increase in lipid levels. daf-16d/f is expressed in multiple tissues such as intestine,
hypodermis and neurons and our results showed that daf-16d/f regulates lipid levels
cell-autonomously in the intestine.
Currently, we are performing a mosaic analysis to find which neurons require
daf-11 activity to regulate lipid storage in the intestine. After identifying the neurons, we
will study their necessity and sufficiency via cell ablation and optogenetic activation,
respectively.
Contributors: Ozseker, Ayse Sena; Wang, Meng
252
A MATHEMATICAL MODEL OF TEBS REVEALS CELLULAR DYNAMICS
INVOLVED IN DUCTAL MORPHOGENESIS
Ingrid Sophie Paine
Advisor: Michael Lewis, Ph.D.-Department of Molecular & Cellular Biology
Mathematics is often used to model biological systems. In mammary gland
development, mathematical modeling has been limited to branching morphogenesis
and breast cancer, without reference to normal duct formation. There are currently no
published models of mammary ductal elongation, a critical process required for, and
intimately coupled with, branching morphogenesis. We sought to fill this gap by
generating a mathematical model of mammary ductal elongation that incorporates the
structure responsible for the majority of pubertal development, the terminal end bud
(TEB).
We present a model of ductal elongation that exploits the geometricallyconstrained shape of the terminal end bud (TEB), the growing tip of the duct, and
incorporates morphometrics, region-specific proliferation and apoptosis rates.
Iterative model refinement and behavior analysis compared with biological data
indicated that the traditional metric of percent fat pad filled when used to evaluate
ductal elongation can be misleading, as it disregards branching events that can reduce
its magnitude. Further, activated caspase-3 staining revealed the clearance of cap cells
present in the body cell layer, thus mitigating their contribution to the luminal population.
Additionally, results from the model suggest that 54% of new born cap cells are not
contributing to the mature duct, raising questions about the stemness of the cells.
Finally, CC3-mediated apoptosis of true body cells, postulated to serve as a mechanism
of lumen formation, occurs at lower levels than previously reported. We performed a
validation by using a fitting process and found that our experimentally derived cap cell
flux values fall within error or those derived from model fitting and that the fit process
again suggests higher levels of cell clearance exist in the TEB.
We conclude that our model contains all the necessary parameters to accurately
predict ductal elongation and is robust compared to a model fit method. Future work will
be to expand our model to include branching phenomena as well as incorporate
additional levels of detail, including molecular signaling, cell type-specific activities, and
stromal influences
Contributors: Paine, Ingrid Sophie1; Chauviere, Arnaud2; Landua John D.1; Sreekumar,
Amulya1; Cristini, Vittorio3; Rosen, Jeffrey M1.; Lewis, Michael T. 1
253
THE TWIK-2 POTASSIUM CHANNEL’S ROLE IN THE DEVELOPMENT
PULMONARY HYPERTENSION IN A HYPOXIC MOUSE MODEL
Lavannya M. Pandit
Advisor: Robert Bryan, Ph.D.-Department of Anesthesiology
BACKGROUND: Pulmonary hypertension (PH) is a complex pathologic state in
which the molecular mechanisms are not well understood. The recently discovered
two-pore potassium channels (K2P) , including the Tandem of P-domains in a Weakly
Inward Rectifying Potassium (TWIK-2) , exist in the pulmonary vascular smooth muscle
cells (PVSMCs) and may regulate pulmonary vascular tone and contractility, thus
contributing the development of pulmonary hypertension.
HYPOTHESIS: We hypothesized that loss of function of the TWIK-2 channel
produces PH in mice, and that 3 weeks 10% hypoxic exposure exacerbates a PH
phenotype in TWIK-2 knockout mice.
METHODS: We performed right-sided cardiac catheterization, lung
histopathology, and electrophysiology studies in 20-24 week old male TWIK-2 KO mice
on a mixed SV129/BL6 background as well as TWIK-2 KO mice C57/BL6 backgrounds,
as well as wildtype age-matched controls on the same backgrounds.
RESULTS: The TWIK-2 KO mice on the SV129/BL6 background developed PH
at 20 weeks but the TWIK-2 mice on the C57/BL6 background did not, as measured by
right–sided cardiac catheterization. Histopathology in both the C57/BL6 and SV129/BL6
background did reveal smooth muscle layer (medial) hypertrophy suggestive of vascular
remodeling seen in PH. Under hypoxic conditions, all the TWIK-2 KO mice (both
SV129/BL6 and C57/BL6 backgrounds) developed PH as measured by right-sided
cardiac catheterization, but these elevations in pulmonary pressures did not differ from
the age-matched wildtype controls of similar genetic background. Electrophysiology
studies revealed that proximal PVSMCs from TWIK-2 KO mice on a SV129/BL6 appear
to be more depolarized compared to wildtype controls, but PVSMCs from TWIK-2 KO
mice on the C57/BL6 background do differ in membrane potential compared to WT
controls from the same genetic background.
CONCLUSIONS: the development of PH in the TWIK-2 KO mice appear to be
dependent on the genetic background. The contribution of TWIK-2 channel to the
development of PH and regulation of PVSMC contractility appears to be strain
dependent which could provide clues to the development of PH in humans.
Contributors: Kitagawa, Melanie; Reynolds, Julia; Marrelli, Sean; Bryan, Robert
254
MACROMOLECULAR THERAPY FOR LIVER DISEASE
Francis Peter Pankowicz
Advisor: Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
While gene therapy holds great potential for treating a wide variety of
diseases, vector delivery remains a significant obstacle to overcome. Viral vector
delivery systems are among the most promising, but are hindered by varied tissuespecificity and immunogenic effects. Adeno-Associated Virus (AAV) is a nonintegrating virus that can infect both dividing and non-dividing cells, and is currently
being evaluated for gene therapy potential due to its diminished immune response in
humans. Tissue-specificity for AAV is mediated by its capsid serotype, some of which
have been studied and tested in a clinical trial setting. Our work focuses on identifying
the most suitable AAV serotype for gene transfer in the human liver. Using AAV with
known serotypes as well as recently isolated novel serotypes, we have highlighted the
dramatic differences between in vitro and in vivo transduction efficiency. Furthermore,
by utilizing a human liver chimeric mouse, we have demonstrated the preferential
infection of hepatocytes from a species-specific perspective. Finally, we have identified
AAV9 as the serotype most suitable for transduction of human hepatocytes in vivo
(human liver chimeric mouse).
Contributors: Pankowicz, Francis; Wang, Lili; Bell, Peter; Bissig- Choisat, Beatrice; Barzi
Dieguez, Mercedes; Legras, Xavier; Wieland, Stefan; Bissig, Karl-Dimiter
255
CONTRIBUTION OF APICAL DENDRITES TO ORIENTATION SELECTIVITY IN
LAYER 2/3 OF MOUSE V1
Ji Young Park
Advisor: Stelios Smirnakis, M.D./Ph.D.-Department of Neurology
Although slice recordings and model studies provide a lot of information about
how neurons integrate their inputs to overall responses, it is still not clear how neurons
in the brain integrate their synaptic inputs to derive their functional properties in vivo.
Pyramidal neurons in layer 2/3 of mouse V1 receive input from LGN via layer 4 and
feedback from higher visual areas. Here, we focus on the function of apical dendrites,
which play an important role in communicating with higher visual areas. We ablate
apical dendrites of layer 2/3 pyramidal neurons in area V1, in vivo, and monitor their
responses to visual stimuli by two photon microscopy. We compare orientation
selectivity of L2/3 pyramidal neurons before and after selective apical dendritic ablation.
This approach provides information on how different dendritic inputs contribute to
neuronal receptive field properties of L2/3 pyramidal neurons in mouse V1.
Contributors: Park,Jiyoung; Smirnakis,Stelios M.
256
CHARACTERISTICS OF ELDERLY AND YOUNGER PATIENTS WITH COPD IN THE
COPGENE AND ECLIPSE COHORTS
Amit Dilip Parulekar
Advisor: Nicola Hanania, M.B.,B.S./M.S.-Department of Medicine
Rationale: The prevalence of chronic obstructive pulmonary disease (COPD)
and its associated comorbidities increase with age. However, little is understood about
the disease in patients over 65 years of age.
Objective: To determine disease characteristics of COPD and its impact in older
patients compared with younger patients.
Methods: A cross-sectional analysis was performed of participants with COPD
and GOLD class II-IV airflow limitation in 2 cohorts: Genetic Epidemiology of COPD
Study (COPDGene) and Evaluation of COPD Longitudinally to Identify Predictive
Surrogate Endpoints (ECLIPSE). We compared demographics, indices of disease
severity, prevalence of comorbidities, exacerbation frequency, and quality of life scores
in patients ≥ 65 versus patients < 65 years old. We tested for associations of age with
exacerbation frequency, quality of life, six minute walk distance, and radiographic
evidence of emphysema and air trapping.
Measurements and Main Results: In the COPDGene cohort, older patients
(n=1663) had more severe disease as measured by FEV1 (1.22L vs. 1.52L, p<0.001),
oxygen use (35% vs. 22%, p<0.001), six minute walk distance (355m vs. 375m,
p<0.001), and radiographic evidence of emphysema (14% vs. 8%, p<0.001) and air
trapping (47% vs. 36%, p<0.001) and were more likely to have comorbidities compared
with younger patients (n=2027). Similarly, in the ECLIPSE cohort, older patients
(n=1030) had lower FEV1 (1.22L vs. 1.34L, p<0.001), greater oxygen use (7% vs. 5%,
p=0.02), lower six minute walk distance (360m vs. 389m, p<0.001), and more
radiographic evidence of emphysema (17% vs. 14%, p=0.009) than younger patients
(n=1131). In both cohorts, increasing age was associated with decreased frequency of
exacerbations (OR = 0.52, 95%CI = 0.43-0.64 in COPDGene, OR=0.79, 95%CI = 0.640.99 in ECLIPSE) and a lower SGRQ score (β=‒8.7, 95%CI = ‒10.0- ‒7.4 in
COPDGene, β=‒4.4, 95%CI = ‒6.1- ‒3.2).
Conclusions: Despite greater severity of illness, older patients with COPD had
better quality of life and reported fewer exacerbations than younger patients. This may
result from the fact that older patients change their expectations and learn to adapt to
the disease.
Contributors: Parulekar, Amit; Tsai, Chu-Lin; Locantore, Nick; Wise, Robert; Demeo, Dawn;
Regan, Elizabeth; Make, Barry; Atik, Mustafa; Mohsin, Ali; Sharafkhaneh, Amir; Kao, Christina;
Hanania, Nicola
257
CHARACTERIZATION OF THE GLOBAL STABILIZING MUTATION A77V AND ITS
ROLE IN THE EVOLUTION OF THE CTX-M CLASS OF β-LACTAMASE ENZYMES
Maha Praful Patel
The introduction of new β-lactam antibiotics into clinical use drives the evolution
of β-lactamase enzymes. β-lactamases gain the ability to hydrolyze new antibiotics by
acquiring amino acid substitutions within their active site. The introduction of thirdgeneration cephalosporin antibiotics led to the emergence of the CTX-M family of βlactamases. These enzymes are frequently identified in antibiotic-resistant clinical
samples and are named for their enhanced ability to hydrolyze cefotaxime over
ceftazidime. Recently, CTX-M enzymes carrying the active site-associated adaptive
mutations P167S and D240G have been identified in resistant isolates. CTX-M variants
carrying these substitutions have an enhanced ability to hydrolyze ceftazidime at the
cost of stability. Proteins can offset a loss in stability by acquiring compensatory
secondary mutations. The A77V mutation has been identified in combination with the
P167S and D240G substitutions and its prevalence emphasizes its role in the evolution
of CTX-M β-lactamases.
We demonstrate that the A77V mutation acts as a stabilizing mutation to offset
the stability defects caused by the P167S and D240G substitutions, allowing the
enzyme to retain stability while acquiring the ability to hydrolyze ceftazidime. Sitedirected mutagenesis was used to introduce A77V, P167S and D240G as single and
double mutations into the CTX-M-14 model enzyme. The mutant enzymes were
characterized using kinetic analysis, competition assays, immunoblot analysis and
thermostability assays. Overall, when combined with the P167S or D240G substitution,
A77V increases the thermal stability and in vivo steady-state expression level of the
enzyme, resulting in increased resistance to cefotaxime and ceftazidime without a
change in catalytic efficiency for the hydrolysis of substrates. The increased thermal
stability of enzymes containing A77V may provide an evolutionary advantage by
allowing the accumulation of destabilizing mutations that broaden their substrate profile.
The results suggest that the effect of drug resistance mutations on protein stability is
important in the evolution of β-lactamases.
Contributors: Patel, Maha; Fryszczyn, Bartlomiej; Palzkill, Timothy
258
AN ALTERED DIVISIVE NORMALIZATION MODEL OF AUTISM
Jaclyn Sky Patterson
Autism is a neurodevelopmental disorder marked by a diverse set of symptoms
including perceptual, social, and cognitive atypicalities. This heterogeneity presents a
significant challenge to establishing a comprehensive characterization of the disorder.
The widespread effect of the disorder on neural systems suggests that autism may
broadly impact neural computations as opposed to isolated systems. As such, we
hypothesize that alterations in canonical computations that occur throughout the brain
may underlie the behavioral characteristics of autism. Here we focus on one
computation in particular, divisive normalization, which balances a neuron’s net
excitation with inhibition reflecting the combined activity of a population of neurons.
Divisive normalization inherently reflects the ratio of neural excitation to inhibition, which
is believed to be abnormally elevated in autism. In the present work, we show that an
altered divisive normalization signal which elevates the excitatory/inhibitory ratio can
account for perceptual findings in autism. Specifically, we develop a neural network
model of primary visual cortex (V1) in which individual units are selective for stimulus
location and orientation. An increased E/I ratio is simulated in the model by reducing
the strength of the inhibitory divisive normalization signal reflecting the population
activity. To examine how this alteration might give rise to perceptual autism
symptomatology, we simulate two perceptual studies comparing the behavior of
typically developing controls and individuals with autism on tasks that strongly engage
V1. The first, a motion discrimination task employing stimuli of different sizes and
contrasts, revealed reduced surround suppression and overall better discrimination
performance in autism for high contrast stimuli, but equivalent performance across the
groups at low contrasts. The second, a feature detection task investigating the
facilitating effect of an attentional cue, reported a sharper gradient of attention in autism
than in controls. Interestingly, we find that the results of both studies could be
accounted for by the same alteration in divisive normalization. Our results suggest that
the divisive normalization framework can provide novel insights into the neural basis
autism and generate hypotheses that are readily testable by psychophysics
experiments. In future work, it should be possible to adapt this framework to other
sensory modalities as well as more complex operations such as facial processing which
require hierarchical processing.
Contributors: Patterson, Jaclyn; Rosenberg, Ari; Angelaki, Dora
259
ESR1 MUTATIONS CONFER CELL-TYPE SPECIFIC RESISTANCE THROUGH
ACTIVATION OF IGF1R
Sasha M Pejerrey
The rediscovery of estrogen receptor (ESR1) mutations in human metastatic
breast cancer is a current clinical scenario. We have modeled the three most frequent
hormone binding ESR1 (HBD-ESR1) mutations using stable lentiviral vectors in multiple
human breast cancer cell lines. We have determined that these mutations confer
relative resistance to tamoxifen (Tam) in a cell-type specific manner due to distinct
epigenetic changes introduced through these mutations. Resistance has only been
observed with concomitant engagement and activation of the insulin growth factor
signaling pathway (IGF1R). The ESR1 mutant-expressing cells also exhibited enhanced
binding with the insulin growth factor receptor beta (IGF1Rβ). Mutant-expressing cells
have also demonstrated a more migratory and invasive phenotype in a cell-type specific
manner. The selective estrogen degrader, fulvestrant, significantly reduced the
anchorage-independent growth of ESR1 mutant-expressing cells, while combination
treatment with the mTOR inhibitor, everolimus, restored Tam sensitivity. Since we
detected relatively high frequencies of these three mutations in primary breast tumors,
our results suggest that clinical targeted sequencing of both primary and metastatic
tumors may be justified, and combination therapies considered. Further
characterization of these mutant-expressing cells is also warranted due to their overall
more aggressive and invasive phenotype, and their relative-resistance to traditional
therapies.
Contributors: Pejerrey, Sasha; Gelsomino, Luca; Gu, Guowei; Rechoum, Yassine; Fuqua,
Suzanne
260
EFFECT OF ALTERNATIVE 3’ UNTRANSLATED REGION SECONDARY RNA
STRUCTURE ON DENGUE VIRUS PATHOGENESIS
Kassandra Leigh Peterson
Advisor: Rebecca Rico, M.P.H.-Department of Molecular Virology & Microbiology
Dengue virus (DENV) is a positive-sense, nonsegmented, single-stranded RNA
flavivirus that causes disease in approximately 50-100 million people annually. The
virus is subdivided into four serotypes: DENVs -1, -2, -3, and -4. Together, these
viruses are the causative agents of dengue fever (DF), dengue hemorrhagic fever
(DHF) and dengue shock syndrome (DSS). DHF and DSS have more severe clinical
manifestations and higher mortality rates. DENV serotypes 2 and 3 are reported to
cause the most cases of DHF. Despite previous circulation of DENV-2 American
Genotype (AMG), no DHF cases were observed in the Americas until introduction of
DENV-2 Southeast Asian Genotype (SEAG).
SEAG differs from AMG at several points in the viral genome. Sequence and
secondary RNA structure variance is observed in the 3’ untranslated region (3’UTR)
across genotypes. This study proposes to determine the differences in translation and
replication efficiencies between the SEAG and AMG. It is critical to determine
differences in replication and translation rates and how this affects the kinetics of early
host immune response. Following investigation of 3’UTR primary sequence variance,
the ability to form secondary RNA structure pseudoknots (PKs) will be compared across
genotypes. PK structure and location determine a flavivirus’ ability to form subgenomic
flaviviral RNAs (sfRNAs). sfRNAs are 3’UTR fragments that interfere with host cell
processes during infection, though the exact mechanisms and extent of the effects are
unknown. This study aims to characterize how each of these 3’UTR elements affect
viral replication, translation, and pathogenesis. Characterization of these 3’UTR
elements can advance vaccine development efforts by determining mechanisms for
viral attenuation. I hypothesize that SEAG causes more severe disease than AMG
because PK structures in the 3’UTR promote more efficient viral protein translation,
enable more efficient viral replication, and generate different sfRNAs.
Contributors:
261
CLINICAL INTERVENTIONS ABROGATE LETHAL STRUCTURAL AND
FUNCTIONAL PULMONARY DEFECTS IN NEONATAL ERK3-/- KNOCKOUT MICE
Braden Kyle Pew
Dennis Bier, M.D.-Department of Pediatrics
Neonatal respiratory distress syndrome (NRDS) is associated with preterm birth
and is the leading cause of death in the neonatal period. Rates of infant mortality due to
pulmonary insufficiency have improved with the introduction of antenatal glucocorticoid
treatment and surfactant replacement. However, the mechanism by which
glucocorticoids promote fetal lung maturation remains poorly understood despite
decades of clinical use. We have developed an Erk3-/- murine model of neonatal
respiratory distress syndrome, in which pups expire shortly after birth due to pulmonary
immaturity without immediate intervention. We have previously found alterations in
expression of genes critical to lung function associated with ERK3 loss as well as
histological changes in response to glucocorticoid therapy. Here we have sought to
further characterize the structural changes associated with neonatal lethality due to
respiratory distress using high resolution micro-CT analysis of murine fetal lungs.
Dexamethasone (0.4 mg/kg) or saline was administered to dams on days E16.5
and E17.5 of gestation, during the saccular stage of development, and fetal lungs were
extracted at E18.5. Lungs were fixed in paraformaldehyde and embedded in stabilizing
gel to preserve pulmonary structure. After overnight staining in 1% iodine, tissues were
imaged on the SkyScan 1272 MicroCT at 70 kV, 142 uA, and 0.2° step angle, for a
voxel size of 3 micrometers and 3K resolution. Images were analyzed using CTAn by
Bruker and ImageJ.
Relative to wild-type littermates, Erk3-/- mice exhibited smaller lung tissue
volume, with decreased porosity and saccular space as well as areas of atelectasis.
These pulmonary defects are partially reversed by glucocorticoid treatment.
Using a murine model of NRDS and high resolution tomography, we have
observed gross morphological alterations in lung structure associated with ERK3 loss
which is abrogated by standard clinical treatments in neonatal care. Additional
characterization of structural alterations in pulmonary development and architecture
associated with NRDS are underway to determine the effect of perinatal clinical
interventions on postnatal lung structure.
Contributors: Pew, Braden; Cuevas-Guaman, Milenka; Shope, Cindy; Dickinson, Mary;
Aagaard, Kjersti
262
ASCC2 IS REQUIRED FOR CARDIAC SPECIFICATION AND CELL CYCLE
REGULATION IN THE POST-IMPLANTATION MOUSE EMBRYO
Victor George Piazza
Advisor: Mary Dickinson, Ph.D.-Department of Molecular Physiology & Biophysics
Background: Activating Signal Co-integrator Complex 1 subunit 2 (Ascc2) is a
transcriptional co-activator known to enhance SRF, NF-KB, and AP-1 transactivation.
Through our involvement in the Knock-Out Mouse Phenotyping (KOMP) project we
have identified Ascc2 as essential for embryonic viability at the post-implantation stage.
The KOMP project utilizes a gene targeting strategy that promotes expression of an
IRES:LACZ under the control of the endogenous promoter, thus allowing us to track
Ascc2 spatiotemporal expression patterns. We aim to determine how disruption of
Ascc2 expression affects cardiac specification and cell cycle during development. The
main aims of this project are to: 1) Characterize the loss of Ascc2 in post-implantation
development, 2) Determine when and where Ascc2 is normally expressed during postimplantation, and 3) Determine the consequences of Ascc2 loss on cardiac marker
spatiotemporal expression patterns.
Materials and Methods: We are utilizing the benefits of the IRES:LACZ in mice
carrying the Ascc2Tm1b allele to analyze spatiotemporal Ascc2 expression in the
mouse embryo, with a focus on stages E7.5-E8.5. We are performing RNA in situ
hybridization to confirm expression patterns of Ascc2 as well as cardiac markers Nkx2.5, MesP1, and Islet1. We are also using lightsheet fluorescence microscopy to
generate 3D images of these markers in wild type and knock-out embryos.
Results and Conclusions: Ascc2-null embryos die before E9.5 with
developmental delay and defects, exhibiting hypoplastic heart and forebrain at this
point, with a thinned heart wall. LacZ data indicates ubiquitous expression of Ascc2 in
the embryo and extra-embryonic yolk sac from E6.5 to E12.5. Analysis at E8.5 has
shown us that Ascc2 loss affects heart tube formation and function as the Ascc2 heart
tubes are smaller and a heart beat cannot be detected in the most severe null embryos.
Interestingly, analysis of mesoderm and early cardiac marker expression using qRTPCR performed on E8.5 embryos show an increase in Mesp1 and Brachyury. Given
this result it is possible that mesoderm cells are not properly specified or fail to
contribute to heart formation. We are currently using lightsheet microscopy to analyze
the localization of mesodermal and cardiac markers in whole embryos and we are
performing in situ hybridization to determine if mesoderm markers are upregulated or
the expression domain has expanded.
Contributors: Piazza, Victor; Wong, Leeyean; Li, Jade; Pang, Paul; Poche, Ross; Dickinson,
Mary
263
ACTIVITY-DEPENDENT TRANSCRIPTIONAL AND CHROMATIN DYNAMICS IN
MECP2 DISORDERS
Amy E Pohodich
Methyl-CpG-Binding Protein 2 (MeCP2) was first described as a transcriptional
repressor that binds to methylated DNA and became the focus of much research after
the discovery that mutations in MECP2 cause Rett syndrome (RTT). RTT is a postnatal
neurodevelopmental disorder characterized by a period of seemingly normal
development followed by a progressive loss of acquired speech, cognitive abilities, and
motor coordination. Stereotyped hand movements replace purposeful hand use, and
seizures and autonomic dysfunction develop within a few years of disease onset. The
importance of having the correct level of MeCP2 in the brain is underscored by the fact
that duplications and triplications spanning MECP2 produce syndromic intellectual
disability, progressive motor dysfunction, and seizures. In RTT patients, the delayed
onset of disease coincides with the timing of peak stimulus-driven synaptic
reorganization required for appropriate circuit formation and neuronal maturation, which
led to the hypothesis that MeCP2 is important for establishing, refining, and/or
maintaining synapses. Consistent with this hypothesis are the numerous abnormalities
observed in MeCP2-deficient mice, including changes in basal transmission,
presynaptic function, and alterations in the balance between excitatory and inhibitory
signaling. Currently, however, the precise mechanisms by which MeCP2 can contribute
to neuronal plasticity are unclear. To gain insight into the role of MeCP2 during
neuronal activity in the mature brain, this project utilizes deep brain stimulation (DBS) to
elicit activation of the dentate gyrus (DG) in awake, freely moving mice. Using this
paradigm, we are assessing the genome-wide transcriptional requirements for MeCP2
upon neuronal activation by analyzing activity-dependent gene expression in wild-type,
MeCP2-deficient, and MeCP2 over-expressing mice. In all three genotypes, we see
robust activity-dependent gene expression following DBS, and we have identified 31
genes whose increased expression after stimulation may be MeCP2-dependent,
including tribbles homolog 1 (Trib1) and immediate early response 2 (Ier2). Additionally,
to determine whether changes in MeCP2 binding partners might contribute to activitydependent MeCP2 functions, we are analyzing MeCP2 protein interactions at baseline
and following DBS in wild-type mice. Preliminary results have revealed 5 novel MeCP2binding partners that show increased binding with neuronal activity, and these proteins
are involved in transcription regulation, histone modification and RNA processing.
Ultimately, we will pair the transcriptional and protein interaction data with analysis of
changes in MeCP2 localization to yield insight into MeCP2 function in neurons.
Contributors: Pohodich, Amy; Raman, Ayush; Liu, Zhandong; Zoghbi, Huda
264
OBESTATIN ENHANCES GLUCOSE-STIMULATED INSULIN SECRETION
THROUGH GHRELIN RECEPTOR
Geetali Pradhan
Advisor: Yuxiang Sun, M.D./Ph.D.-Department of Pediatrics
Susan Samson, M.D.-Department of Medicine
Background: Obestatin, a 23 amino acid peptide derived from the ghrelin gene,
is expressed in various tissues including the stomach and pancreas (1). Obestatin is
known to reduce food intake and body weight, improve memory and regulate sleep, but
no effect on secretion of growth hormone and corticosterone. Obestatin is also
suggested to increase mass and survival of pancreatic β cells (2) but it’s effect on
insulin secretion remains unclear. In studies using CHO cells expressing GHS-R, it has
been demonstrated that obestatin failed to increase Gs mediated cAMP unlike ghrelin
(3), suggesting no binding. Further, GPR 39 (3) and GLP-1R (2) have been suggested
to be the endogenous receptor of obestatin. To date, the receptor of obestatin is
unclear and controversial.
Methods: We studied the effect of obestatin on insulin secretion under glucosestimulated condition both in vitro and ex vivo using rat insulinoma (β cell) INS-1 cells
and mouse pancreatic islets (WT, Ghrelin-/- and Ghsr-/- islets). To determine whether
the effect of obestatin on insulin secretion is mediated through the ghrelin receptor,
Growth Hormone Secretagogue Receptor (GHS-R), we used islets from our GHS-R
knockout mouse model. We also transiently knocked down GHS-R in INS-1 cells and
pharmacologically blocked GHS-R using antagonists YIL 781 and JMV2959.
Results: Obestatin treatment of INS1 cells and obestatin deficient Ghrelin-/islets significantly increased glucose stimulated insulin secretion (GSIS) compared to
no obestatin treatment. Interestingly, incubation of pancreatic islets from GHS-R KO
mice with obestatin had significantly lower insulin response compare to controls.
Furthermore, treatment of obestatin in GHS-R knockdown INS-1 cells also showed
similar effect on insulin secretion under glucose-stimulated condition. Even
pharmacologically blocking GHS-R in INS-1 cells using antagonists also exhibited lower
effect on GSIS compared to scramble. Overall, our results indicate that obestatin has
profound stimulatory effect on insulin secretion in both INS-1 cells and mouse
pancreatic islets; this effect is potentially mediated via GHS-R in β cells.
Conclusions: Our studies indicate that obestatin is a potent insulin secretagogue
under glucose-stimulated condition. Obestatin-stimulated GSIS is likely mediated via
GHS-R in pancreatic β cells. Obestatin’s stimulatory effect on insulin secretion and
promoting effect on β cell survival together make obestatin a powerful therapeutic
candidate for Type 2 diabetes.
Contributors: Pradhan, Geetali; Lee, JongHan; Sun, Yuxiang
265
PROFILE OF INTESTINAL MICROBIOME IN INFANTS WITH SHORT BOWEL
SYNDROME
Muralidhar H Premkumar
Purpose of study: SBS is a common cause of gastrointestinal mortality and
morbidity in newborn infants. Though microbiome-altering interventions such as
antibiotics or probiotics are commonly used, the exact profile of intestinal microbiome in
infants with SBS is unknown. We describe the profile and evolution of intestinal
microbiome in infants with SBS.
Methods used: SBS included those infants who underwent an abdominal surgical
intervention and if they needed total parenteral nutrition for over 4 weeks. Controls were
gestationally age-matched infants without gastrointestinal morbidities. Stool samples
from the diapers or from the stoma were collected once a week. DNA was extracted
using PowerMag Microbiome isolation kit (MoBio) and the bacterial V4 region of the 16s
rRNA gene was amplified by PCR and sequenced on the MiSeq (Illumina) platform.
Sequencing reads were analyzed using QIIME, and the SILVA database was used for
operational taxonomic unit (OTU) classification.
Summary of results: 255 stool samples were analyzed from 16 cases and 21
controls. Cases included 4 gastroschisis, 5 small intestinal perforations, 4 with NEC &
perforation, 1 NEC, 1 volvulus and 1 jejunal atresia.
Infants with SBS showed
decreased diversity and abundance of microbiome. Decreased diversity of microbiome
was associated with Cesarean section, absence of oral feeds and use of antibiotics.
Infants fed with artificial formula demonstrated higher diversity compared to breast fed
infants. infants with complicated gastroschisis and NEC demonstrated the least
diversity.
In SBS there were decreased lachnospiracea, lactobacillus, while
pseudomonas, Escherichia, Shigella were more abundant.
Conclusions: This is the first ever description of intestinal micriobiome profile in
infants with SBS. We have demonstrated that infants with SBS have decreased
abundance, diversity and evenness of microbiome. More significantly, within the group
of SBS, the diversity, evenness and abundance decreased with complications. Defining
the microbiome in infants with SBS, will refine existing gut microbiome altering
therapies and promote development of new strategies.
Contributors: Premkumar, Muralidhar, MBBS, DCH, MRCPCH, Ajami, Nadim, PhD, Gordon,
Pamela, RNC-NIC, Chakkalakal, Smrithi, Petrosino, Joseph, PhD and Lee, Brendan, MD, Phd.
266
Antibiotic Cross Resistance Mutation is Induced by Ciprofloxacin in a General
Stress-Response-Controlled Mutagenic Break Repair
John P Pribis
Antibiotic resistance is a leading health threat worldwide estimated to cost >$20
billion annually, with de novo mutations a leading cause. We discovered a large a gene
network controlling mutagenic repair of DNA breaks (MBR), with stress response
regulators as main hubs, provoking mutations preferentially when cells are maladapted
to their environment. The SOS DNA-damage and general (RpoS) stress responses
switch DNA break repair to mutagenic by upregulating error-prone DNA polymerases
and allowing their use in repair. Here, we show that DNA-damaging quinolone antibiotic
ciprofloxacin (cipro) induces mutagenesis to cross resistance in E. coli dependently on
key MBR proteins. First, sub-MIC cipro levels increase mutation rate to ampicillin
(AmpR)- and rifampicin-resistance (RifR) 14±4 and 29±8-fold. These result from knockout and alteration of ampD and rpoB genes, respectively, confirmed by sequencing.
Second, the cross-resistant mutants have no survival advantage in cipro, indicating that
cipro induces mutagenesis, rather than selecting cross resistance. Third, cipro-induced
AmpR and RifR mutagenesis requires MBR proteins including SOS-response
regulators LexA and RecA, error-prone DNA polymerases IV, II and V, and
recombinational break-repair proteins RecB and RuvC, required for survival of cipro
(break repair/SOS) and accumulation of cipro-resistant mutants—though whether
mainly via selection was unclear. The cross-resistance assays demonstrate induction
of mutagenesis.
Fourth, surprisingly, cipro-induced mutagenesis also requires
functional RpoS, activator of the general stress-response, not known previously to be
induced by cipro/quinolones, but by starvation and many other stressors. The data
show that cipro induces mutations per se and cross-resistance to unrelated antibiotics,
and support a MBR mechanism underpinned by SOS and general stress responses.
Ampicillin also induces RpoS-dependent Pol IV-dependent mutagenesis, with
similarities to MBR, shown previously. The stress response regulators are attractive
candidates for proposed novel “anti-evolvability” drugs to block mutagenesis and inhibit
evolution of antibiotic resistance.
Contributors: Pribis, John P; García-Villada, Libertad; Rogers, Elizabeth; Rosenberg, Susan M
267
UNDERSTANDING THE ACTIVATION MECHANISM OF CGMP-DEPENDENT
PROTEIN KINASE (PKG) I
Liying Qin
Advisor: Choel Kim, Ph.D.-Department of Pharmacology
Type I cGMP-dependent protein kinase (PKG I) is a serine/threonine kinase that
regulates smooth muscle tone, vasorelaxation and platelet aggregation. It is a
homodimer and each monomer contains an N-terminal regulatory domain (R-domain)
and a C-terminal catalytic domain (C-domain). The R-domain includes a leucine zipper
(LZ) domain which mediates dimerization of the protein, an autoinhibitory (AI) sequence
which binds to the C-domain and inhibits the kinase activity, and two tandem cGMPbinding domains (cNBDs). Based on solution structural studies of PKG I and crystal
structure of cAMP-dependent protein kinase (PKA), the putative working model of PKG
I is that the release of R-domain from C-domain caused by cGMP binding to the cNBDs
results in kinase activation. However, the molecular details of PKG activation
mechanism remains unknown.
To study the activation mechanism of PKG I, we decided to crystalize its
holoenzyme and determine its crystal structure. However, the holoenzyme with both Rand C-domain on the same polypeptide is too flexible to crystalize and the full-length
protein purified from insect cells suffers from cAMP and cGMP contamination.
Therefore, we divide the kinase into two fragments: the R-domain and the C-domain.
To obtain a true apo state of the R-domain, we have chosen a special E. coli strain
TP2000 for protein expression because it is adenylate cyclase deficient, thus can
express nucleotide-free R-domain. To determine the boundary of C-domain for cocrystallization with R-domain, we have cloned a few constructs of the C-domain. One of
the constructs has been optimized for baculovirus expression and the purified protein
has been crystallized. The crystals diffracted to 2.6 Å. We are currently optimizing the
crystallization and cryo-protection conditions for the crystals. At the same time, we are
cloning the R-domain for crystallization of the R:C complex.
Contributors: Qin, Liying; Sankaran, Banumathi
268
SPEG (STRIATED MUSCLE PREFERENTIALLY EXPRESSED GENE) FACILITATES
PROPER CARDIAC CONTRACTILITY BY REGULATION OF RYANODINE
RECEPTOR TYPE 2 MEDIATED CALCIUM RELEASE
Ann Pepper Quick
Advisor: Xander Wehrens, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
BACKGROUND Heart failure is one of the leading causes of hospitalization, yet there
remains a lack of targeted therapeutic options. Calcium (Ca) dysregulation through disruption of
Ca handling proteins including Ryanodine receptor type 2 (RyR2) and Juntophilin-2 (JPH2) has
been implicated in heart failure. So, to better understand the regulation of cardiac Ca release
we implemented co-immunoprecipitation (coIP) of RyR2 and JPH2 from mouse heart lysate
followed by mass spectrometry. This revealed only one protein (SPEG) that pulled down with
each of these proteins respectively. SPEG is necessary for cardiac development, but it is
unknown whether SPEG is essential for adult cardiac function. Based on the preliminary data
and the literature, we hypothesize that SPEG maintains proper adult cardiac function by
regulating RyR2 Ca release through binding and phosphorylating RyR2 and JPH2. Gene
therapy with adeno-associated virus serotype 9 (AAV9) is a new strategy to target potentially
therapeutic agents to the heart. We propose to use AAV9 gene therapy with “miniSPEG” to
rescue SPEG loss of function.
METHODS Recombinant SPEG was co-transfected with RyR2 or JPH2 in HEK293
cells. Co-IP was performed to confirm SPEG binding to RyR2 and JPH2. This process was
repeated using truncations of SPEG to determine which portion of SPEG binds RyR2 and
JPH2. Alanine mutagenesis was used to determine the binding region. Transthoracic
echocardiography was performed on anesthetized SPEG inducible, cardiomyocyte-specific
conditional knockout (cKO) mice at baseline and 4 and 8 weeks post tamoxifen injection.
Echocardiograms were analyzed using Vevo 2100 to determine wall dimensions and cardiac
function. At 8 weeks post tamoxifen injection, mice were humanely euthanized and
cardiomyocytes were isolated and used for either a) Ca imaging with fluo-4AM b) T-tubule
staining with di-8-anneps, or c) co-localization by immunofluorescence using antibodies to JPH2
and RyR2. Cells were imaged by confocal microscopy and analyzed using Image J.
RESULTS. In vitro studies confirmed that full length SPEG binds RyR2 and JPH2. RyR2
pulled down a myc-tagged SPEG amino acid 1-861 (out of 3262 amino acids), which was
confirmed by reciprocal coIP with anti-myc. Further truncations narrowed the binding site to aa:
187-712 a region which contains many proline rich binding motifs. Alanine mutagenesis of
several proline residues did not disrupt SPEG-RYR2 binding. Jph2 binds SPEG toward the
Cterminal in a region tnear the kinase domains. The in vivo studies showed that eight weeks
post tamoxifen injection, cKO mice developed heart failure with an average ejection fraction of
27.1%+/-17.7% compared to 66.3% in the control. SPEG cKO cells exhibited > 2-fold increased
Ca spark frequency and deceased transient amplitude. T-tubule power was decreased by 50%
in SPEG cKO cells and JPH2-RyR2 co-localization was decreased in SPEG cKO
cardiomyocytes (R= .19) compared to controls (R=.42).
CONCLUSIONS. SPEG is necessary for adult cardiac function in mice. Cellular studies
confirm that SPEG cKO mice develop Ca dysregulation, T-tubule disruption, and decreased
JPH2-RyR2 co-localization indicative of heart failure. These studies will need to be repeated
before the development of heart failure in cKO mice in order to determine whether loss of
SPEG or heart failure itself is the cause of disrupted Ca homeostasis. Molecular studies
indicate that RyR2 binds to a proline rich SPEG Nterminal. However, further fragmentation and
mutagenesis is necessary in order to determine a binding region. This information will be used
to study the kinase activity of SPEG on RyR2. Further studies will focus on AAV9-mediated
rescue of SPEG cKO mice. Contributors: Quick, Ann; Wang, Qiongling; Chiang, David;
Reynolds, Julia; Buxton, Sam; Showell, Jordan; Wehrens, Xander HT
269
DISCOVERY OF PLASMODIUM FALCIPARUM EXP1 AS A GLUTATHIONE
TRANSFERASE INHIBITED BY ARTESUNATE
Joel Patrick Quiros
The malaria parasite Plasmodium falciparum causes the most deadly form of
malaria in humans. According to the World Health Organization, an estimated 198
million cases of malaria infection were reported in 2014 with 584,000 resulting in death.
Before the discovery of artemisinin, widespread use of the antimalarial chloroquine has
lead to the emergence of drug resistant P. falciparum strains. In these resistant strains,
one up-regulated and important, yet functionally unknown gene is the exported antigen
1 (EXP1), a human antigen and vaccine candidate that is exported to the parasital
vacuole membrane and food vacuole. EXP1 is essential and may play an important role
in malaria and in its human immune response. Here, we functionally predict and
biochemically characterize EXP1 as a membrane glutathione S-transferase using a
computational network method. EXP1 efficiently degrades cytotoxic hematin, is potently
inhibited by artesunate, and is associated with artesunate metabolism and susceptibility
in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action
of a frontline antimalarial drug.
Contributors: Lisewski, Andreas; Ng, Caroline; Adikesavan, Anbu; Miura, Kazutoyo; Putluri,
Nagireddy; Eastman, Richard; Scanfeld, Daniel; Regenbogen, Sam; Altenhofen, Lindsey;
Llinás, Manuel; Sreekumar, Arun; Long, Carole; Fidock, David; Lichtarge, Olivier
270
ESTIMATE OF THE REBINDING RATE DURING RUPTURE OF MEMBRANECYTOSKELETON BONDS IN A HNSCC CANCEL CELL
Vivek Rajasekharan
Advisor: Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
Introduction. The Rho family of Ras-related guanosine triphosphate (GTP)
binding proteins (e.g., Rho A) when active contribute directly to the magnitude and
dynamics of membrane-cytoskeleton linkages by binding both to the membrane and
effector proteins (e.g., Arp2/3) to form a membrane – F-actin linkage. Method. To
determine the contribution (direct or indirect) of active Rho GTPases to the linkages at
the cytoskeleton-membrane we measure the time course of the membrane-cytoskeletal
rupture force in HN31 cells with an optical tweezers at slow loading rates (< 60 pN/s) in
the presence and absence of drugs that inhibit cycling of Rho GTPases. We use a cell
line derived from a metastasis-derived head and neck squamous cell carcinoma
(HNSCC) as it exhibits a missense mutation in HRAS, and the Rho GTPases (e.g.,
Ras, Rac 1, RhoA and Cdc42) are persistently active. Results. At a constant loading
rate a time delay, tdelay is observed before the bonds start to fail after which the force
increases monotonically for time (t rupturing at force, f*. Comparing events from all
cells we find the delay exhibits an exponential distribution with a time constant (15 s-1)
and that (t increases monotonically with f*. We extract bond characteristics by
comparing the experimental data with theory that calculates the lifetime of a cluster of
bonds under shared constant loading with and without rebinding (J. Chem. Phys. 2004
121. 8997), and estimate the maximum number of bonds at four (4), off-rate:
<koff(f=0)>: 1 s-1, bond length, <xb>: 0.25 nm and <(>: kon(f=0))/koff(f=0) at 6-15.
Similar results are observed when the cells are treated with microtubulin-disrupting
drug, nocodazole. When HN31-cells are incubated in Clostridium difficile toxin B active
Rho GTPase is reduced by up to 50% and tdelay is observed much less frequently
which suggests a different cluster of bonds is present at the membrane-cytoskeleton
interface. Conclusions. Detection of a delay suggests the loading rate is below the
mean equilibrium value for bond rupture, i.e., rebinding is faster than unbinding as
described (e.g., Proceedings of the Royal Society of London. Series B, Biological
Sciences, 1988 234: 55-83). This provides a way to experimentally estimate the
intractable <kon(f=0)> of the membrane-cytoskeleton bonds which is at least 15 s-1 in
this system. Further experiments at slower loading rates (< <60 pN/s) will confirm
whether this is close to f →0 limit.
Contributors: Rajasekharan, Vivek; Sreenivasan, Varun; Myers, Jeffrey N; Pereira, Fred A;
Farrell, Brenda;
271
NEUROENDOCRINE REGULATION OF LYSOSOMAL FUNCTION IN C. ELEGANS
AND MICE
Prasanna V Ramachandran
Neurodegenerative disorders are devastating conditions that impose significant
burden on a growing aging population. Although degeneration is limited to the nervous
system, serious disease manifestations often affect peripheral metabolic tissues
through unknown mechanisms. Our research focuses on the neuronal regulation of
peripheral metabolic processes by the evolutionarily conserved protein Tubby.
Mutations in Tubby, a neuronally expressed gene, result in blindness and deafness as
well as obesity in mammals. We found that in C. elegans, loss of function of the Tubby
homolog tub-1 also resulted in analogous phenotypes. Interestingly, neuronal tub-1
deficiency led to abnormal lysosomal activity in the intestine, the C. elegans fat storage
tissue. tub-1 mutants showed misregulated expression of lysosomal genes, and
excessive intestinal accumulation of lysosomal markers. Lysosomal and metabolic
derangements were also observed in mice mutant for Tubby – we discovered that
livers of homozygous Tubby mice exhibited misregulated expression of lysosomal
enzymes and steatohepatosis. These findings reveal a neuroendocrine signaling axis
wherein neuronal Tubby/tub-1 regulates peripheral lysosomal function and metabolism.
We sought to elucidate both upstream and downstream components of this novel axis.
A candidate-based approach revealed ser-5, a G protein-coupled receptor, as the
upstream activator of tub-1 in neurons. IP-mass spectrometry analysis identified a G
protein subunit, gpc-1, as a putative direct TUB-1 interactor in neurons. Additionally, we
discovered that a majority of neuropeptides are downregulated in tub-1 mutants.
Further in silico analysis revealed sptf-1 as a putative tub-1 dependent master regulator
of neuropeptide synthesis.
Contributors: Ramachandran, Prasanna V.; Wang, Meng C.
272
BATCH CORRECTION ALGORITHM FOR NEXT-GEN SEQUENCING DATASETS
Ayush Raman
Advisor: Zhandong Liu, Ph.D.-Department of Pediatrics
Batch effects are sub-groups of unwanted variations that have different behavior
across conditions and are unrelated to the biological or scientific variables in a study.
Many of the genomics studies have showed that many of the significant results like
differential expression are confounded with sample preparation date, samples prepared
by different individuals or sample sequenced using different platforms. It is one of the
one of the most common problems in analyzing Next Gen Sequencing (NGS) datasets.
The three types of batch correction algorithms are:
Batch Correction with known batch effects – Eg: ComBat
Batch Correction with unknown batch effects, where the algorithm estimates the
batch effects – Eg: sva/svaseq
Using database of previous measurements and try to estimate the batch effects
– Eg: barcode
Moreover, we don’t have a good statistical model for the analysis of RNA-Seq
data after removing batch effects. We present our batch effects algorithm based on
linear regression model with known batch effects. We estimate the biological variables
like conditions and treatment using our model. We also use our method to correctly
predict the batch based on the regression coefficients. Our initial findings on RNA-Seq
datasets related to MeCP2 syndromes (from Dr. Zoghbi’s group) have showed that our
algorithm works better than ComBat and sva, -- the two most common batch removal
software. Furthermore, we use our method to obtain a batch free dataset that could be
use for down stream analysis like differential gene and transcript expression analysis of
RNA-seq datasets.
Contributors:
Raman, Ayush; Pohodich, Amy; Zoghbi, Huda; Liu, Zhandong
273
DOT1L AS A THERAPEUTIC TARGET FOR THE TREATMENT OF DNMT3AMUTANT ACUTE MYELOID LEUKEMIA
Rachel Rau
Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute
myeloid leukemia and portend a poor prognosis, thus new therapeutic strategies are
needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis
is altered DNA methylation and the attendant gene expression changes, however our
current understanding is incomplete. We observed that murine HSCs in which Dnmt3a
had been conditionally deleted markedly overexpress the histone 3, lysine 79 (H3K79)
methyltransferase, Dot1l. We demonstrate that Dnmt3-null HSCs have increased
H3K79 methylation relative to wild type HSCs, with the greatest increases noted at DNA
methylation canyons, which are regions highly enriched for genes dysregulated in
leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led
us to explore DOT1L as a therapeutic target for the treatment of human DNMT3Amutant AML. We show that pharmacologic inhibition of DOT1L suppressed the
expression of oncogenic canyon-associated genes and led to dose- and timedependent inhibition of cellular proliferation, induction of apoptosis, cell cycle arrest and
terminal differentiation in DNMT3A-muant cell lines in vitro. We also show in vivo
efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3Amutant AML. DOT1L inhibition was also effective against primary patient AML samples
with DNMT3A mutations, reducing colony forming capacity and inducing terminal
differentiation in vitro. These studies suggest that DOT1L may play a critical role in
DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L being already in
clinical trials, DOT1L could be an immediately actionable therapeutic target for the
treatment of this poor prognosis disease.
Contributors: Luo, Min; Rodriguez, Benjamin; Jeong, Mira; Rosen, Allison; Rogers, Jason;
Campbell, Carley; Daigle, Scott; Deng, Lishing; Song, Yongcheng; Sweet, Steve; Chevassut,
Timothy; Andreeff, Michael; Li, Wei; and Goodell, Margaret A.
274
COLLABORATIVE FILTERING FOR PRECISION MEDICINE: INTEGRATING AND
PREDICTING MULTI-ENTITY INTERACTIONS
Sam Julian Regenbogen
Modern medicine produces copious information, but currently lacks the ability to
fully capitalize on it. Specifically, there is considerable need to integrate knowledge from
disparate studies to discover connections across domains. We used a Collaborative
Filtering approach, inspired by online recommendation algorithms, in which nonnegative matrix factorization is used to predict interactions among chemicals, genes,
and diseases utilizing only information about how they connect to one another. Our
approach, applied to matrices derived from the Comparative Toxicogenomics
Database, successfully recovered Chemical-Disease, Chemical-Gene, and DiseaseGene interactions in 10-fold cross-validation experiments. Additionally, we successfully
predicted each of these interaction matrices from the other two. Integrating all three
CTD interaction matrices, we used NMF to predict STRING, an external network of
protein-protein interactions. Finally, our approach was able to integrate the CTD and
STRING interaction data to improve Chemical-Gene cross-validation performance
significantly, and to retrospectively predict new information added to CTD. Our
approach, which can integrate new data with little modification, will be a useful addition
to the precision medicine and drug repurposing toolkits.
Contributors: Wilkins, Angela; Lichtarge, Olivier
275
TRANSPORT MECHANISM OF THE EIIC GLUCOSE SUPERFAMILY OF
TRANSPORTERS
Zhenning Ren
Advisor: Ming Zhou, Ph.D.-Department of Biochemistry & Molecular Biology
Carbohydrate
transporters
of
the
phosphoenolpyruvate:carbohydrate
phosphotransferase system are crucial for sugar uptake in bacteria; however, little is
known concerning how these proteins recognize and transport carbohydrates
across the cell membrane. Here we report the 2.55 Å structure and functional
characterization of a maltose transporter, bcMalT. The bcMalT structure is in an
outward-facing conformation, in contrast with the previous structure of an Ndiacetylchitobiose transporter in an inward-facing conformation. These structures
identified a mobile transport domain that may undergo a roughly 20 Å rigid-body
movement to provide alternating access to the bound substrate from either side of the
membrane. Crosslinking of pairs of cysteine residues that are distant in the crystal
structures but are predicted to move close to each other in the alternate conformation
provides further support for the large-scale movement of the transport domain. These
results provide a mechanistic framework for understanding substrate recognition and
translocation.
Contributors: McCoy, Jason; Stanevich, Vitali;
276
COMPARING K-MEANS CLUSTERING TO GRAPH DISSECTION IN A HIGH
DIMENSIONAL BIOLOGICAL DATA SET
Alexander Renwick
Sophisticated methods exist for high dimensional biological data, but they are not
widely used. Clear demonstration of their superiority to more familiar methods would
encourage their adoption. This paper carriers out a transcriptome clustering analysis
using data derived from an influenza vaccination study. It compares clusters generated
by k-means to those generated by a dissection of a graphical model (the Graphical
Lasso). The resulting clusters are evaluated in terms of biological significance by testing
for KEGG pathway enrichment. Out of a data set of 631 transcripts (96 subjects), kmeans identified ten clusters and the graph dissection method identified eleven. The
two methods showed similar enrichment patterns, both finding pathways related to
immune system function and to Influenza A specifically.
Contributors: Renwick, Alexander; Shaw, Chad; Belmont, John
277
THE ROLE OF ESTROGEN IN METASTASIS
Natalie Michelle Reyes
Background: Although 75% of breast tumors are ER(-postive, there are no ERpositive preclinical models which reliably metastasize to study the effects of hormones
on invasion and metastasis. We have studied the role of estrogen in metastasis in an
ER(-positive MCF-7 sub-line (MCF-7 Met) which has spontaneously acquired the ability
to metastasize in vivo.
Experimental design and methods:
Microarray analysis was performed
comparing MCF-7 Met to parental, non-metastatic MCF-7 cells. The differential gene
expression was compared to genes differentially expressed when cells expressed
K303R ER alpha. We performed soft agar growth assays, mammosphere assays,
signal transduction assays and ERE luciferase assays. We injected MCF-7 Met and
MCF-7 cells into athymic nude mice to study in vivo growth properties.
Results: We found that the MCF-7 Met line shared 66% gene expression
similarity with cells expressing K303R ER alpha. We also found that upon hormone
treatment the MCF-7 Met cells had higher levels of pS118 and pS305 ER alpha, which
was observed in cells expressing K303R ER alpha. The K303R ER alpha model also
had an increased in anchorage independent growth under estrogen treatment and we
observed similar results in the MCF-7 met line. The MCF-7 Met line was able to form
more spheres during a mammosphere assay when compared to MCF-7. In preliminary
results we also observed estrogen hypersentivity in the MCF-7 Met line during ERE
luciferase assay, which is another phenotype that was observed in cells expressing the
K303R ER alpha mutation. 94% of the mice injected with the MCF-7 Met line
metastasized to distant sites regardless of hormonal treatment but their was no
difference in primary tumor growth when treated with Tam or estrogen withdrawal.
Conclusion: I have determined that many of the genes differentially expressed in
MCF-7 Met cells are related to estrogen signaling and tamoxifen resistance using
bioinformatic approaches. This suggests that the processes of invasion and metastasis
may be influenced by the hormonal mileau. I have also determined that the MCF-7 Met
line may share a similar phenotype to cells expressing the K303R ER alpha mutation.
Although MCF-7 Met cells remain hormone-dependent in vivo, they have acquired the
ability to metastasize, potentially suggesting that hormone resistance is not a
prerequisite for ER-positive breast cancer cells to metastazise to distant sites.
Contributors: Reyes, Natalie M; Corona, Arnoldo; Covington, Kyle; Gu, Guowei, Rechoum,
Yassine; Fuqua, Suzanne AW
278
A NOVEL NON-SECOSTEROIDAL VDR AGONIST, VDRM2, INHIBITS PROSTATE
TUMOR GROWTH
Justin Michael Roberts
Advisor: Nancy Weigel, Ph.D.-Department of Molecular & Cellular Biology
The role of vitamin D signaling in prostate cancer (PCa) is controversial. Some
studies find an inverse correlation between levels of vitamin D metabolites and PCa
risk, but others do not. The active metabolite 1α,25-dihydroxyvitamin D3 (1,25D), a
ligand for the vitamin D receptor (VDR), inhibits the growth of PCa cells in vitro. 1,25D
or less calcemic analogs also inhibit growth in some pre-clinical models, but clinical
trials have been disappointing. A majority of PCas contain a genomic rearrangement
that links the androgen and 1,25D regulated TMPRSS2 promoter to the coding region
of an ETS transcription factor, typically ERG, termed T/E. VCaP cells are the only
commonly used cell line that contains the T/E fusion. We have shown previously that
VDR agonists inhibit VCaP cell growth in vitro despite inducing T/E. However, EB1089,
a 1,25D analog, which inhibited growth of LNCaP xenografts, had no effect on VCaP
xenografts. VDR and ERG cooperate to hyper-induce the vitamin D metabolizing
enzyme, CYP24A1. Thus, T/E may limit VDR signaling through inactivation of 1,25D.
To counteract the induction of CYP24A1 in VCaP cells, we tested a novel nonsecosteroidal VDR agonist, VDRM2, developed by Eli Lilly & Co. VDRM2 inhibits
LNCaP and VCaP cell growth and is resistant to metabolism. Using a subcutaneous
VCaP xenograft model, we saw a significant reduction in tumor volume and tumor mass
in mice treated with 3 ug/kg of VDRM2 compared to vehicle. Importantly, we achieved
this effect without causing hypercalcemia as indicated through serum calcium levels.
Additionally, an RNA-Seq was performed in both LNCaP and VCaP cells to further
elucidate the mechanism of VDR action and ligand-specific biology. By utilizing a novel
VDR agonist, we have overcome two of the principle limitations of VDR agonist use
clinically.
Contributors: Roberts, Justin; Mackrell, James; Rocha, Guilherme; Krishnan, Gary; Weigel,
Nancy
279
PROTEIN SUMOYLATION IS ESSENTIAL FOR OOCYTE DEVELOPMENT AND
FEMALE FERTILITY
Amanda Rodriguez
Advisor: Stephanie Pangas, Ph.D.-Department of Pathology & Immunology
1-2% of women suffer from early menopause or primary ovarian insufficiency
(POI), a common cause of infertility. Women with POI suffer from long-term health
risks, which include dementia, increased heart disease, osteoporosis, and premature
death. Strikingly, in 90% of POI cases, the cause is idiopathic. Two major mechanisms
that contribute to POI are ovarian follicle dysfunction or oocyte depletion. Mouse
models have been instrumental in identifying POI candidate genes, which include
oocyte-specific transcription factors such as newborn ovary homeobox gene (Nobox)
and spermatogenesis and oogenesis basic helix-loop helix gene (Sohlh1 and Sohlh2).
Although it is well established that these transcription factors are necessary for the
formation, activation, and development of oocytes, surprisingly little is known about how
their activity is regulated. We performed an in silico analysis of the oocyte-specific
transcription factors and found a conserved consensus site for a post-translational
modification called SUMOylation. In recent years, SUMOylation has emerged as an
important regulator of transcription, as well as other cellular processes including protein
localization (cytoplasmic or nuclear), protein-protein interactions, or protein stability. A
central component of the SUMOylation cascade is the SUMO-conjugating enzyme,
UBC9. Conventional knockout of Ubc9 in mice leads to embryonic lethality during the
early post-implantation stage, therefore, we generated a novel oocyte-specific knockout
of Ubc9 (termed Ubc9 cKO). Ubc9 cKO female mice are sterile and have almost
complete depletion of oocytes by two months of age. Prior to depletion, these mice
have compound ovarian defects in follicle development, ovulation, and oocyte meiotic
maturation. Our results also demonstrate that SUMOylation regulates oocyte-specific
transcription factors through several different mechanisms, including altered stability,
activity and nucleo-cytoplasmic shuttling. Additionally, RNA sequencing results
comparing wild-type and Ubc9 cKO oocytes identified distinct gene expression patterns
in both primary and secondary follicles, indicating a defective transition between these
two stages in Ubc9 cKO ovaries. Understanding how oocyte-specific proteins are
regulated by SUMOylation may hold the key to managing fertility in women with POI, or
uncovering the basis of their disease. Our novel mouse model provides a key discovery
tool for deciphering candidate pathways involved in ovarian dysfunction, and
establishes SUMOylation as an essential process for during oocyte development.
Contributors: Rodriguez, Amanda; Tripurani, Swamy; Demarque, Maud; Dejean, Anne; Pangas,
Stephanie
280
IN VIVO AND IN VITRO METABOLISM OF NEO HDL, A NOVEL HDL-LIKE
PARTICLE
Perla Janet Rodriguez
Advisor: Henry Pownall, Ph.D.-Department of Medicine
Serum opacity factor (SOF), a protein produced by S. pyogenes, causes
clouding of human plasma. In vitro, SOF catalyzes the disproportionation of plasma
high density lipoproteins (HDL), giving lipid-free
apo A-I, a small neo HDL that is apo A-II-rich and cholesterol poor, and a
cholesteryl ester rich microemulsion (CERM). In vitro tests showed that the neo HDL
supports several reverse cholesterol transport (RCT) steps and that it has improved
atheroprotective qualities over HDL. Mice injected a low dose (4 mg) rSOF had reduced
plasma cholesterol ~50% in three hours. Size exclusion chromatography reveals that
the CERM is prominent in the plasma profile in vivo. However, lipid-free apo A-I and
neo
HDL in vivo occur at <1% of their in vitro levels. The in vivo fate of neo HDL is
not yet known.
Neo HDL cholesterol ester (CE) [3H] was incubated with human plasma and
each isolated lipoprotein, with or without lipoprotein deficient serum (LPDS), which
contains lipoprotein modifying enzymes cholesteryl ester transfer protein (CETP),
phospholipid transfer protein (PLTP), and LCAT. Over time, CE transferred to all
lipoproteins in the presence of LPDS. Neo HDL-CE transferred to HDL in the absence
of LPDS but not to the other lipoproteins. In mouse plasma, the CE transferred to HDL
at a much faster rate. These results suggest a potential fusion between neo HDL and
HDL. Torcetrapib and DTNB, CETP and LCAT inhibitors, were added to similar
experiments which resulted in partial inhibition of neo HDL-CE transfer to all
lipoproteins suggesting involvement in neo HDL catabolism. Adding an LCAT inhibitor
alone to these studies had little effect, suggesting LCAT does not contribute to neo HDL
catabolism or fusion to HDL.
Neo HDL-CE [3H] uptake studies were performed using three CHO cell lines, two
of which express low endogenous levels of SR-BI and one overexpressing murine SRBI. CE uptake from neo HDL only occurred when SR-BI was overexpressed. Similar
experiments in human hepatocytes showed an increased uptake of neo HDL-CE
compared to CHO cell lines, suggesting uptake of neo HDL might be by a different
mechanism. WT mice treated with either HDL or neo HDL show that unlike HDL, neo
HDL is immediately taken up by the liver, consistent with in vitro data. Our hypothesis is
that Neo HDL fuses to nascent HDL or becomes lipidated quickly, potentially interacting
with SR-BI and other hepatic receptors
Contributors: Rodriguez, Perla; Gillard Baiba; Rosales, Corina; Pownall Henry
281
THE ROLE OF GLIAL ACTIVATION IN BLOOD-BRAIN BARRIER PERMEABILITY
Ethan Roy
Claudia Robertson, M.D.-Department of Neurosurgery
Alzheimer disease (AD) is a progressive form of neurodegenerative dementia.
The prevalence of AD is expected to dramatically increase in the coming decades, and
yet there has been little progress toward any effective therapy. This fact is mostly due to
a lack of knowledge of the pathophysiology that causes cognitive decline and
neurodegeneration in AD.
One hypothesis of AD pathogenesis that is currently gaining scientific support is
known as the “vascular hypothesis.” It suggests that the primary pathologies of AD may
first affect the blood vessels in the brain and their ability to provide needed metabolic
resources. The healthy brain possesses blood vessels that are highly specialized, an
anatomical feature known as the blood-brain barrier (BBB). This complex structure
disallows most substances entry from the circulation into the brain. The BBB is known
to be functionally diminished in the brains of AD patients. It is thought that the
neurovasculature is negatively impacted early in AD progression, preceding other
pathology, and that neurons are eventually killed by both chronic starvation from
nutrients and by exposure to toxic blood plasma components.
Though the mechanisms behind the pathophysiology of AD are not yet fully
understood, there are a few conserved features, including amyloid β plaques as well as
the activation of glial cells, primarily astrocytes. The overall goal of this project is to
elucidate mechanisms by which integrity of the neurovasculature can be modulated by
inflammatory signals from activated glial cells in the brain.
We have generated a mouse model wherein NFκB signaling is upregulated in
astrocytes, mimicking a state of inflammatory activation in these cells. One prominent
phenotype observed in vivo is increased BBB permeability. This finding may provide a
link between Aβ pathology, which is known to cause NFκB activation in glial cells, and
BBB dysfunction. Here we are investigating one possible mediator of the NFκBdependent BBB disruption, namely the complement system, which was shown to be
increased in our mouse model, and is known to be aberrantly active in AD. We
hypothesize that, in the context of an AD mouse model, Aβ stimulation of astrocytes
causes BBB permeability through an NFκB/complement-dependent pathway, and that
blocking the pathway will rescue BBB integrity.
Contributors: Roy, E; Lian, H; Guo, Q; Zheng, H
282
DICTYOSTELIUM DISCOIDUEM SOCIALITY DURING GROWTH WITH GRAMNEGATIVE BACTERIA
Michelle Jenny Rubin
Dictyostelium discoideum is a eukaryotic amoeba that consumes bacteria during
growth. Upon starvation, Dictyostelium cells cooperate with one another while
developing into multicellular structures in which some of the cells sacrifice themselves
while supporting the sporulation of the others. Currently, sociality is defined as
intercellular interactions during Dictyostelium development, but we propose that social
interactions occur during vegetative growth as well. We have generated many
Dictyostelium mutants that do not grow on Gram-negative bacteria and we tested the
cell-autonomous properties of the phenotype. Interestingly, when we mixed just 10%
wild type Dictyostelium cells with one of the mutants, we observed that the mutant grew
on Gram-negative bacteria. Additionally, when we plated the mutant at different cell
densities on Gram-negative bacteria, the cells were able to grow at high cell densities.
Therefore, we hypothesize that Dictyostelium growth on bacteria is a social process that
is mediated by cooperative predation. We will determine whether social interactions
take place during growth on Gram-negative bacteria and we will use biochemical and
genetic tools to determine the identity and the roles of the secreted factors in the
process.
Contributors: Rubin, Michelle; Kuspa, Adam; Shaulsky, Gad
283
CHARACTERIZATION OF LIGHT ADAPTATION INDUCED ALTERATIONS IN
MOUSE RETINAL GANGLION CELL SPATIOTEMPORAL TUNING
Jasdeep Singh Sabharwal
Advisor: Samuel Wu, Ph.D.-Department of Ophthalmology
Reverse correlation methods such as spike-triggered averaging consistently
identify the spatial center in the linear receptive fields (RFs) of retinal ganglion cells
(GCs). However, the spatial antagonistic surround observed in classical experiments
has proven more elusive. Tests for the antagonistic surround have relied on models
that make simplifying assumptions such as space-time separability and radial
homogeneity/symmetry. We circumvented these, along with other common
assumptions, and sought to develop a more accurate model which would account for
the antagonistic surround and inseparability predicted to exist in GCs.
We observed a linear antagonistic surround in 754 of 805 mouse GCs. By
characterizing the RF’s space-time structure, we found the overall linear RF's
inseparability could be accounted for both by differences between the center and
surround and differences within the surround. By using a model with five RF space-time
subcomponents we were able to parameterize properties of the RGC while maintaining
space-time inseparability. We then probed the space-time STA with a model prediction
of the surround we identified considerable asymmetry in the RF surround. These results
shed new light on the spatiotemporal organization of GC linear RFs and highlight a
major contributor to its inseparability.
Contributors: Sabharwal, Jasdeep; Cowan, Cameron; Wu, Samuel
284
EXPERT SYSTEM: AN INTEGRATED APPROACH TO THE DETERMINATION OF
CAUSATIVE VARIANTS IN RETINAL EYE DISEASE.
Jason Scott Salvo
Purpose: With the decreasing cost of genomic sequencing, data driven analysis
has become an increasingly important aspect of molecular diagnosis. Larger data
sources have vastly improved post-sequencing processes such as annotation and
filtering, yet ultimate determination of molecular causality is still left to experts of the
field. Our goal was therefore, to design an algorithm whereby a list of patient variants
could be scored and ranked according to likeliness of disease causality using
information gleaned from large stores of genomic data. Such a system would need to
learn from ever growing datasets, continuously evolving and optimizing.
Methods: We have developed a statistical framework that integrates gene-disease
association, inheritance pattern, and functional prediction, to rank mutant genes in a
patient. Taking advantage of previously published mutant alleles and our internal
database, we estimated the prior probabilities of each gene associated with a specific
disease. This study focused on common retinal diseases of which many genes were
associated with multiple diseases. The algorithm scores genes as likely to be causative
based on known disease associations, and ability to fit a given inheritance pattern.
Functional prediction scores were integrated as a means to define variant potency, with
normalization
based
on
genic
level
analysis
of
common
variants.
Results: The algorithm was trained with gene-disease association data from more than
1000 patients with various retinal diseases. We tested the algorithm on a separate
cohort of 30 patients and compared the results to molecular diagnoses determined by
human experts. Strikingly, our results showed over 85% correlation between the
algorithm and human analysis. Of 20 high confidence human calls, 18 algorithmic calls
where correlated. The algorithm was similarly unable to call 3 of 4 samples with no
human determination, while discovering a possibly overlooked causative gene in the
fourth
of
RP1L1.
Conclusions: RES represents a significant step toward streamlining NGS based
molecular diagnosis as it prioritizes the most likely disease causing genes through
quantification, allowing researchers and diagnosticians alike to work efficiently and
effectively. More importantly, this tool will continue to improve with the accumulation of
larger data sets. Similar methods can be implemented for other human diseases as
well.
Contributors: Salvo, Jason S.; Zaneveld, Eric; Chen, Rui
285
TENASCIN C AS AN EFFECTOR OF PROSTATE CANCER DERIVED BONE
METASTASIS
Rebeca San Martin
Advisor: David Rowley, Ph.D.-Department of Molecular & Cellular Biology
The purpose of this study is to characterize the microenvironment changes in
prostate cancer bone metastasis in the context of a reactive stroma response, and to
evaluate how these changes affect metastatic colonization and proliferation.
The stroma plays an important role in the maintenance of tissue homeostasis.
Stroma associated with secretory epithelium initiates an efficient wound repair response
in the event of a breach in the epithelial layer. This “reactive stroma” response is
characterized by the accumulation of myofibroblasts and the remodeling of the
extracellular matrix at the site. The reactive stroma response initiates early in prostate
cancer, co-evolves with the disease, and is predictive of recurrence. One way reactive
stroma remodels the microenvironment is by deposition of tenascin-C. Tenascin-C is an
extracellular matrix protein that is expressed during development where is critical for
neurogenesis and osteogenesis. In contrast, expression of tenascin C in adult tissues is
restricted to regions of wound repair, tissue remodeling and pathological conditions,
such as cancer.
To assess whether the bone exhibits a reactive tissue phenotype in the context
of metastasis, human prostate cancer metastasis tissue arrays were evaluated using
immunohistochemistry and spectral deconvolution. This work identified a tenascin-C
expression pattern at trabeculae-associated metastatic sites, suggesting the evolution
of a reactive endosteum.
In order to evaluate the mechanisms involved, we developed an in vitro 3D
osteogenic organoid, using human mesenchymal stem cells induced to osteoblastic
differentiation, which exhibits a reactive endosteum phenotype. Co-culture with the
metastatic prostate cancer cell line VCaP showed preferential binding at sites high in
tenascin C deposition. Metastatic cells also adhere to osteo mimetic surfaces coated
with tenascin C in vitro, showing an accelerated growth rate and forming 3D colonies.
We have determined that

cell adhesion to tenascin C–rich surfaces. Preliminary data has also identified signaling
through WNK1 and STAT6 as additional candidate mechanisms that mediate tenascinC induced biology in prostate cancer cells that are metastatic to bone surfaces.
These studies characterize a reactive endosteum phenotype at sites of
metastatic prostate cancer foci and suggest that elevated tenascin-C at these sites
mediates adhesion and other biological properties of cancer cells. This study will help
provide data from which to develop novel therapeutic approaches to treat metastatic
disease.
Contributors: San Martin, Rebeca; Rowley, David.
286
inte
MIR-130B TARGETS ARHGAP1 INCREASING CDC42 ACTIVITY AND METASTATIC
POTENTIAL IN EWING SARCOMA CELLS
Laura Satterfield
Advisor: Jason Yustein, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma (OS) and Ewing’s sarcoma (EWS) are the most common bone
tumors in the pediatric population. While significant progress has been made with
towards increasing patient outcomes, our success in eradicating these two malignances
is significantly less than most other pediatric malignancies. One reason for continued
treatment failure is due to high risk disease states such as the presence of metastatic
lesions. Metastasis is of the utmost clinical relevance as it accounts for more than 90%
of solid tumor deaths. Improvements in treatment regimens have been stagnant over
the past 20-30 years most likely due to our lack of understanding their molecular
pathogenesis. Overall survival rates are approximately 65-70% for localized disease
and less than 20% for aggressive, disseminated states. Thus, the clinical need to
understand high risk disease, such as metastasis is significant in order to develop novel
therapeutics.
miRNAs are highly conserved short 18-22 single stranded non-coding RNAs that
have the ability to repress target translation and affect key cell signaling pathways often
deregulated in cancer. Recently, miRNAs have been implicated in numerous cancers;
however the functional role of miRNAs in sarcomas is limited. In order to identify novel
alterations in miRNAs, we have utilized our analysis of microRNA expression from
localized and metastatic tumors derived from novel genetically engineered mouse
models of osteosarcoma. Among the miRNAs most significantly dysregulated, miR130b was shown to be significantly upregulated in the metastatic lesions. Furthermore,
it has been shown that miR-130b is overexpressed in OS and EWS patient samples,
and this event is significantly associated with poor patient outcome in the latter
sarcoma. Furthermore, we have verified miR-130b overexpression in both mouse and
human sarcoma cell lines and in metastatic EWS tumor samples obtained from Texas
Children’s Hospital. Functional analysis shows overexpression of miR-130b increases
migration and invasion in vitro
In order to identify novel targets of miR-130b contributing to metastasis, we
performed gene expression analysis. We identified Cdc42GAP as a novel target of
miR-130b. Cdc42GAP is a negative regulator of Cdc42 which hydrolyzes the active
GTPase. ELISA analysis of Cdc42 activity reveals that Cdc42 activity is increased with
overexpression of miR-130b.Further microarray and pathway analysis suggest a
mechanism by which activation of Cdc42 leads to positive regulation of the MAPK
cascade. Subsequent western blot analysis reveals that overexpression of miR-130b
leads to the activation of SAPK/JNK and subsequent phosphorylation of c-JUN
suggesting activation of AP-1 transcription factor. Taken together, these findings
suggest that overexpression of miR-130b promotes sarcoma cell migration and invasion
through targeting of Cdc42GAP and modulation of the Cdc42 and SAPK pathways.
Contributors: Satterfield Laura, Shuck Ryan, Kurenbekova Lyazat, Donehower Lawrence,
Yustein Jason
287
EPIGENETIC CHANGES ASSOCIATED WITH IN UTERO NICOTINE EXPOSURE IN
THE RHESUS MACAQUE
Stephen Michael Saylor
The prevalent use of e-cigarettes and other nicotine products is increasing
among women of reproductive age, making their effect on pregnancy an important
public health concern. Nicotine is associated with adverse effects on the fetus including
reduced placental blood perfusion and decreased fetal pulmonary function. Utilizing a
rat model of in utero nicotine exposure, we have recently demonstrated epigenetic
alterations in the lung and brain of offspring at postnatal day 1. Moreover, previous
studies in non-human primates show effects of nicotine on fetal pulmonary function and
oxidant radical production, and these effects are mitigated by treatment with the antioxidant Vitamin C (VC). Studies have also shown that nicotine is a potent histone
deacetylase inhibitor. A clinical trial involving VC treatment on a population of smoking
and non-smoking pregnant mothers indicates that VC has an impact on pulmonary
function, but little remains known about the placenta. Based on these observations, we
sought to determine the impact of nicotine and VC on differential histone posttranslational modification (PTMs) in the placenta, and the molecular impact of these
changes. We hypothesized that differential site-specific histone PTMs would be
detected in fetal lung and placental tissues in response to nicotine and tobacco, which
may be further modulated upon VC treatment. Nicotine and VC demonstrate a
significant synergistic effect on the fetal placental and lung epigenome in the Rhesus
macaque, as well as the human placenta. Notable site-specific histone PTMs were
observed, with effects that were insult and treatment specific, with differential effects
seen in nicotine exposed macaques and tobacco exposed human placental tissue.
These findings suggest that while Vit C may abrogate the clinical impacts of nicotine
exposure, the mechanisms are distinct.
Contributors: S. SAYLOR, M. SUTER, L. SHOREY-KENDRICK, C. MCEVOY, M. HU, A.
FRIAS, E. SPINDEL, K. AAGAARD
288
ISL1 DIRECTS CELL FATE DECISIONS IN THE PANCREAS BY SPECIFYING
PROGENITOR CELLS TOWARDS DIFFERENT ENDOCRINE LINEAGES
Marissa Ann Scavuzzo
Advisor: Malgorzata Borowiak, Ph.D.-Department of Molecular & Cellular Biology
During pancreatic development, five different mature hormone-producing cells
that play critical roles in regulating energy homeostasis are specified from common
endocrine progenitor cells (EPs). The basic helix-loop-helix transcription factor
Neurogenin-3 (Ngn3) is required for the development of all EPs; while it is currently
thought that these EPs are homogenous with equal potential to differentiate into all five
endocrine lineages, we have evidence indicating that the potential of EPs depends on
the transcriptional landscape of the cell, such as the presence or absence of the LIM
homeodomain transcription factor Islet-1 (Isl1). To understand the impact of Isl1 on the
maturation of progenitors into functional endocrine cells, Isl1 dependent transcriptional
targets were analyzed through shRNA knockdown and overexpression using the
pINDUCER system during different developmental windows in human embryonic stem
cell (hESC)-derived pancreatic cells. We show that Isl1 directs cell fate decisions
during pancreatic development by modulating Ngn3 expression and shifting temporal
competence windows. When Isl1 is lost, Ngn3 expression increases 86 fold while
overexpression of Isl1 represses Ngn3. This shift in expression also led to a shift in
endocrine lineage commitment, with more alpha and delta cells as well as ghrelin
expression when Isl1 is lost and more beta and gamma cells when Isl1 is
overexpressed. Furthermore, single cell analysis of murine cells in the EP stage
supports EP heterogeneity by revealing distinct progenitor populations. We will further
dissect the cell fate decisions by differentiating a novel hESC line with inducible
dCas9:BFP/Isl1-GFP and tracing the lineage of cells deficient in Isl1, overexpressing
Isl1, and ectopically expressing Isl1 using CRISPRi-RFP and scaffold sgRNA-RFP. Isl1
dependent changes will be analyzed by RNA-sequencing, while novel EP populations
will be determined by Fluidigm single cell RNA-sequencing. Additionally, we find that
Isl1 is alternatively spliced in a developmentally regulated pattern in various mouse
tissues, including the pancreas and the brain, resulting in two distinct protein isoforms,
Isl1a and Isl1b. For the first time in the human context, we show that Isl1 is alternatively
spliced in differentiating hESC-derived pancreatic cells and the mature human
pancreas. The noncanonical Isl1, Isl1b, lacks a cognate binding site for LIM interacting
proteins, therefore we are analyzing the transcriptional distinctions, differential LIM
interactions, and ultimately cell fate decisions between Isl1a and Isl1b by re-expressing
the alternative mRNA variants in differentiating Isl1-null hESCs. This information is
critical to in vitro differentiation of stem cells into beta cells or other pancreatic
endocrine cells for regenerative therapies, as understanding how to direct cell fate,
specify progenitors to have the proper potential, and efficiently produce beta cells is
essential. Contributors: Scavuzzo, Marissa A.; Yang, Diane; Sharp, Robert; Wamble, Katrina;
Chmielowiec, Jolanta; Mumcuyan, Nirva; Borowiak, Malgorzata
289
THE ROLE OF CONSTITUTIVE ANDROSTANE AND Β-CATENIN IN
HEPATOCELLULAR CARCINOMA
Jessica Diane Scott
Advisor: David Moore, Ph.D.-Department of Molecular & Cellular Biology
Hepatocellular carcinoma is a devastating liver cancer that is on the rise in the
developed world. Constitutive Androstane Receptor (CAR) is a nuclear receptor which
is located primarily in the liver and mediates xenobiotic response. CAR is also essential
to mouse liver tumor development induced by phenobarbital. 80% of tumors induced by
phenobarbital and the genotoxic carcinogen diethylnitrosamine (DEN) contain β-catenin
mutations, usually in exon 3, while tumors induced by DEN alone do not contain βcatenin mutations. This suggests that β-catenin and CAR may cooperate to induce liver
hyperproliferation and tumor formation. Our lab discovered that activating CAR and βcatenin simultaneously in mice results in hepatomegaly in the short term, and tumor
development in the long term. Activating either component alone results in transient,
relatively mild liver growth.
Though most cells in the body are diploid, mature liver cells are more often
tetraploid and sometimes octoploid or higher. In adult rodents, the proportion of 4N:8N
hepatocytes is about 2:1, but the ratio in CAR-activated mice is 1:1, indicating a shift
toward higher ploidy. High polyploidy does serve as a proliferation checkpoint, but in
CAR/β-catenin activated hepatocytes, the normal senescence response is bypassed
and cells proliferate unchecked. The PI3K/Akt pathway has been implicated in insulinmediated liver polyploidy and is a commonly disrupted pathway in many types of
cancer, including hepatocellular carcinoma. Short-term CAR activation in mice results in
reduced Akt phosphorylation, whereas phosphorylation is restored in dual-activated
mice. After 8 months of CAR/β-catenin activation, however, Akt phosphorylation is
completely eliminated. Future steps in this project will focus on further elucidating the
connections between CAR/β-catenin activation, polyploidy, and the Akt pathway.
Contributors: Scott, Jessica; Dong, Bingning; Moore, David.
290
A BIOPHYSICAL MODEL OF THE PHOTORECEPTOR NETWORK IN THE TIGER
SALAMANDER RETINA
Robert Lionel Seilheimer
Program
Advisor: Samuel Wu, Ph.D.-Department of Ophthalmology
Purpose: In addition to voltage-dependent currents in the inner segment, rod
responses are shaped by electrical coupling with adjacent rods. While the physiology of
single rods has been studied extensively, the function of the coupled rod network has
not been systematically characterized. In this study we investigate the photoreceptor
network with dual cell voltage clamp experiments and simulations using a quantitative
biophysical model.
Methods: Tiger salamander retinas were whole-mounted and two adjacent rods
were voltage-clamped. Recordings were made using an Axon Instruments Multiclamp
700A. One rod was stepped through a set of voltages while the other was clamped at
its resting potential. Ionic currents were blocked pharmacologically using TEA, Cs+, and
Co2+. Simulations were performed in MATLAB using custom-written software. Voltagedependent ion currents (Ih, IKx, IKv, ICa, and Ileak) were modeled using HodgkinHuxley-like equations. The rod network was modeled as a square array coupled by
linear, symmetric resistors using the measured conductance values.
Results: Membrane currents of rod pairs were measured by dual whole-cell
voltage clamp techniques. We found that rods in the flat-mounted salamander retina
are strongly coupled with a mean junctional conductance of 1100 pS. In addition,
membrane currents in rods are shaped by voltage-gated currents such as Ih, Ikx, Ikv,
and ICa. We created a quantitative biophysical model of the coupled rod network
incorporating these currents and the measured coupling conductance. Using this
model, we performed simulations of the dual voltage clamp experiments and our results
were consistent with the experiments. To verify the model, we performed additional
experiments in which one or more voltage-dependent current was blocked
pharmacologically. Our model adequately predicted the changes in the current
waveform caused by this blockade.
Conclusions: Our results demonstrate the importance of electrical coupling
between rods in shaping rod signals. Furthermore, our quantitative, biophysical model
of the coupled rod network serves as a useful tool for predicting the behavior of the rod
network. This will in turn allow us to better understand how inputs from the
photoreceptor layer are transmitted to bipolar cells, retinal ganglion cells, and the rest of
the visual system.
Contributors: Seilheimer, Robert; Gao, Fan; Wu, Samuel
291
NOTCH SIGNALING AND STEM CELL-PROGENY INTERPLAY
Fatih Semerci
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D.-Department of Pediatrics
Formation of new neurons in the adult hippocampus represents an adaptive
response of the brain to our environment and/or internal needs. The balance between
underproduction and overproduction of newborn neurons is achieved by whole-cell
means of altering functional circuitry based on the demand. This whole-cell plasticity
suggests that some form of feedback signaling from the progeny to the primary neural
stem cell (NSC) may exist. A known mechanism for cell-cell communication involves the
Notch pathway. Thus, we hypothesized that amplifying neuroprogenitors (ANPs), which
are in direct contact with the primary NSCs, may participate in regulating the NSC
quiescence vs. active proliferation. Indeed, the key components of the Notch pathway
are expressed in the neurogenic niche in a cell type specific manner. Notch receptor is
expressed in the NSCs, while Notch ligands, Jag1 and Delta1, are expressed in ANPs
and granule cells, respectively. Moreover, utilizing a comprehensive bioinformatics
approachdatabase query, we have discovered that Lunatic Fringe (Lfng), a key modifier
of Notch receptor, is selectively expressed in NSC. Further characterization of the LfngeGFP expressing cells confirmed that they areshowed that they carry the unique
properties of NSCs:, as they gave rise to neurogenic progeny, decreased in
numberTheir numbers decrease with increased age, and , they escape from
antimitogenic drug (Temozolomide) treatment, and they responded to the electrical
stimuli known to increase NSC proliferation. We also generated an Lfng-CreERT2
mouse that allowed us to trace the lineage of Lfng expressing cells starting from NSCs
and verified that they can give rise both to granule neurons and astrocytes and verified
that indeed Lfng expressing cells are NSCs. Functionally, removal of Lfng resulted in
increased NSC proliferation followed by decrease in NSC number and neuron
production, suggesting the necessity of Lfng for proper NSC function. On the other
hand, constitutive heterozygote deletion of Jag1 resulted in increased NSC cell cycle
duration with no change in their absolute numbers. These results strongly implicate
Notch signaling in the control of NSCs and their progeny, and suggest a potential
communication and feedback mechanism between the NSCs and their progeny to fine
tune adult neurogenesis. Our data are very significant as we present Lfng-eGFP and
Lfng-CreERT2 as a new mouse models that permits comprehensive and specific
studies of NSC properties and propose a novel mechanism that may operate in the
hippocampal neurogenic niche.
Contributors: Semerci, Fatih; Choi, William Tin-Shing; Encinas, Juan Manuel; Thakkar, Ani;
Depreux, Frederic; Segil,Neil; Groves, Andrew Kelton; Maletic-Savatic, Mirjana
292
EXPLORING THE ROLE OF UBIQUILIN IN PROTEOSTASIS AND
NEURODEGENERATION
Mumine Senturk
Our lab is interested in understanding the molecular mechanisms that underlie
neurodegeneration. To identify essential genes involved in neuronal maintenance, we
performed an EMS-induced unbiased forward genetic screen on Drosophila X
chromosome. One of the loci we identified is ubiquilin (ubqn) and one of its human
homologs UBQLN2, has been shown to cause dominant X-linked amyotropic lateral
sclerosis (ALS).
Ubiquilin is predicted to function in the ubiquitin-proteasome system-mediated
degradation by recruiting poly-ubiquitinated proteins to the proteasome. Furthermore, it
functions in ER-associated degradation (ERAD) where Erasin, an ER-membrane
protein, acts as a platform to recruit both Ubiquilin and VCP to ER membrane. VCP
helps in translocation of misfolded proteins from ER to cytoplasm for ubiquitination.
Ubiquilin in turn interacts with poly-ubiquitinated misfolded proteins and recruits them to
proteasomes. More intriguingly, Vapb, an ER transmembrane protein, also control
ERAD and may be present in complex with VCP. Hence, the functions of Ubqn, VCP
and Vapb seem to converge on the ER and proteostasis. Interestingly, mutations in
VCP and VapB also cause ALS, suggesting that ER homeostasis and proteostasis play
a prominent role in ALS.
I showed that loss of ubqn in fly eye causes neurodegeneration and glial death
upon aging. Loss of ubqn also leads to ER stress and defects in protein degradation.
The link between Ubqn, VCP and Vapb and their involvement in proteostasis and
neuronal maintenance is intriguing and I am therefore exploring these links using
Drosophila as a model system.
Contributors: Senturk, Mumine; Yamamoto, Shinya; Jaiswal, Manish; Mao, Dongxue; Bellen,
Hugo
293
KCTD13 GENE DOSAGE CHANGES ARE ASSOCIATED WITH ANOLOMOUS
LOWER URINARY TRACT DEVELOPMENT
Abhishek Seth
Objective: Hypospadias is a common congenital anomaly of the penis, occurring
in approximately 1 in 250 live births. The molecular basis for hypospadias and lower
urogenital defects is poorly understood. Using comparative genomic hybridization
arrays (aCGH), we have identified a novel candidate gene, potassium channel
tetramerization domain containing 13, KCTD 13 at 16p11.2 in patients with
hypospadias, and cryptorchidism. Copy number variants at 16p11.2 are the most
common syndromic genomic variants identified to date. KCDT13 encodes a substratespecific adapter of a BCR E3 ubiquitin-protein ligase complex, which regulates the
cytoskeleton and cell migration via ubquitination and degradation of RHOA. Given its
role in cellular architecture and motility, KCTD13 likely represents an important factor in
the embryogenesis of the urethra and lower urinary tract. We hypothesize that defects
in KCTD13 are common in patients with hypospadias, cryptorchidism and lower GU
tract anomalies and gene-dosage defects in KCTD13 result in aberrant development of
the lower urinary tract.
Methods: Genomic DNA from pediatric patients with hypospadias,
cryptorchidism, ambiguous genitalia and control patients was analyzed by aCGH using
720K NimbleGen arrays (Roche). PCR was performed using CNV-taqman assays to
validate putative regions of duplication or deletions. In-situ hybridization (ISH) of mouse
embryos was performed.
Results: A de novo 16p11.2 deletion encompassing KCTD13 was identified in a
child with hypospadias by aCGH. A ~600 kb paternally (asymptomatic) inherited
duplication in the same region was identified in a second patient with hypospadias,
cryptorchidism and micropenis. We identified 31 other patients in the literature and
public databases (DECIPHER) with defects in KCTD13 and concomitant GU anomalies.
These GU anomalies range from ectopic testes to ambiguous genitalia. In situ
hybridization shows robust expression of KCTD13 in the entire urinary tract, particularly
in the genital tubercle. Furthermore, immunofluorescent staining of mouse urethra
shows robust expression of KCTD13 in the urotheleum. Using CRISPR genome
editing, a kcdt13 null mouse was produced and currently undergoing phenotypic
analyses.
Conclusion: KCTD13 gene-dosage changes are found in a subset of children
with hypospadias, cryptorchidism and lower urinary tract anomalies. Phenotypic
analysis of kctd13 null mouse model is underway to prove causation beyond
association.
Contributors: Abhishek Seth, In-Seon Choi, Shaye Lewis, Carolina J. Jorge, and Dolores J
Lamb
294
COCHLEAR PERIVASCULAR INTEGRITY AND AUDITORY FUNCTION REQUIRE
CHOLESTEROL HOMEOSTASIS
Michelle L. Seymour
Advisor: Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
Animal models and human studies support a link between altered cholesterol
homeostasis and sensorineural hearing loss. However, mechanistic understanding of
how circulating lipids modulate cochlear cellular activities is lacking. The rising
prevalence of obesity and hypercholesterolemia demands a clearer model of how
systemic cholesterol homeostasis influences cochlear cholesterol homeostasis and
hearing function. We seek to define the relationships between systemic and cochlear
cholesterol homeostasis during hearing development and adulthood, as well as how
elevations of systemic cholesterol affect cochlear and hearing function.
We have shown that systemic cholesterol levels influence levels of cholesterol
within cochlear tissues, yet they do not correspond exactly, partially due to regulated
permeability of the cochlear blood-labyrinth barrier (cBLB). We discovered that lowdensity lipoprotein receptor knock-out (LDLR KO) mice display elevated hearing
thresholds, indicative of hearing loss, concomitant with elevations in serum cholesterol
by 1 month. The cBLB is mostly intact at this age and only becomes completely
permeable by 4 months in LDLR KO mice, allowing circulating cholesterol to enter the
cochlear duct. However, cochlear cholesterol levels do not significantly increase in
LDLR KO mice until 8-12 months of age, which reflects a complex mechanism involving
cochlear homeostasis in combination with integrity of the cBLB. Vascular organization
within the stria vascularis (SV), the metabolic engine of the cochlea, is also altered in
LDLR KO mice. Perivascular-resident macrophage-like melanocytes (PVM/Ms), which
contribute to maintenance of the structural integrity of the cBLB in the SV vessels, are
decreased in LDLR KO mice. The loss of PVM/Ms and alterations in SV vascular
organization occur prior to elevations in cochlear cholesterol, suggesting that
cholesterol-induced hearing loss may initially result from strial damage and compromise
of cBLB integrity.
Funded by NIH/NIA 5T32 Training Grant AG00183 (MLS, 2010-2012) and
NIDCD/NIH NRSA award number F31DC012503 (MLS, 2012-2015).
Contributors: Seymour, Michelle L.; Pereira, Fred A.
295
INCREASED ABUNDANCE OF COLONIC MUCOSAL FAECALIBACTERIUM
PRAUSNITZII IN PEDIATRIC TREATMENT NAÏVE ULCERATIVE COLITIS
Rajesh Rasik Shah
Background
Ulcerative colitis (UC) pathogenesis likely involves a dysregulated interaction
between host genes, intestinal microbes and environment. Faecalibacterium prausnitzii
has been observed to exhibit anti-inflammatory properties, is less abundant in patients
with ileal Crohn’s disease and reductions are associated with worse clinical outcomes.
In UC, conflicting observations have been made about F. prausnitzii abundance. The
aim of our study was to characterize the colonic mucosal microbiome of treatmentnaïve pediatric UC patients.
Methods
The first cohort (discovery) consisted of 6 UC patients and 26 controls. The
second cohort, the validation cohort, consisted of biopsy samples from 12 treatment
naïve UC patients and 13 controls. All biopsy samples were taken from the left side of
the colon in areas of inflammation. 16S rRNA gene sequencing was performed,
processing of sequence data, assignment of operational taxonomic units (OTUs) and
calculation of alpha-diversity were performed in QIIME. The Galaxy web-based platform
implementation of PICRUSt was used for functional genome determination. STAMP
was used to analyze the high-throughput metagenomic data.
Results
From both cohorts, UC patients had a comparable Shannon diversity index
compared to controls. In the discovery cohort, multiple species level taxa were
differentially abundant between UC patients and controls. Specifically, F. prausnitzii
was significantly (p=0.01) more abundant in UC patients compared to controls. From
the validation cohort, a similar trend was seen for a higher abundance of F. prausnitzii
in UC patients compared to controls, but this did not reach statistical significance
(p=0.24). Clinically, a higher abundance of a F. prausnitzii was associated with clinical
improvement (p=0.042). Functionally, significant differences were seen in benzoate,
biotin, glutamine and fatty acid metabolism between UC patients and controls.
Conclusions
Our study demonstrated limited, but detectable differences between the colonic
mucosal microbiome of treatment naïve UC patients and controls. Despite a small
sample size, significantly more UC patients had increased abundance of F. prausnitzii
compared to controls.
Contributors: Shah Rajesh, Cope Julia, Nagy-Szakal Dorottya, Versalovic James, Hollister
Emily, Kellermayer Richard
296
INVESTIGATING THE ROLE OF SKAP2 IN RETINOBLASTOMA
Perris S. Shaw
Advisor: Richard Hurwitz, M.D.-Department of Pediatrics
Patricia Chevez-Barrios, M.D.-Department of Ophthalmology
Purpose: SKAP2 expression has been associated with cancer cell proliferation.
We have found that SKAP2 gene expression is increased in invasive retinoblastoma. I
am investigating the role of SKAP2, a protein associated with Src kinase that has a role
in actin polymerization, to determine if it is responsible for cell growth, migration and/or
invasive potential in retinoblastoma.
Hypothesis: I hypothesize that the expression of SKAP2 and SKAP2 regulation
of Src family kinases will drive the metastatic potential in retinoblastoma cells.
Methods: SKAP2 protein expression in retinoblastoma cell lines was
demonstrated by Western blot. A Lentiviral vector system delivering an shRNA targeting
SKAP2 that expressed GFP and the puromycin resistance gene was used to
knockdown SKAP2 expression in Rb cell lines. GFP expressing retinoblastoma cells
were sorted and further selected by puromycin resistance.
Results: SKAP2 expression in both metastatic and non-metastatic Rb cell lines
was demonstrated. GFP expressing retinoblastoma cells transduced with three shRNAs
targeting SKAP2 had reduced growth as compared to transduced cells that did not
express GFP or with cells transduced with a control vector not targeting SKAP2.
Conclusion: Retinoblastoma cells that had decreased expression of SKAP2
exhibited decreased proliferation in vitro. Future experiments will determine if the same
effects are observed in an in vivo murine model of retinoblastoma and whether the
metastatic potential of the disease is affected. Furthermore, the effect on Src kinase
phosphorylation of proteins in retinoblastoma cells with decreased SKAP2 expression
will be examined.
Contributors: Shaw, Perris; Akinfenwa, Patricia; Hurwitz, Mary; Hurwitz, Richard
297
DESIGN AND GENERATION OF MOUSE MODELS EXPRESSING ECTOPIC CGG
REPEATS TO STUDY FRAGILE X-ASSOCIATED PRIMARY OVARIAN
INSUFFICIENCY (FXPOI)
Katharine Elizabeth Shelly
Advisor: David Nelson, Ph.D.-Department of Molecular & Human Genetics
Fragile X-associated Primary Ovarian insufficiency (FXPOI) is the most common
known genetic cause of premature ovarian failure, affecting roughly 20% of women
heterozygous for an FMR1 premutation allele. Characteristics of this disorder in
humans include alterations in hormone levels, increased follicular atresia, and
premature cessation of menses. A subset of these features was reported in previous
investigations of Fmr1 premutation mouse models, coupled with aberrations in
granulosa cell populations. These two premutation models were both constructed with
an expanded CGG tract in the context of the Fmr1 transcript and cannot distinguish the
effects of Fmr1 alterations from the effects of CGG RNA. We hypothesize that these
phenotypes result predominantly from the expression of premutation-sized CGG repeat
tract RNA (rCGGs) that limit the normal function of rCGG-binding proteins within the
ovary.
To determine whether ectopically expressed premutation-sized rCGG repeats
are sufficient to confer phenotypes associated with FXPOI, we designed a construct to
enable tissue-specific expression of 87 CGG repeats fused to either FMR1 or
fluorescent ZsGreen cDNA, targeted to the Rosa26 locus. We predict that expression of
expanded CGG repeats in vivo will recapitulate phenotypes previously associated with
FXPOI. Ultimately, these mouse models will enable us to determine whether ectopically
expressed CGG repeat tracts are sufficient to confer features of FXPOI, and will
potentially allow us to assess the contributions of specific tissues to these phenotypes.
Contributors: Shelly, Katharine; Nelson, David
298
CELL TYPE-SPECIFIC FEEDBACK TO V1 ENFORCES A NARROW TEMPORAL
WINDOW FOR SUPRA-LINEAR ENHANCEMENT OF FEED-FORWARD INPUTS
Shan Shen
Cortical sensory hierarchies are linked by extensive top-down feedback
connections whose function remains elusive. We combined multiple whole-cell
recordings with optogenetics to examine the connectivity of feedback from secondary
visual area LM (lateromedial area) to primary visual cortex (V1) in mice. We found that
LM provided strong monosynaptic excitation to Layer 2/3 parvalbumin and somatostatin
expressing interneurons and weaker input to all other cell types. Activation of feedback
from LM elicited biphasic membrane potential responses in V1 excitatory neurons, with
an initial brief depolarization followed by a longer-lasting hyperpolarization. Feedback
activation alone rarely elicited spikes on V1 principle cells, but when paired with visual
stimuli, firing rates were increased three-fold within a narrow temporal window. Our
results suggest that feedback enforces a narrow window of opportunity for supralinearly enhancing feed-forward information, consistent with models of vision where topdown priors and bottom-up evidence are combined for optimal inference.
Contributors: Shen, Shan*; Jiang, Xiaolong*; Reimer, Jacob; Sinz, Fabian; Tolias, Andreas
299
INACTIVATION OF KLF4 RELEASES REPRESSION OF THE MAP2K7/JNK
PATHWAY IN PEDIATRIC T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Ye Shen
Advisor: H Lacorazza, Ph.D.-Department of Pathology & Immunology
Acute lymphoblastic leukemia is the most common hematological malignancy in
pediatric patients, and disease relapse is the leading cause of cancer-associated death
in children. Despite steadily improved outcomes in patients with newly diagnosed
disease, little progress has been made to treat relapse leukemia and to reduce the
incidence of relapse. We found that low levels of the transcription factor KLF4 in
pediatric T-ALL samples was caused by hypermethylation of its promoter. Transformed
KLF4-deficient bone marrow cells with the mutant Notch1-L1601P-ΔP resulted in a
more aggressive T-ALL due to increased proliferation of leukemic cells and expansion
of leukemia initiating cells. Loss of KLF4 enhances MAP2K7-JNK pathway in both
murine and pediatric T-ALL cells and inhibition of JNK pathway suppresses leukemic
cell growth. Thus, KLF4 emerges as a novel tumor suppressor in T-ALL and inhibition
of activated JNK pathway can be used as alternative therapy.
Contributors: Shen, Ye; Suppipat, Koramit; Park, Chun Shik; Mistretta, Toni-Ann; Horton,
Terzah; Rabin, Karen, Lacorazza, Daniel
300
A HIGHLY SENSITIVE AND PCR-BIAS FREE SINGLE CELL RNA-SEQ METHOD
ALLOWS QUANTITATIVE ASSESSMENT OF GENE EXPRESSION AT SINGLE
CELL RESOLUTION
Kuanwei Sheng
Advisor: Chenghang Zong, Ph.D.-Department of Molecular & Human Genetics
Single-cell technologies have emerged as advanced tools to study various
biological processes, and have revolutionized our view on whole-organism science.
However, even with the broad applications, we still face the technical challenges of the
sensitivity and accuracy in the state of art of single cell transcriptome profiling. Current
methods have only 40-50% capture efficiency and have strong 3’ bias. The popular
methods such as SMART-seq can only use PolyA transcripts for cDNA synthesis.
Therefore, signal of the premRNA and some noncoding RNA will be completely lost.
With the limitations described above, a highly sensitive and quantitative single cell
RNA-seq method is highly desired in the field.
Here, we present a single cell RNA-seq method—Multiple Annealing and Tailing
Based Quantitative RNA-Seq (MATQ-Seq) that significantly improves the sensitivity and
reproducibility compared to other methods. This allows the researchers to detect the
gene expression changes at a quantitative level. Moreover, we showed that MATQ-Seq
can detect not only polyA mRNA but also nonpolyA transcripts like histones and premiRNAs.
We show that this method can be used to study a variety of biological processes
including tumorigenesis and cancer metastasis. Interestingly, partially degraded RNA
samples can also be efficiently amplified by our method, which demonstrates a great
potential for the applications with clinical samples.
Contributors: Sheng, Kuanwei; Deng, Qing; Cao, Wenjian; Zong, Chenghang.
301
OMEPRAZOLE POTENTIATES HYPEROXIA-INDUCED DEVELOPMENTAL LUNG
INJURY IN NEWBORN MICE VIA ARYL HYDROCARBON RECEPTOR DEPENDENT
MECHANISMS
Binoy Shivanna
Advisor: Bhagavatula Moorthy, Ph.D.-Department of Pediatrics
Background: Hyperoxia contributes to bronchopulmonary dysplasia in preterm
infants. We showed that omeprazole (OM) protects adult mice against hyperoxic lung
injury via aryl hydrocarbon receptor (AhR)-dependent mechanisms. Additionally, we
observed that newborn AhR dysfunctional mice are more susceptible to hyperoxiainduced lung inflammation and alveolar simplification. Whether OM activates AhR and
protects newborn mice against hyperoxia-induced developmental lung injury is
unknown. The objective of our study was to test the hypothesis that omeprazole will
decrease hyperoxia-induced developmental lung injury in newborn mice via AhR
activation.
Design/Methods: One day old wild type C57BL/6J pups while exposed to either
air or 85 % O2 (hyperoxia), were administered 10 or 25 mg/kg of OM or the vehicle,
polyethylene glycol, i.p. once daily from birth for 14 d. The dosing regimen was based
on our dose-response studies that suggested at least 25 mg/kg/d of i.p. OM for 3-6 d is
required to activate the AhR. Additionally, we used OM at a dose of 10 mg/kg/d to
determine its AhR independent effects. Following experiments, the mouse lungs were
harvested to determine AhR activation, inflammation, oxidative stress, angiogenesis,
and alveolarization.
Results: OM treatment (10 to 25 mg/kg) increased oxygen toxicity in a dosedependent manner. OM increased hyperoxia induced lung macrophage influx.
Interestingly, mice treated with high dose OM had persistent acute lung inflammation
upon exposure to hyperoxia. Additionally, higher dose OM: i. Increased lung
malondialdehyde and nitrotyrosine levels and alveolar simplification; and ii. Decreased
the expression of vascular endothelial growth factor and its receptor and vonWillibrand
factor positive lung blood vessels, compared to the vehicle-treated mice exposed to
hyperoxia. Surprisingly, CYP1A1 protein, a marker of AhR-activation, was degraded
with prolonged treatment with OM. Furthermore, OM decreased the expression of
NADP(H) quinone oxidoreductase, an antioxidant enzyme, which is also regulated by
the AhR.
Conclusions: Contrary to our hypothesis, prolonged OM therapy decreases
functional AhR activation and potentiates inflammation, oxidative stress, and alveolar
simplification in a model of hyperoxia-induced lung injury in newborn mice. These
findings suggest the need to be cautious before instituting OM therapy in neonates.
Contributors: Shivanna Binoy, Zhang Shaojie, Patel Ananddeep, Jiang Weiwu, Wang Lihua,
Moorthy Bhagavatula
302
CONSTITUTIVE DELIVERY OF IL-7 SIGNALING ENHANCES ANTIGEN-MEDIATED
EXPANSION OF T-CELLS EXPRESSING A GD2-SPECIFIC CHIMERIC ANTIGEN
RECEPTOR
Thomas Shum
Advisor: Cliona Rooney, Ph.D.-Department of Pediatrics
Stephen Gottschalk, M.D.-Department of Pediatrics
Adoptive lymphocyte therapy has shown success against lymphomas but not
against solid tumors. Using the 3 signal paradigm of T-cell activation where Signal 1 (Tcell receptor activation), Signal 2 (co-stimulation), and Signal 3 (cytokines activation)
work together to expand adoptively transferred lymphocytes in-vivo and drive their
elimination of tumors, it is evident that tumors are not conducive to T-cell activation.
Higher generations of tumor specific chimeric antigen receptors (CARs) can provide
Signal 1 and 2 upon antigen ligation, as well as Signal 3 from IL-2 secretion. However,
Signal 3 remains incompletely activated due to the T-cell’s inability to produce other
beneficial cytokines such as IL-15 and IL-7. We aim to correct this Signal 3 deficiency
through genetic modification of T-cells with a genetic construct IL7RP2 that
constitutively supplements T-cell function with IL-7 signaling. We show that in the
absence of exogenous cytokine supplementation, T-cells transduced with a GD2specific CAR (GD2-CAR) and IL7RP2 expanded ~57-fold after 4 weeks when
repeatedly co-cultured with irradiated GD2-expressing LAN-1 neuroblastoma cells,
compared to 3-fold expansion in T-cells transduced with GD2-CAR alone within the
same timeframe. We also find that the IL7RP2 mediates only antigen-dependent
expansion and does not confer resistance to the iCaspase9 suicide gene. In NSG mice
bearing subcutaneous LAN-1 neuroblastoma tumors, GD2-CAR T-cells + IL7RP2
demonstrated faster in-vivo expansion kinetics post-adoptive transfer but similar antitumor efficacy compared to GD2-CAR T-cells transduced a with a mOrange construct.
In conclusion, triggering Signal 3 activation with IL7RP2 results in sustained GD2-CAR
T-cell expansion against GD2-expressing target cells.
Contributors: Shum, Thomas; Omer, Bilal; Tashiro, Haruko; Kruse, Robert; Castillo, Paul;
Hyunh, Mai; Gottschalk, Stephen; Rooney, Cliona
303
A CRITICAL DEVELOPMENTAL WINDOW FOR 17Β-ESTRADIOL ANTIEPILEPTOGENIC EFFECT IN A MOUSE MODEL OF X-LINKED INFANTILE
SPASMS
Meagan Siehr
Advisor: Jeffrey Noebels, M.D./Ph.D.-Department of Neurology
X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic epilepsy syndrome.
Few treatments exist that effectively reduce infantile spasms, prevent epilepsy and
improve developmental outcomes in ISSX. The Arx(GCG)10+7 mouse model carries
the most common mutation in ISSX and exhibits many phenotypic features of ISSX
including epilepsy, motor spasms in early life, and loss of GABAergic interneurons, a
population responsible for synaptic inhibition in the CNS. Recent work has shown that
treatment with 17β-Estradiol (E2) in Arx(GCG)10+7 neonates (P3-10) reduced neonatal
spasms and seizures in adults. However, E2 treatment in adult mice (P33-40) had no
effect on these phenotypes. This suggests that the antiepileptogenic effect of E2 in
Arx(GCG)10+7 is developmentally confined and in order to effectively translate this
therapy to the clinic, it is crucial to further investigate the temporal boundary for E2
efficacy in Arx(GCG)10+7. In this work, we examined the effect of delaying treatment
initiation by several days. Little is understood about the molecular and cellular
mechanisms underlying the anti-epileptogenic actions of E2. As estrogen receptor beta
(ERβ) is expressed in cortical interneurons, we explored whether activation of this
estrogen receptor subtype is sufficient to reproduce the effect seen with E2. Moreover,
as E2 is known to have neuroprotective and anti-apoptotic effects in the brain, we
tested whether E2 may reduce apoptotic markers in the Arx(GCG)10+7 neonatal brain.
Delaying E2 treatment until the second postnatal week (P7-13), using either
40ng/g/day or 80ng/g/day, had no effect on neonatal spasm frequency, seizure and
interictal spike frequency (a second measure of cortical hyperexcitability). These data
suggest a critical developmental window for the antiepileptogenic effect of E2 in the
Arx(GCG)10+7 model of ISSX. During this critical window, selective activation of ERβ
using LY500307 (Eli Lily & Co.) at 200ng/g/day from P3-10 was sufficient to produce a
partial antiepileptogenic effect in Arx(GCG)10+7. Treatment with LY500307 significantly
reduced seizures but not neonatal spasms or interictal spikes. Full effect may require
activation of other ERs or a higher dosage of LY500307. Furthermore, we found that
within this critical widow, E2 reduces total apoptotic cells marked by CC3 expression in
Arx(GCG)10+7 neocortex. However, this effect was not observed in WT suggesting E2
does not reduce general apoptosis in the neonatal brain and this is specific to the
Arx(GCG)10+7 mutation. Future work will examine how specific neuronal types, such
as cortical GABAergic interneurons, are influenced by the anti-apoptotic effects of E2 in
Arx(GCG)10+7 neonates.
Contributors: Meagan S. Siehr, Rocco D. Lucero, Joshua W. Lalonde, Jeffrey L. Noebels
304
HEART ATTACK VARIANT MARKS PLATELET-SPECIFIC, ALTERNATIVE START
SITE OF MFN2 AND IS ASSOCIATED WITH EXPRESSION LEVELS
Lukas Mikolaj Simon
A single nucleotide polymorphism (SNP) rs1474868 in the MFN2 gene was
previously associated with Myocardial infarction (MI). We conducted expression
quantitative trait locus (eQTL) analysis in human platelets and identified rs1474868 as
the leading eQTL for MFN2 (P<1e-45). Integration of human platelet RNA-seq data
revealed a novel, platelet-specific alternate start site of MFN2 near rs1474868. This
finding was validated using 5’ Rapid amplification of cDNA ends. Analysis of ChIP-seq
data profiled in K562 cells from the Encyclopedia of DNA Elements (ENCODE)
database showed a cluster of transcription factors binding near rs1474868. The SNP
rs3766744 was in strong linkage disequilibrium with rs1474868 and overlapped a large
number of transcription factor binding sites. We validated the regulatory function of
rs3766744 by showing differential transcriptional activity between the A and G alleles of
rs3766744 (P<0.05) using luciferase reporter assay. This is the first study to connect
the MI-associated SNP rs1474868 to a platelet-specific regulatory mechanism in MFN2.
Contributors: Simon, Lukas; Chen, Edward; Edelstein, Leonard; Bray, Paul; Shaw, Chad
305
COORDINATED CYTOSKELETAL FUNCTIONS REGULATE PHYSIOLOGIC NK
CELL LYTIC GRANULE MOTILITY AFTER ARRIVAL AT THE IMMUNOLOGICAL
SYNAPSE
Papiya Sinha
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system
that kill virally infected and malignant cells. They contain specialized secretory
lysosomes called lytic granules, which mediate target cell death by the secretion of
perforin and granzymes onto a diseased cell for host defense. This process of
cytotoxicity depends upon the ability of NK cells to transport their granules to the
immunologic synapse (IS) with the help of microtubules. Until recently, it was suggested
that microtubule organizing center and lytic granule polarization to the IS directly
preceded granule docking and membrane fusion. However, using total internal
reflection fluorescence microscopy (TIRFm), we recently defined a lengthy
multidirectional movement of granules at the IS prior to arrest and subsequent
degranulation. In this study, we performed single particle tracking of lytic granules at the
IS to understand the nature, mechanism and ultimately the purpose of this unexpected
movement. The motility of granules within the synaptic region were evaluated in the
NK92 and YTS human NK cell lines as well as in ex vivo human NK (eNK) cells. In all
three, granule motility was mostly sub-diffusive (constrained) at the IS with average
mean square displacements of 0.04 to 0.1μm2 at 20s. A small percentage of stationary
movement and directed migration were also observed. In an effort to dissect the
contributions of cytoskeletal elements, small molecule inhibitors were added following
the initiation of imaging and granules were tracked and analyzed. F-actin
depolymerization with Latrunculin A (LatA) did not change synaptic granule
displacement in NK92 and eNK cells, but increased in YTS cells. However, in all the 3
NK cells, microtubule depolymerization using nocodazole resulted in reduced granule
displacement, an effect that was further reduced by addition of LatA in combination. To
discern the relative contribution to granule motility derived from facilitated movement
along microtubules vs. concordant movement of the entirety of the microtubule network,
paclitaxel was used to stabilize microtubules. Granules still maintained their subdiffusive character with some still depicting directed motility. With these experiments we
have defined the synaptic movement of lytic granules and identified contributing roles
of, and interactions between, the actin and microtubule networks. This study is
important because it underscores a new and unforeseen step that facilitates NK cell
killing. In addition to filling the gap in knowledge of granule motility at the IS, these
results identified previously unknown requirements for effective cytotoxicity. Our results
broaden the understanding of NK cell biology with the use of advanced microscopy and
multifaceted quantitative analysis, which will be beneficial for therapeutic intervention of
NK cell function to treat immunological disorders.Contributors: Sinha, Papiya; Tsao, David;
Mace, Emily; Carisey, Alex; Kolomeisky Anatoly; Diehl Michael; Orange, Jordan
306
A NOVEL CROSSOVER HOTSPOT INSTIGATOR FOR RECOMBINATION IN
ESCHERICHIA COLI
Priya Sivaramakrishnan
Advisor: Christophe Herman, Ph.D.-Department of Molecular & Human Genetics
Homologous recombination is a universal mechanism used by all growing cells to
repair DNA double-strand breaks (DSBs) with high fidelity. The foremost step in this
pathway is the processing of DSB ends to create DNA substrates suitable for homology
search and strand exchange. In Escherichia Coli, RecBCD, a multi-subunit complex
performs this step using its translocase, helicase and endonuclease activities to move
along the template as it unwinds and degrades the DNA. This DNA resection by
RecBCD continues until it encounters a correctly oriented sequence called a chi
(crossover hotspot instigator) site. Chi sites modify RecBCD function resulting in the
production of single-strand DNA onto which the major recombination protein RecA is
loaded. As it translocates, RecBCD is likely to encounter other DNA metabolic
processes such as replication and transcription. Chi sites occur more frequently on the
mRNA synonymous strand, but the functional significance of this distribution bias is
unknown. We hypothesized that modulation of transcription elongation can affect the
repair of DSBs and found that that the transcription factor GreA blocks DNA repair.
GreA belongs to a family of transcription factors that directly bind RNA polymerase
(RNAP). When RNAP is stalled and cannot continue elongation, it enters into a
‘backtracked’ state. GreA regulates transcription processivity by rescuing backtracked
RNAP complexes and restarting elongation. Loss of function greA mutants ((greA)
survive much better compared to wild-type when DSBs are induced by exogenous
damaging agents. Examination of DNA damage by pulsed-field gel electrophoresis
revealed that (greA mutants have an improved DSB repair capacity. This enhanced
repair is dependent on RecA and RecB. (greA mutants also show increased viability
when a single DSB is created at a specific position in the E. coli genome. To
understand the mechanism by which GreA impedes repair, we developed an assay
using massively parallel sequencing to examine the kinetics of DSB repair. Our results
show decreased resection in (greA suggesting that RNAP in the presence of GreA
interferes with DNA processing by RecB. We propose a model where RNAP
encountering RecBCD undergoes backtracking and remains in this state in the absence
of GreA. Since backtracked RNAP complexes are highly stable, RecBCD is unable to
continue resection and is forced to cease DNA processing and switch to recombination
mode. Thus, without GreA, a backtracked RNAP mimics a chi site, promoting
recombination. Since DSBs are a debilitating form of DNA damage capable of triggering
chromosomal rearrangements, our discovery of a novel instigator of recombination
contributes to identifying ways to maintain cellular genome stability.
Contributors: Sivaramakrishnan, Priya; Bravo, Maria; Halliday, Jennifer A.; Rosenberg, Susan
M.; Herman, Christophe
307
PREDICTING MENDELIAN DISEASE GENES BASED ON THE LACK OF
VARIATION OBSERVED IN CONTROL INDIVIDUALS
Zachry Tore Soens
There are two primary obstacles a geneticist faces when attempting to provide a
molecular diagnosis to a patient with a Mendelian disorder. The first obstacle is on the
variant level, and can be described as the problem of variants of unknown significance.
Determining which rare variants out of the thousands within an individual are
deleterious and have a detrimental impact on a gene’s function is an ongoing challenge
in the field of genetics. The second obstacle is on the gene level. After narrowing
down the list of variants from a diseased patient to only the ones which are potentially
deleterious, a geneticist still has to determine which gene with a deleterious variant
could be responsible for the phenotype of the patient. This task usually relies on what
is known about the gene from biomedical literature, but many genes have never been
studied so little information is available about their function. ~3,500 out of ~19,500
protein coding genes are currently associated with at least one human disease. This
means the relevance of ~16,000 genes to human disease has yet to be established.
The aim of this project is to create a gene-level annotation tool which can predict
the likelihood that any given gene is either a haploinsufficient disease gene, recessive
disease gene, or dispensable gene based on the amount of variation (or lack thereof),
type of variation, and frequency of that variation as observed in control individuals. We
hypothesize that genes important for human health have a reduction in the amount of
functional protein-altering variation when compared to genes that are dispensable genes whose biallelic loss-of-function does not result in an obvious phenotype.
Variants were compiled from 17,500 control individuals without a Mendelian
disease who’ve had whole exome sequencing performed. Gene-level variant-based
covariates were generated based on the compiled variant data for machine learning
use. Known Mendelian disease genes were used as positive controls while manually
chosen dispensable genes were used as negative controls for machine learning
predictive model training. Trained predictive models perform well achieving an AUC of
~0.90 as evaluated by 5-fold cross validation, and when the predictions are applied to
~450 disease genes not used during predictive model training, we found that ~70% are
correctly predicted as disease genes which perfectly matches the sensitivity assessed
during cross validation. We conclude that the relative depletion in variation in any given
gene correlates with the likelihood that that gene is in fact a disease gene to the point of
being able to predict which human genes are disease genes with fair accuracy.
Contributors: Soens, Zachry; Chen, Rui
308
OLFACTORY REGULATION OF C. ELEGANS REPRODUCTIVE AGING
Jessica N Sowa
As women age, they experience both a decline in fertility and an increased risk of
miscarriage and birth defects. Although predicted to be a regulated process, little is
known about the mechanisms regulating the onset and progression of reproductive
aging. In addition to purely genetic contributions, reproductive aging is thought to be
regulated by the complex interactions of genetic signaling pathways and environmental
conditions. However, the molecular mechanisms integrating environmental and genetic
signals to regulate reproductive aging remain unknown.
Caenorhabditis elegans experience their diet of bacteria as a source of both
metabolic and sensory input. We took advantage of this relationship as a convenient
method of introducing environmental variation by raising C. elegans on different
bacterial diets. We found that C. elegans exposed to different bacterial environments
show significant differences in the duration of their reproductive span. To further dissect
the molecular mechanisms underlying these reproductive span differences we chose to
focus on two strains of Escherichia coli (OP50 and HB101) that gave drastically
different effects on C. elegans reproduction. We found that worms raised on OP50
reproduce longer and maintain their fertility later than worms raised on HB101. This
effect is mediated by a pair of olfactory neurons which perceive a volatile odorant signal
from the HB101 E.coli. The presence or absence of this environmental cue affects
germline proliferation and maintenance in C. elegans, ultimately contributing to the
timing of reproductive senescence. We have identified a pair of olfactory neurons, the
AWB neurons, that are specifically required for this olfaction-mediated reproductive
adaptation as well as for chemotaxis of C. elegans to the smell of the HB101 bacterial
diet. Optogenetic activation of the AWB neurons confirmed that AWB activation was
sufficient to induce reproductive span shortening in the absence of HB101. Finally, we
identified neuropeptide release as the mechanism of AWB neurotransmission.
Together, our results reveal a novel pathway for the regulation of reproductive aging in
C. elegans, and suggest the relevance of environment-sensitive signaling mechanisms
in regulating the onset and progression of reproductive aging.
Contributors: Sowa, Jessica; Mutlu, Ayse Sena; Wang, Meng
309
THE ROLE OF C/EBPß ISOFORMS IN MAMMARY STEM/PROGENITOR CELLS
AND TUMOR INITIATING CELLS
Aaron Jonathon Spike
The goal of these studies is to elucidate the mechanisms by which specific
isoforms of the transcription factor C/EBPβ regulate mammary stem cells and tumor
initiating cells. There are three translationally regulated C/EBPß isoforms, the full length
LAP1, the slightly truncated LAP2, and the short isoform LIP, but little is known about
their individual functions in mammary gland stem cells and development. The ratio of
the C/EBPβ isoforms is thought to act in mammary gland development to regulate the
equilibrium between proliferation and differentiation. The precise function of individual
C/EBPß isoforms in mammary stem cells (MaSC) is unclear; however it has been
shown that transplantation after deletion of C/EBPβ in pooled mammary epithelial cells
results in severe defects in lobuloalveolar development and a significant reduction in
MaSC repopulating units as shown by limiting dilution and serial transplantation
experiments. Gene expression analysis performed on outgrowths showed a number of
genes of interest whose expression was modulated in both the luminal and basal
compartments. Several specific genes of interest known to function in stem cells
maintenance are; Eya1, Notch3, Stat3, and ∆Np63. C/EBPß has been studied
extensively in cancers such as glioblastoma multiforme and has been shown to be a
master regulator of mesenchymal transformation in that disease. Isoform specific
effects of C/EBPß in breast cancer however are relatively understudied. It has been
shown that expression of LIP in human patients correlates with poor prognosis in
ER/PR-/- breast cancer. Our overall hypothesis is that: C/EBPß regulates the
expression of key genes in an isoform specific manner in mammary stem or progenitor
cells and breast cancer tumor initiating cells
Contributors: Spike, Aaron; Rosen, Jeffrey
310
STREAMLINED PROTOCOL FOR TCR SEQUENCING, CLONING, AND IN VIVO
FUNCTIONAL ANALYSIS OF ISLET ANTIGEN-REACTIVE HUMAN T CELLS
Maran Lee Sprouse
Advisor: Maria Bettini, Ph.D.-Department of Pediatrics
Type 1 diabetes (T1D) is a T cell mediated autoimmune disease characterized
by the selective destruction of insulin secreting beta cells present in the pancreatic
islets. The goal of our research is to develop a system for isolation and characterization
of islet antigen-specific CD4+ T cells from the peripheral blood of new onset T1D
patients. Through a novel, multiplex-nested PCR protocol, we cloned paired alpha and
beta TCR chains from individual GAD65-responsive T cells and tested the resulting
chimeric-TCR constructs in vitro for surface expression in HEK293T cells. Additionally,
we analyzed TCR variable region gene usage from individual cells and found 3
common genes for beta and 4 common genes for alpha. Currently we are testing the in
vitro specificity of cloned TCRs as well as assessing their functional affinities. Next,
utilizing a humanized TCR/HLA retrogenic mouse system, we will test human islet
antigen-reactive TCRs for their in vivo function, their ability to escape thymic selection,
infiltrate the pancreas and cause spontaneous diabetes.
Contributors: Maran Sprouse1, Pinaki Banerjee2, Matthew L. Bettini1, Maria Redondo1 and
Maria Bettini1.
311
FATE DETERMINATION IN MAMMARY STEM CELLS
Amulya Sreekumar
Stem cells orchestrate epithelial tissue homeostasis by balancing the diverse
processes of cell division, differentiation and apoptosis. This fine balance is often
disrupted as a consequence of dysregulated signalling pathways during
tumourigenesis. Stem cell driven, highly proliferative pubertal mammary epithelial
structures termed terminal end buds (TEBs) serve as a dynamic model to study the
complex processes underlying homeostasis. We hypothesize that targeted disruption of
the stemness-maintaining Wnt pathway components results in an abrogation of
homeostatic processes that leads to tumourigenesis. These studies are relevant to our
understanding of breast cancers as multiple models of the aggressive basal-like
subtype of breast cancers show aberrant Wnt pathway hyperactivity.
To elaborate the processes of basal homeostasis in the TEB, we employ the use
of an s-SHIP:eGFP mouse model that marks cap cells in the pubertal mammary gland
(putative stem cells in the TEB). Finally, we perturb Wnt signalling locally in the TEB by
administering Wnt pathway ligands and antagonists in the vicinity of TEBs or globally,
utilizing the MMTV-Wnt1 overexpression model.
We demonstrate that cap cells invaginate from their stromal adjacent location in
the TEB and intermix with inner TEB cells where a large percentage undergoes
apoptosis. Apoptosis potentially acts as a mechanism of lumen formation and balances
out excessive cap cell divisions. Our laboratory and others have previously described
that these cap cells in the TEB reside in a Wnt responsive niche. Preliminary data
suggest that global hyperactivation of Wnt signalling in the MMTV-Wnt1 model results
in defective TEB lumen formation and decreased apoptosis. Future studies will focus on
studying the mechanisms by which the Wnt signalling pathway co-ordinates these
homeostatic processes.
Our results demonstrate that the Wnt pathway finely tunes homeostatic
processes in the normal mammary gland. Current studies are focused on studying the
contribution of the disrupted Wnt pathway at different stages of tumourigensis and
inhibiting Wnt pathway components locally to study their short-term effects on
homeostasis.
Contributors: Sreekumar, Amulya; Toh, Eajer; Roarty, Kevin; Lee, Dong-kee; Xu, Jianming;
Rosen, Jeffrey
312
EFFECTS OF CONSTITUTIVELY SILENCING PURKINJE CELL
NEUROTRANSMISSION ON THE PROCESSING OF VESTIBULAR SELF-MOTION
SIGNALS IN MICE
Trace L Stay
Roy Sillitoe, Ph.D.-Department of Pathology & Immunology
Movement activates vestibular sensors which provide feedback about the
movement. For these signals to convey useful information, the brain must fit them in a
frame of reference. Extracellular recordings in macaque monkeys have shown that
specific cell types in the hindbrain can provide that reference, by transforming net
acceleration signals into component acceleration relative to gravity (‘tilt’) or acceleration
from linear inertia (‘translation’). However, which particular circuit elements are
necessary for this computation is currently unknown. We sought to determine whether
Purkinje cell signaling, ultimately the final output of the cerebellar cortex, is required to
separate tilt and translation in alert animals. To do so, we first confirmed that selective
neural representation of tilt and translation is a fundamental process across different
species, by adapting macaque motion testing paradigms for alert mice. Using single
unit extracellular recordings in cerebellar lobules 9c and 10, we find that significant
populations of Purkinje cells respond to tilt or translation. Next, we utilized a conditional
L7Cre;Vgatflox/flox genetic mouse model developed in our lab to examine self-motion
processing when Purkinje cell output is constitutively eliminated in the cerebellum. Our
preliminary data suggest that loss of Purkinje cell output does not interfere with
selectivity to tilt or translation. These data argue that cerebellar cortical output is not
necessary for maintaining normal vestibular sensation, which implicates a process of
direct signal transformation within the cerebellum rather than obligatory closed loop
feedback involving the cerebellar cortical microcircuits. My ongoing work seeks to fully
elucidate the acute and long-term relationships between the cerebellum and vestibular
nucleus during typical vestibular processing.
Contributors: Trace L. Stay, Michael E. Shinder, Jean Laurens, Dora E. Angelaki*, and Roy V.
Sillitoe*
*Co-mentors
313
INTERLEUKIN 6 (IL-6) EXPOSURE REDUCES CHEMOTHERAPY-INDUCED
APOPTOSIS IN ACUTE MYELOID LEUKEMIA
Alexandra McLean Stevens
Advisor: Michele Redell, M.D./Ph.D.-Department of Pediatrics
Background: Increased levels of circulating IL-6 are associated with poor clinical
outcome in multiple malignancies; this is believed to occur through increased activity of
the anti-apoptotic IL-6-induced Stat pathway. Our group has previously shown that
increased IL-6-induced tyrosine phosphorylated Stat3 (pY-Stat3) at relapse, compared
to the response at diagnosis, is associated with poor clinical outcome in pediatric AML
patients. Additionally, we have shown that isolated IL-6-induced pY-Stat3 at diagnosis
portends a poor outcome in pediatric AML. These results support a possible
relationship between chemoresponsiveness and IL-6-induced Stat3 response. We
hypothesized that for a subset of pediatric AML cases, IL-6 provides a survival
advantage for the AML blasts.
Methods: AML cells lines demonstrating IL-6-induced pY-Stat3 where analyzed.
Cell lines examined included NB4, THP1, and KG1. Cells were plated on fibronectincoated plates and exposed to IL-6 and soluble IL6 receptor
 (s IL50 ng/mL and
-6Rα;
100 ng/mL respectively) or vehicle control (VC) for 24 hours prior to treatment with
escalating doses of chemotherapy (mitoxantrone, etoposide, or cytarabine). To
examine the effects of blockade of the IL-6/Stat3 pathway on apoptosis, a monoclonal
antibody against the IL-6 receptor, Tocilizumab, was used. For that experiment, NB4
cells were exposed to 100uM Tocilizumab for 1 hour prior to IL-6/sIL-6Rα. For all
experiments, after 24 hours of chemotherapy or VC, cells were harvested and analyzed
for apoptosis by FACS with Annexin V-FITC and Propidium Iodide. For each cell line
and at each chemotherapy dose, differences in %Annexin V-FITC+ cells between the
IL6 and control conditions were compared for significant differences by ANOVA.
Results: For NB4 cells, IL-6 treatment prior to etoposide (100 nM-10 uM) and
mitoxantrone (10 nM-1 uM) reduced apoptosis at all tested doses when compared to
VC (n=3). For example, at 100 nM mitoxantrone, 31% of cells were Annexin V+ in the
VC condition, compared to 19% of cells pre-treated with IL-6. THP1 and KG1 cells
pretreated with IL-6 also demonstrated protection from mitoxantrone (KG1 n=3; THP1
n=2). For NB4 cells treated with mitoxantrone, exposure to Tocilizumab 1 hour prior to
treatment with IL-6 restored the rate of apoptosis towards control. For example, at the
100nM dose of mitoxantrone, the % AnnexinV+ cells were 26%, 15%, and 21% for VC,
IL-6/sIL-6Rα, and IL-6/sIL-6Rα +Tocilizumab, respectively (means, n=3).
Conclusions and Future Plans: We have found that for some AML cell lines, IL-6
protects AML blasts from chemotherapy induced apoptosis in vitro. Our data suggest
that in a subset of patients, the IL-6-induced Stat3 pathway may be promoting the
chemoresistance often seen in relapsed AML. Our results provide support for further
development and evaluation of targeted agents against the IL-6/Stat3 pathway for AML.
Contributors: Stevens, Alexandra MD; Shi, Xin; Kreuger, Michael; Long, Xin PhD; Sison,
Edward MD; Redell, Michele MD, PhD
314
A NOVEL ASSAY FOR THE IN VIVO DETECTION OF REACTIVE OXYGEN SPECIES
USING MRI
Gary R. Stinnett
Advisor: Robia Pautler, Ph.D.-Department of Molecular Physiology & Biophysics
Background: Under normal physiological conditions there is a fine regulation
between reactive oxygen species (ROS) and antioxidants within the body. However, in
multiple disease states, increases in ROS or a decrease in endogenous antioxidants
can cause a perturbation in redox homeostasis. When this balance is disrupted
damage can occur to major cellular components such as the nucleus, mitochondrial
DNA, membranes, and cytoplasmic proteins. Because of the profound role increases in
ROS plays in multiple diseases, including liver, diabetes and neurodegenerative
diseases, technologies that enable the ability to detect ROS levels in live animals would
undoubtedly provide unprecedented insights into multiple preclinical models of human
disease. Recently, our collaborators have designed nano-antioxidant PEG-HCCs, which
not only rapidly quench superoxide but also have been shown to enter cells.
Furthermore, the PEG-HCCs accumulate in the liver where they are eventually broken
down.
An additional important feature of superoxide and hydroxyl radicals are they
have an unpaired electron and thus are paramagnetic, resulting in a shortening of the
spin-lattice relaxation time constant, T1, rendering them MRI detectable. Therefore, in
tissues where there are significant increases in superoxide and hydroxyl radicals, there
should be a concomitant decrease in T1. We therefore hypothesize that it is possible to
use MRI in conjunction with the PEG-HCCs to measure in vivo ROS levels. That is,
based upon the inherent paramagnetic properties of superoxide and hydroxyl radicals,
we should be able to perform a quench assay by measuring the T1 values before and
after the addition of the PEG-HCCs.
Results: Baseline T1 values in the liver of KO mice (n=2) were found to be lower
than those of WT littermates (n=2). After PEG-HCC administration T1 values in WT
mice stayed relatively unchanged while the T1 in the liver of the KO mice increased
after 2.5hrs and returned to baseline levels after 1 week (Fig 1). Two way ANOVA
analysis of the percent change in normalized liver T1 shows that the percent change
curves for WT and KO mice pre, 2.5, hours, 24 hours, and 1 week after Peg-HCC
injection are statistically different. Further analysis demonstrates that the percent
change in normalized liver T1 is significantly larger for the KO mice than the WT mice
(Fig 2).
Discussion: Preliminary data demonstrates our ability to measure differences in
T1 relaxation due to superoxide and hydroxyl radicals. This is further validated after
administration of nano-antioxidant PEG-HCCs when decreased T1 levels in mice
exhibiting increased oxidative stress are returned to WT levels.
Contributors: Moore, Kelly; Loïc, Samuel, Ming Ge; Graham, Brett; Tour, James; Pautler, Robia
G.
315
STRUCTURAL BASIS FOR DIFFERENT SUBSTRATE PROFILES OF TWO
CLOSELY RELATED CLASS D Β‑ LACTAMASES AND THEIR INHIBITION BY
HALOGENS
Vlatko Stojanoski
Background: Serine β-lactamases are enzymes that hydrolyze β-lactam
antibiotics. OXA-163 is a class D serine β-lactamase that was isolated from Klebsiella
pneumoniae and Enterobacter cloacae and has the ability to hydrolyze extendedspectrum cephalosporins especially ceftazidime. OXA-163 differs from the OXA-48
carbapenemase by one substitution (S212D) in the active site β5-strand and a fouramino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands.
Methods: In this study, we performed enzyme kinetic analysis, inhibition assays,
X-ray crystallography, and molecular docking.
Results: The results have confirmed the different substrate profiles of OXA-48
and OXA-163 and determined that both enzymes are inhibited by halogens with
different IC50 values. In addition, the crystal structure of OXA-163 has been solved at
1.72-Å resolution. The major difference in the OXA-163 structure compared to OXA-48
is expansion of the active site pocket due to the four amino acid deletion. Particularly,
the absence of the Arg214 residue in OXA-163 expands the active site compared to
OXA-48 and allows a bulky substrate such as ceftazidime to be accommodated. The
structural difference from OXA-48, which cannot hydrolyze ceftazidime, provides a
rationale for the change in substrate specificity between the enzymes. Additionally, the
crystal structure of OXA-163 in the presence of iodine was solved at 2.89-Å resolution.
In this structure an iodine ion was observed in the active site. The presence of the
iodine in the active site inhibits carbamylation of Lys73 and rearranges the active site of
OXA-163. The most prominent differences are distortion of the β5-strand and occlusion
of the substrate-binding site. Conclusion: Our findings provide a molecular basis for
how OXA-163 is able to confer resistance to ceftazidime. More broadly, these results
give an insight of how an expansion of the active site changes the substrate profile of
an enzyme.
Contributors: Stojanoski, Vlatko; Chow, Dar-Chone; Fryszczyn, Bartlomiej; Hu, Liya; Nordmann,
Patrice; Poirel, Laurent; Sankaran, Banumathi; Prasad, B. V. Venkataram and Palzkill, Timothy.
316
ASSESSMENT OF COMBINATORIAL GENE THERAPY FOR THE TREATMENT OF
OSTEOARTHRITIS
Adrianne Elayne Stone
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Osteoarthritis (OA) is a progressively debilitating condition involving joint
degeneration that causes substantial joint stiffness and pain, decreased mobility, and
increased health care costs. Currently, any one of the 27 million individuals in the US
suffering from OA will merely be proscribed pain management medication, physical
therapy with lifestyle modifications, or in end-stage disease a joint replacement surgery
will be advised. Injections of cytokines and proteoglycans have shown some promise in
clinical and pre-clinical trials, yet due to the rapid degradation and turnover of these
compounds repeated injections are necessary to obtain any long-lasting results,
potentially leading to an increase in adverse events. Major obstacles to the
development of new treatments for OA have include both the difficulty of identifying
quantifiable endpoints for progression in animal models, and the complex cell
autonomous and non-cell autonomous components of the pathophysiology of the joint.
To overcome these obstacles, our lab developed a novel imaging technique combining
staining, phase-contrast optics, and microCT analysis of the joint, which allows for the
robust quantification of murine cartilage volume, surface area, and osteophyte
formation. To address the issue of the complex etiology of OA development, our lab
has focused on a few secreted molecules as potential therapeutic targets – namely
Insulin-Like Growth Factor 1 (IGF1), Proteoglycan 4 (PRG4) and Interleukin 1 Receptor
antagonist (IL1Ra). PRG4 is a major component of the cartilage extracellular matrix and
mediates mechanical stress by providing lubrication. Inflammation has also been
attributed to disease progression, and IL1Ra has been proven to function as an antiinflammatory through the inhibition of Interleukin 1 (IL1). Lack of cartilage regeneration
during disease progression has been shown to be ameliorated by IGF1 gene transfer,
and some synergistic effect when combined with IL1Ra has also been seen previously.
Both published work and preliminary data from our lab and others shows that
individually each provides some protection from OA progression. We hypothesize that
combinatorial therapy with the triple combination of genes will provide increased
protection from OA development and progression through addressing cartilage matrix
loss, chondrocyte survival, and inflammation. We aim to test this by comparing the
effects of combinatorial therapy to mono-therapy in a post-traumatic model and in an
age-related model of OA development, as the balance of individual factors contributing
to disease is different in each.
Contributors: Stone, Adrianne; Grol, Matthew; Ruan, MZ; Cela, Racel; Dawson, Brian; Lee,
Brendan
317
P-21 PROTEIN (CDC42/RAC)-ACTIVATED KINASES AS POTENTIAL NEGATIVE
REGULATORS OF P53 FUNCTION
Lauren Elizabeth Straker
Tumor suppressor p53 is the most commonly mutated gene across all tumor
types, and is frequently dysregulated when not mutated. Activated by numerous stress
signals, p53 functions as a transcription factor to enact several tumor suppressor
activities, such as DNA repair, apoptosis, and cell cycle arrest. Given the critical role of
p53 in maintaining genomic integrity, coupled with its widespread perturbation across
numerous cancer types, a deeper understanding of p53 biology will be crucial to the
advancement of cancer therapeutics. In order to ascertain therapeutic leads, we
leveraged computational tools to identify novel p53 kinases. Hypothesis-generating
algorithms Graph Diffusion, Bag of Words, and non-negative matrix factorization
indicated a probable connection between p53 and p21 protein (Cdc42/Rac)-activated
kinases (PAKs). Of the six kinases in the PAK family, four were present in our
predictions.
PAKs have a well-established role in promoting cell survival and are known
drivers of several cancer hallmarks. Mounting experimental evidence supports a role
for PAK signaling in the development and progression of various cancers, and PAK
overexpression has been associated with resistance to therapy and decreased survival
in several tumor types. Additional evidence has shown that Cdc42 (a primary activator
of PAKs) negatively regulates p53, inhibiting its pro-apoptotic function; however, the
precise molecular link between Cdc42 and p53 remains unclear. Given this information,
we hypothesize that PAK(s) negatively regulate p53.
To assess for interactions between PAKs and p53, co-immunoprecipitation (coIP) experiments were performed in HEK293 cells. Our results show a strong interaction
between p53 and two of four tested PAK family members – PAK4 and PAK6.
Additional co-IP testing in HCT116 and MCF7 cell lines is ongoing.
Further
experiments will be performed in order to confirm p53 phosphorylation by PAK(s), as
well as to fully characterize the nature and biological effects of these interactions.
In conclusion, we have uncovered a novel connection between p21 protein
(Cdc42/Rac)-activated kinases (PAKs) and p53. Once further elucidated, this finding
may be highly valuable to the understanding of p53 biology, as well as to the selection
of therapeutics for a large subset of cancer patients.
Contributors: Straker, Lauren; Bachman, Benjamin; Wilkins, Angela; Adikesavan, Anbu;
Donehower, Lawrence; and Lichtarge, Olivier.
318
PROTEIN REFOLDING AND MOLECULAR RECOGNITION IN LACI
Danielle Dee Stuhlsatz
Advisor: B. Pettitt, Ph.D.-Biochemistry
Misfolded and unstructured domains of proteins represent important examples of
disease states where the understanding of the recognition, or folding process has
important potential therapeutic implications. The mechanisms governing molecular
recognition and the transition of proteins from their unfolded state to their native state
remain related but unanswered fundamental biophysical questions. Refolding of protein
domains often occurs during DNA binding. Specifically, LacI contains a hinge region
that is disordered without DNA or when nonspecifically bound, but becomes ordered
when bound to the correct DNA sequence. Experimentally, the hinge region has a
measurable disorder to order transition when binding its specific sequence. This system
has a nontrivial sequence and allows us to test the importance of the protein folding
transition versus that of the contributions of DNA bending and DNA protein interactions
separately. The ions of the system seem to also play an important role in stabilizing this
flexible protein. The hinge-helix sequence of the LacI system will be computationally
tested in various multicomponent aqueous solutions.
Contributors: Pettitt, B. Montgomery
319
JAG1 PLAYS PLEOTROPIC ROLES IN PROSTATE CANCER PROGRESSION
Qingtai Su
Advisor: Li Xin, Ph.D.-Department of Molecular & Cellular Biology
Prostate cancer is a major health threat to US males. Deregulation of the
Notch/Jag1 pathway was closely associated with prostate cancer progression; however,
the mechanistic roles of Jag1 in prostate cancer development are poorly defined.
We took advantage of prostate cancer cell lines and transgenic mouse models to
investigate the role of Jag1 in prostate cancer progression. We injected the Du145 cells
over-expressing Jag1 and relevant control cells into NOD/SCID mice via tail veins.
Surprisingly, Jag1 up-regulation accelerated distal colonization of tumor cells in lung,
reduced latency of metastasis and shortened mouse lifespan. Meanwhile, we
generated a transgenic mouse model that allows conditionally specific over-expression
of Jag1 in prostatic epithelial cells. We found that over-expression of Jag1 alone did not
transformed epithelial cells by 1 year. Epithelium proliferation and apoptosis were not
influenced by Jag1 expression. We crossed this mouse with Pten-null prostate cancer
model to interrogate Jag1 functions during cancer progression. Remarkably, our
preliminary result indicated that Jag1 promotes seminal vesicle tumor growth.
Nevertheless, the growth of epithelial cells within prostate tumor was delayed by Jag1
expression. The seemingly dual functions of Jag1 in this tumor model and biology
implications remain further investigation.
Contributors: Su, Qingtai; Zhang, Boyu; Kwon, Oh-Joon; Wei, Xing; Zhang, Li; Xin, Li
320
THE USE OF KU SEPARATION-OF-FUNCTION MUTANTS TO EXPLORE KU’S
ASSOCIATION AND FUNCTION AT HUMAN TELOMERES
Ann Tabitha Sukumar
The natural ends of linear chromosomes are composed of specialized
nucleoprotein structures called telomeres. Telomeres consist of tandem six nucleotide
repeats bound by the components of the shelterin complex, which prevent chromosome
ends from being fully engaged as substrates for DNA repair. The shelterin protein,
TRF2, plays a crucial role in telomere end protection by facilitating the assembly of the
t-loop structure. When end protection is compromised, telomeres are subjected to
deleterious chromosome end-to-end fusions mediated by nonhomologous end joining
(NHEJ), the major DNA double strand break repair pathway in mammalian cells.
However, Ku, an important NHEJ factor, associates with functional telomeres and
contributes to telomere functions.
The Ku70 and Ku80 subunits of the Ku heterodimer form a ring-like structure,
which allows it to load onto DNA ends at DSBs, where it contributes several activities to
NHEJ. How Ku is repressed from participating in NHEJ at telomeres, particularly when
the t-loop is resolved, is not well understood. Data from our lab suggests that this may
be achieved, in part, by Ku’s interaction with TRF2.
Ku is an essential protein in human cells, not for its role in NHEJ, but to prevent
telomere loss mediated by homology-directed repair. Given this essential role in
humans, we are capitalizing on separation-of-function mutants to better understand
how Ku contributes to telomere maintenance, while its NHEJ activity is repressed. We
have previously published the human Ku70-α5 mutant, which is impaired for its
interaction with TRF2 and for NHEJ. In addition, we have generated a human Ku80
mutant that is defective for DNA end binding (Ku80-DEB), but proficient for
heterodimerization with Ku70 and for interaction with TRF2. Together, they will allow us
to test how the α5 region and end binding contribute to Ku’s association with telomeres
and its role in telomere maintenance.
Contributors: Sukumar, Ann
321
PROTECTING CAR T CELLS FROM IMMUNOSUPPRESSIVE CYTOKINES
PRESENT IN THE TUMOR MICROENVIRONMENT
Sujita Sukumaran
Advisor: Ann Leen, Ph.D.-Department of Pediatrics
Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancerrelated deaths in the US and has proven resistant to all standard therapies, highlighting
the need for novel treatments. Immunotherapy using T cells genetically modified to
express chimeric antigen receptors (CARs) is emerging as a powerful therapeutic to
treat various blood-derived malignancies. Hence, to determine whether we could target
pancreatic cancer, we generated a CAR directed against tumor-expressed prostate
stem cell antigen (PSCA) and demonstrated that CAR-PSCA T cells (73±16%
transduction efficiency, n=5) could specifically kill PSCA-expressing cell lines (CAPAN1
and K562-PSCA) but not 293T (PSCA- target) (74±4%, 73±6% and 9±3% specific lysis,
respectively, 10:1 E:T, n=3). However, while the infusion of CAR T cells may provide
therapeutic benefit, pancreatic tumors use a variety of immune-evasion mechanisms,
including the production of immunosuppressive cytokines (IL4, IL10, TGF
) tha t inhib
effector T cells. Indeed, we observed elevated levels of IL4 in patient serum compared
to healthy individuals (14.3±19.5 pg/mL vs 1.1±1.4 pg/mL, respectively). Hence, to
protect our CAR T cells and exploit IL4 at the tumor site, we generated a chimeric
cytokine “4/7R” receptor, which links the IL4R exodomain with the signaling
endodomain of the IL7R, an immunostimulatory receptor.
To evaluate the protective effects of the 4/7R we transduced T cells to express
both CAR-PSCA and 4/7R (30-72% double positive cells; n=4) and assessed the
expansion of CAR-PSCA and 4/7R/CAR-PSCA T cells exposed to IL4. Although CARPSCA T cells were unable to expand in the presence of IL4 (2x106 cells-day 0 and
6.1±3.8x107-day 28), 4/7R/CAR-PSCA T cells expanded exponentially (2x106 cells to
5.1±3.6x109, n=3). Moreover, culture in IL4 enriched the 4/7R transgenic cells
(45.9±15% to 86±11% over 28 days, n=3). Next, we assessed the impact of IL4
exposure on the cytokine profile of the transgenic cells. With prolonged culture in IL4 (a
prototypic Th2 cytokine), CAR-PSCA T cells Th1 cytokine production was diminished
[IFN 
- 5±2 ng/mL and TNF 
- 1.1±0.1 ng/mL], while their 4/7R counterpart retained a
Th1-polarized profile [IFN
 91±2
the 4/7R/CAR-PSCA T cells expanded only in the presence of both antigen and
cytokine - signals present in combination only at the tumor site. We are currently
investigating the in vivo persistence and cytolytic effects of 4/7R/CAR-PSCA T cells
when used to treat mice engrafted with IL4-producing tumors.
Contributors: Sukumaran, S; Mohammed, S; Watanabe, N; Bajgain, P; Anurathapan, U;
Heslop, H.E; Rooney, C.M; Brenner, M.K; Leen, A.M; Vera, J.F.
322
FUNCTIONAL CHARACTERIZATION OF DEVELOPMENTAL BRAINSTEM
POPULATIONS IN ADULT RESPIRATORY PHYSIOLOGY
Jenny J. Sun
Advisor: Russell Ray, Ph.D.-Department of Neuroscience
The perpetual rhythm of breathing is essential for survival and emerges through
the interactions of a highly redundant and anatomically complex array of brainstem
neuronal networks. Previous studies suggest that proper partitioning of the embryonic
hindbrain into transient, genetically-defined segments called rhombomeres (r) is
required for normal respiratory development. However, because most studies of
rhombomeric mispatterning result in embryonic or neonatal lethality, it remains unclear
if rhombomeres ultimately define distinct circuit modules that regulate unique aspects of
respiration in the adult animal, or if the respiratory network is mapped postnatally or by
activity-dependent cues. Because early gene expression is critical to the organization of
neural circuits, I hypothesize that embryonic rhombomeric patterning persists in the
adult and underlies the functional organization of the respiratory system. To examine
the role of rhombomere-specific neuronal subsets in adult respiration, we used
pharmaco-genetic DREADD-based mouse lines to acutely and non-invasively perturb
(RC::FP_hM4D) targeted neurons in the adult brainstem, bypassing perinatal lethality.
In combination with whole-body plethysmography, we are able to accurately measure
respiratory parameters under room air, hypercapnic (5% CO2), and hypoxic (10% O2)
conditions in conscious and unrestrained mice. Our data demonstrate that various
rhombomere-derived populations (r1, r2, r3&5, r4, and r7&8) differentially affect
respiratory rate, tidal volume, minute ventilation, oxygen consumption, and waveform
patterns when they are perturbed under different respiratory conditions. For example,
under room air conditions, adult perturbation of r1, r2, and r7&8 derived neurons all
resulted in increased respiratory rate and tidal volume while perturbation of r3&5 and r4
neurons resulted in a decrease. These data show the contribution of early embryonic
patterning in defining the functional organization of the adult respiratory network, and
set the stage to use intersectional genetics to further identify neuron subtypes within
each rhombomere that are involved in specific aspects of respiratory homeostasis.
Contributors: Sun, Jenny; Ray, Russell
323
CHARACTERIZING THE DNA METHYLOME OF OSTEOSARCOMA
Jiayi Monika Sun
Advisor: Ching Lau, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma is the most common malignant bone tumor in adolescents and
young adults. There has been no improvement in patient survival in the last 20 years
largely due to the lack of reliable methods to predict response to standard treatment
and identification of novel therapeutic targets. We aim to discover new prognostic
markers and targets by performing a comprehensive characterization of the
osteosarcoma genome with the goal of identifying genetic aberrations indicative of
tumor biology and predictive of patient outcome.
We performed genomic characterization of 89 osteosarcoma samples acquired
from the Children’s Oncology Group, Texas Children’s Hospital and other institutions
around the world. These cases were profiled using gene copy number, mRNA and
microRNA expression, DNA methylation, whole genome sequencing and RNA-seq. I
focused on analyzing the DNA methylation data which was acquired using Illumina’s
HumanMethylation 450K BeadChip which profiles over 485,000 CpG sites across the
human genome and integration with the other platforms.
Using unsupervised clustering we separated the osteosarcoma samples into two
groups with significantly different overall survival rates. We identified two genes,
ZSCAN16 and HIST1H4D, which were differentially methylated between the two groups
and were able to recapitulate the difference in survival using their expression. Using
BumpHunter, we also identified that this region was differentially methylated between
the two groups. This region on chromosome 6 contains many other histone related
genes and is known as histone cluster 1. We also identified differentially methylated
regions in 14q overlapping with a cluster of microRNAs whose aberrant expression has
been linked to osteosarcoma. Integration of methylation with copy number data
revealed that the methylation subgroups which are predominantly hypomethylated also
have a concentration of structural aberrations on several chromosomes including chr17
overlapping TOP3A, a key chromosomal maintenance gene.
Osteosarcoma has several notable methylation abnormalities. HIST1H4D codes
for a subunit of histone 4 which is part of the nucleosome complex responsible for
packaging of DNA. TOP3A is involved with chromosomal maintenance. Aberrations in
genes such as these could be responsible for the genomic complexity we are observing
in osteosarcoma. We are currently in the process of evaluating whether these genes
do indeed affect chromosomal instability and the resulting effect on the tumor’s
response to therapy and overall prognosis.
Contributors: Sun Jiayi; Taylor, Aaron; Shen, Jianhe; Yu Alex; Xu Gufeng; Voicu, Horatiu;
Guerra Rudy; Man Chris; Lau Ching
324
VACUOLAR PROTEASE AND HSP70 CHAPERONES REGULATE SIR2 TURNOVER
DURING AGING IN YEAST
Yu Sun
Advisor: Weiwei Dang, Ph.D.-Department of Molecular & Human Genetics
Sir2, a class III histone deacetylase, is a key regulator of aging in budding yeast.
Overexpression of Sir2 extends replicative lifespan and this longevity effect is
conserved in many eukaryotes. Sir2 protein level is reduced during aging, leading to
high levels of H4K16 acetylation at telomere regions. However, there is no reduction in
Sir2 transcripts in old cells1. We hypothesize that the loss of Sir2 protein during aging is
mainly due to a change in its degradation rate.
We found that neither of the major protein quality control pathways, UbiquitinProteasome System and autophagy, is responsible for Sir2 degradation in old cells. Our
proteomics study comparing protein level changes with age identified Pep4 (vacuolar
protease A, the homolog of mammalian Cathepsin D) as one of the most up-regulated
proteins in old cells. Sir2 protein level was stabilized and this protein was localized to
the vacuole of old pep4∆ cells, indicating the translocation of Sir2 from nucleus to
vacuole during aging. Sir2 purified from pep4∆ cells is more resistant to proteinase K
digestion, while the enzymatic activity of Sir2 is unaffected. By mass spectrometry
analysis of immunoprecipitated Sir2, we identified Ssa2 and Ssb2, members of the
HSP70 family chaperones, as novel Sir2 binding partners, which was confirmed by coIP experiments. Deletion of Ssa1/2 stabilized Sir2 in old cells. Ssa1/2 is also implicated
to regulate the sub-cellular localization of Sir2. Preliminary data suggests that the
lifespan of pep4∆ cells is longer than wild type, possibly through regulating Sir2
abundancy.
Overall, we propose a model in which Sir2, during aging, is translocated to the
vacuole and is degraded in a unique Pep4-dependent pathway. The HSP70
chaperones Ssa2 and Ssb2 may be involved in the targeting and translocation of Sir2 in
this process.
Reference
Dang, Weiwei, et al. "Histone H4 lysine 16 acetylation regulates cellular
lifespan." Nature 459.7248 (2009): 802-807.
Contributors: Sun, Yu; Berger, Shelley L.; Dang, Weiwei.
325
STEROID RECEPTOR COACTIVATOR-2 IS A CRITICAL REGULATOR OF
ENDOMETRIAL CANCER CELL METABOLISM
Maria Magdalena Szwarc
Steroid receptor coactivator-2 (SRC-2) is a member of the SRC family of
coregulators which also includes SRC-1 and -3. SRCs exert a wide-spectrum of
physiological processes ranging from mammary morphogenesis to metabolic
homeostasis. Deregulation of SRC expression is a causal factor for numerous
pathologies. In the case of the endometrium, clinical studies reveal that SRC-2 and -3
levels are elevated in the endometrium of patients diagnosed with polycystic ovary
syndrome who have an increased risk of developing endometrial cancer (EC). Elevated
expression of these two coregulators has also been found in hyperplastic and
neoplastic endometrium. These clinical findings suggest a causal link between SRC
function and the emergence of endometrial disorders. This is supported by our previous
findings showing that increased expression of SRC-2 in the endometrium of a
transgenic mouse model leads endometrial hyperplasia. This study focuses on
determining the mechanism by which SRC-2 promotes endometrial hyperplasia and
cancer progression.
We found that knockdown of SRC-2, but not SRC-1 and -3, leads to a reduction
of EC cell growth and anchorage independence. SRC-2 is also required to maintain
glycolytic capacity and oxygen consumption of EC cells indicating SRC-2’s role in
regulation of EC cell metabolism. Measurements of levels of key glucose metabolites
revealed that SRC-2 is critical for the maintenance of phosphopentose and nucleotide
levels generated by the pentose phosphate pathway which is crucial for cellular
proliferation. This is accompanied by a reduction of a key enzyme for phosphopentose
production, ribose-5-P isomerase (RPIA). Importantly, siRNA mediated downregulation
of RPIA, similarly as in the case of SRC-2, leads to significant reduction of cell growth.
Decrease of SRC-2 levels also attenuates tumor expansion in vivo. Together these
findings indicate that SRC-2 is regulator of cell metabolism critical for EC development
and progression.
Contributors: Szwarc, Maria M; Kommagani, Ramakrishna; Putluri, Nagireddy; Cristian, Coarfa;
Lanz, Rainer B; DeMayo, Francesco J; Lydon, John P; O’Malley, Bert W
326
KCA1.1 CHANNELS ARE CRUCIAL FOR THE DIFFERENTIATION OF HUMAN
MYOBLASTS INTO MYOTUBES
Rajeev Babu Tajhya
Progressive muscle wasting is the main cause of disability in patients with
myotonic dystrophy type 1 (DM1). DM1 is a neuromuscular disorder affecting 1 in 8000
people worldwide and has no cure. The expansion of tri-nucleotide CUG repeats in the
3’-untranslated region of dmpk gene is known to cause errors in alternative splicing of
pre-mRNA leading to multiple systemic disorders in DM1 patients. However, the
downstream pathways contributing to reduced myogenesis remain unclear.
KCa1.1 is calcium-activated and voltage-dependent K+ channel that affects
proliferation and migration by regulating membrane potential and intracellular signaling
pathways. Myogenesis requires proliferation of myoblasts that exit from cell cycle and
fuse together to form multinucleated myotubes. We have identified a decrease in
expression of KCa1.1 channels in proliferative myoblasts from DM1 patients. We
hypothesized that the loss of KCa1.1 channels in DM1 impairs differentiation of
myoblasts into myotubes.
We applied specific inhibitors of KCa1.1 channels in myoblasts from healthy
volunteers. Inhibition of KCa1.1 channels enhanced proliferation by failing to exit from
cell-cycle, impaired migration and decreased matrix metalloproteinase-2 production
required for myotube fusion. Ultimately, myotube fusion was delayed as measured by
reduced myogenic transcription factors and decreased fusion index. In contrast, overexpressing KCa1.1 channels in DM1 myoblasts normalized their proliferation to the
levels observed in healthy myoblasts.
Increased NF-κB nuclear transport is associated with muscle wasting by
regulation of downstream target cyclinD1. We observed increased levels of activatedNF-κB increased in DM1 myoblasts compared to healthy myoblasts. In addition,
inhibiting KCa1.1 channels in healthy myoblasts increased activated-NF-κB alongwith
an increase in cyclinD1 which explains the delay in cell-cycle exit. Furthermore, we
detected increased intracellular Ca2+ in DM1 and inhibiting KCa1.1 channels in normal
myoblasts led to an increase in intracellular Ca2+ suggesting the KCa1.1 channels
might be affecting myogenesis via Ca2+-mediated signaling.
Our studies show that KCa1.1 channels are crucial in maintaining proliferation,
migration and myotube fusion, suggesting KCa1.1 modulation could be utilized as a
novel therapeutic approach to rescue delayed myogenesis in DM1.
Contributors: Tajhya, Rajeev; Tanner, Mark; Hu, Xueyou; Huq, Redwan; Timchenko, Lubov;
Beeton, Christine
327
THE E3 UBIQUITIN LIGASE, ARIADNE-1 (ARI-1), REGULATES MICROTUBULAR
TRANSPORT IN BOTH NEURON AND GLIA IN DROSOPHILA AND IS REQUIRED
FOR PROPER SYNAPTIC TRANSMISSION
Kai Li Tan
Synaptic transmission is a complex process that involves various cellular
activities, including but not limited to, protein synthesis and degradation, vesicular and
protein trafficking, and fusion and fission of membranes. Orchestrating the different
cellular activities is thus important to ensure optimal synaptic function. In a forward
genetic screen on the Drosophila X-chromosome to identify mutations in essential
genes that cause defects in synaptic transmission, we identified mutations in ari-1, the
Drosophila homolog of mammalian HHARI. ari-1 is a highly conserved gene, which
encodes a ring-between-ring E3 ubiquitin ligase. By recording electroretinograms, we
found that homozygous mutant photoreceptors display a loss of ON-transients when
activated by light, indicating defects in neurotransmission. To further characterize this
synaptic transmission defect, we turned to the 3rd-instar larval neuromuscular junction
(NMJ). We performed electrophysiological recordings and found that postsynaptic
muscle cells show an increased excitatory junction potential (EJP) upon stimulation
when compared to control. To identify the cause of the increased evoked response, we
performed immunostaining for different synaptic components. We find that several
synaptic markers, including Bruchpilot (Brp) and Synaptotagmin (Syt) accumulate at the
distal axons of the mutants. Moreover, the mutant axons are abnormally large in
diameter, with glia-positive bulges scattered along the axon. These phenotypes indicate
a defect in microtubule-associated trafficking in both the neuron and the glia, since the
trafficking of synaptic proteins such as Brp and Syt relies on microtubule. Microtubular
mutants such as microtubule associated proteins (MAPs) and motor proteins also
display abnormal axonal and glial morphology. Through MAP staining, we show that
microtubule formation is intact. Since the phenotypes we observe are very similar to
those seen in microtubule motor protein mutnats such as kinesin heavy chain (khc)
mutant, we are currently investigating if the microtubular motor proteins, such as the
khc, are functional, through genetics and biochemical methods. Ultimately, we want to
identify the interacting protein(s) of Ari-1 in regulating the transport of synaptic protein
and to maintain normal morphology and function of the neuron and glia.
Contributors: Tan, Kai Li*; Haelterman, Nele*; Bellen, Hugo
328
KCA1.1 AS A TARGET OF FIBROBLAST-LIKE SYNOVIOCYTES DURING
RHEUMATOID ARTHRITIS
Mark R Tanner
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of largely
unknown etiology that mainly affects diarthrodial joints, leading to joint destruction, pain,
and decreased mobility. The fibroblast-like synoviocyte (FLS) is a resident joint cell that
has a central role in RA pathogenesis. In healthy individuals, FLS compose a layer of
the synovium and function to maintain homeostasis within the joint. During RA, FLS
develop a pathogenic phenotype with a hallmark feature being a high degree of
invasiveness, in which in vitro invasiveness is correlated with disease severity. This
altered phenotype of FLS contributes to joint destruction during RA. Currently, no RA
therapy has been developed to specifically target FLS.
We have found that FLS from patients with RA and from rats with a model of RA
have increased expression of the KCa1.1 potassium channel at their plasma membrane
when compared to FLS from patients with osteoarthritis or from healthy rats,
respectively. Selectively blocking this channel reduces the in vitro invasiveness of FLS.
This is accomplished through the role of KCa1.1 as a regulator of calcium homeostasis
in RA-FLS, with downstream effects on Akt phosphorylation and the plasma membrane
expression and activation of β1 integrins.
We have also found that inhibiting KCa1.1 with a selective small-molecule
blocker after onset of clinical signs of arthritis significantly reduces joint inflammation,
bone and cartilage damage, and synovial hyperplasia in multiple rat models of RA. FLS
from blocker-treated animals also have reduced ex vivo invasiveness compared to
those from vehicle-treated animals. Furthermore, in vivo treatment with a peptide
venom KCa1.1 blocker reduces disease severity in a rat model of RA with fewer side
effects than small-molecule blocker treatments. These studies indicate the importance
of KCa1.1 as a novel target for RA and emphasize the potential efficacy of directly
inhibiting FLS in reducing the severity of this debilitating disease.
Contributors: Tanner, Mark; Huq, Redwan; Tajhya, Rajeev; Gulko, Percio; Beeton, Christine
329
EXPRESSION PROFILING OF INTRACRANIAL GERM CELL TUMORS REVEALS
UPREGULATION OF RAS THROUGH MRNA-MICRORNA SIGNALING PATHWAY
Aaron Michael Taylor
Advisor: Ching Lau, M.D./Ph.D.-Department of Pediatrics
Intracranial germ cell tumors (iGCTs) account for 3 percent of intracranial tumors
in children domestically and 11 percent of brain neoplasms in Asian countries. iGCTs
are separated into two clinically- and biologically-distinct subtypes based on histology:
germinomas and non-germinomatous germ cell tumors (NGGCTs). The delicate
location of these tumors makes surgical resection or biopsy very difficult and makes
subtype distinction more challenging. Current treatments have significant side effects
on the brain and negatively impact quality of life. In this study, we aim to discover
molecular signatures that can identify tumor subtype to facilitate treatment stratification
or serve as targets for therapeutic intervention. To this end, we performed mRNA
expression profiling (Human Genome U133 Plus 2.0 Array) and miRNA expression
profiling (ABI TaqMan Platform) with 36 and 49 iGCTs, respectively. The cohort
represented a broad spectrum of iGCT from patients between 1 to 41 years of age that
were treated at Texas Children’s Hospital or various hospitals in Japan. Forty-two
percent of these cases were germinoma and 58% NGGCTs. Stratification of samples
using unsupervised non-negative matrix factorization (NMF) clustering based on mRNA
or miRNA expression revealed two distinct subgroups that delineated germinomas from
NGGCTs. 1777 genes were found to be differentially expressed between subtypes.
Employing stepwise model building in each data set separately, we were able to
separate these groups using only two mRNA, LIN28B and L1TD1, and two miRNA,
miR-26a and miR-373. LIN28B is a marker for undifferentiated stem cells often
expressed in testicular and ovarian cancers. miR-26a suppresses the LIN28B gene
and is down-regulated in germinoma. LIN28B directly binds and suppresses the let-7
microRNA family, which are responsible for suppression of the KRAS oncogene,
previously found to be mutated in ~19% of iGCTs. LINE-1 Type Transposase Domain
Containing 1 (L1TD1) is required for human stem cell renewal and directly interacts with
LIN28B for its RNA binding function. LIN28B and L1TD1 are both known to be
upregulated in other systemic germ cell tumors, but this has not yet been documented
in iGCTs. miR-373 has been documented to be upregulated in testicular germ cell
tumors. In conclusion, our results show that intracranial germinoma have similar
expression to systemic germinoma, and may be responsive to similar treatments. Our
findings suggest an oncogenic mechanism in iGCTs by which activation of LIN28B and
L1TD1 results in downregulation of the let-7 miRNA and subsequent upregulation of
KRAS. Our data suggests that treatment aimed at downregulating the KRAS pathway in
germinoma may be effective, and highlights the need for further specific studies of
NGGCTs.Contributors: Taylor, Aaron; Ren, Liangzuo; Terashima, Keita; Shen, Jianhe; Sun,
Jiayi; Adesina, Adekunle; Su, Jack; Nakamura, Hideo; Nishikawa, Ryo; Lau, Ching C.
330
THE ROLE OF NOTCH SIGNALING ON OLFACTORY BULB CIRCUIT
MAINTENANCE
Burak Tepe
The mammalian brain has a remarkable capacity for continued neurogenesis
throughout life. Every day, thousands of adult-born neurons are generated in the
subventricular zone and migrate anteriorly where they eventually integrate into the
existing neuronal circuitry of the olfactory bulb (OB). During this process, only fifty
percent of adult-born neurons will survive long-term while the rest are eliminated
through apoptosis. This dramatic reduction suggests that certain selection events act on
these neurons to determine which ones will be integrated.
In order to find molecular mechanisms that regulate this selection, we performed
a microarray analysis on mutants that we have previously shown have deficits in adultborn neuron circuit integration and found that Notch signaling is altered between
mutants and control animals. Using a Notch reporter mouse, we found that Notch
signaling is highly active in the OB. Detailed analysis of these reporter animals revealed
that increased Notch levels are correlated with increasing maturity of adult-born
neurons. Therefore we hypothesize that Notch signaling is involved in the maintenance
of olfactory bulb circuits by promoting adult-born neuron integration and neuronal
function. By investigating the function of Notch in the OB, we aimed to find novel
functions for Notch signaling in the regulation of neuronal plasticity and circuit
maintenance.
Contributors: Tepe, Burak; Kim, Cynthia J. ; Garcia, Isabella; Arenkiel, Benjamin R.
331
ALLOSTERIC REGULATION OF D2 DOPAMINE RECEPTOR (D2R) TO GENERATE
BIAS TOWARDS THE β-ARRESTIN2 PATHWAY USING EVOLUTIONARY TRACEGUIDED POINT-MUTATIONS
María Elisa Terrón-Díaz
One third of all available medications target G-Protein Coupled Receptors
(GPCR). Therefore, specificity towards ligands and downstream effectors of GPCRs is
important for understanding protein function and for the development of therapeutic
drugs with fewer side effects. The serotonin (5-HT2AR) and D2 dopamine (D2R)
receptors are both part of the bioamine subfamily of GPCRs and have similar sequence
identity. However, they discriminate stringently in their cellular responses between
endogenous agonists. Using Difference Evolutionary Trace (ET) analysis functional
determinants were previously identified and rationally targeted by swapping residues
from 5-HT2AR to D2R. The sixteen residues swapped altered D2R ligand potency,
affinity, and specificity. Beyond the ligand bias, we aim to utilize these sixteen D2R
mutants to determine the effector bias with respect to β-arrestin2, a key player in D2R
desensitization and resensitization pathway. Although it is well established that βarrestin2 is recruited to D2R in an agonist-dependent matter, it is not known how these
16-point mutations in D2R regulate β-arrestin2 recruitment to the receptor in an
allosteric manner. β-arrestin2 dependent D2R internalization will be measured in
wildtype as compared to mutant D2R after agonist treatment using cell-ELISA assays.
We hypothesize that one or more of the 16 D2R mutants, that were previously shown to
have ligand bias, will also have β-arrestin2 bias. It is known that β-arrestin2 is recruited
to plasma membrane in presence of 5-HT2AR in an agonist independent matter
suggesting that agonist-dependent 5-HT2AR internalization is lead in a β-arrestin2
independent matter. Therefore, D2R mutants that have decreased β-arrestin2
dependent internalization as compared to wildtype after agonist treatment will suggest a
loss-of-function phenotype in D2R similar to 5-HT2AR. This project will shed light as to
how allosteric regulation of D2R can influence effector bias aiding in the understanding
of the pathway and improving rational approaches to drug design.
Contributors:
332
UNCOVERING NUTRITIONAL VIRULENCE MECHANISMS OF BACILLUS
ANTHRACIS
Austen Lee Terwilliger
Bacterial infections can be thought of as a “tug-of-war” between the host and
pathogen over essential nutrients like sugars, metals, and amino acids. The host limits
the pathogen’s access to these nutrients as a growth restriction strategy termed
nutritional immunity. Conversely, the pathogen subverts these host defenses through
nutritional virulence mechanisms. Previous work demonstrated that Bacillus anthracis,
the causative agent of anthrax, secreted the InhA1 metalloprotease to degrade
hemoglobin and incorporate its peptides as an essential amino acid source in vitro.
Here, we report that InhA1 is required for scavenging essential amino acids from
several serum proteins as well as whole serum in vitro. We also develop a transposon
library screen that identified several transporters and regulators as potential nutritional
virulence factors. Future work will focus on characterizing mutants in a mouse model of
anthrax. These studies will broaden our understanding of bacterial pathogenesis
mechanisms and provide new avenues for targeted treatment of bacteremia.
Contributors: Terwilliger, Austen; Maresso, Anthony
333
CD62L+ SUBSET OF NKT CELLS HAS SUPERIOR IN VIVO PERSISTENCE AND
ANTI-TUMOR ACTIVITY
Gengwen Tian
Advisor: Leonid Metelitsa, M.D./Ph.D.-Department of Pediatrics
Va24-invariant Natural Killer T cells (NKTs) effectively localize to tumors and
have natural antitumor properties that make them highly attractive as a carrier of
chimeric antigen receptors (CARs) for redirected cancer immunotherapy. However, the
mechanisms responsible for NKT-cell numeric expansion and in vivo persistence are
unknown, impeding the clinical application of NKTs or CAR-NKTs. We show that
antigen-induced in vitro expansion of primary NKTs is associated with the accumulation
of a CD62L+ subset, while CD62L- cells undergo early exhaustion. Only CD62L+ NKTs
survived and proliferated in response to secondary stimulation. After transfer to NSG
mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs and when transduced
with a CD19-specific CAR had higher therapeutic efficacy in a Raji lymphoma model.
Proliferating CD62L+ cells downregulated or maintained CD62L expression when they
were activated via TCR alone or in combinations with co-stimulatory receptors,
respectively. We then tested 161 clones of K562 cells genetically modified to express
CD1d and co-stimulatory ligands. We found that the B-8-2 clone
(HLAnullCD1dmedCD86high4-1BBmedOX40Lhigh) induced the highest rate of NKTcell expansion with the preservation of CD62L expression. Compared with CAR-NKTs
expanded with autologous PBMC, B-8-2-expanded cells exhibited prolonged in vivo
persistence and superior therapeutic activity in a Raji lymphoma model. Therefore, we
have identified CD62L as a marker of a functionally distinct NKT-cell subset endowed
with high proliferative potential and have developed artificial antigen-presenting cells for
the generation of CD62L-enriched NKTs for effective cancer immunotherapy.
Contributors: Gengwen Tian1, Amy N. Courtney1, Bipulendu Jena2, Andras Heczey1, Daofeng
Liu1, Ekaterina Marinova1, Linjie Guo1, Xin Xu1, Hiroki Torikai2, Dean Lee2, Gianpietro Dotti3,
Laurence J. Cooper2, and Leonid S. Metelitsa1
334
THE ROLE OF ADAPTIVE IMMUNE SYSTEM IN THE ESCAPE OF BREAST
CANCER CELLS FROM PRIMARY TUMORS
Lin Tian
Metastasis is one of the hallmarks of cancer, and is the direct cause of more
than 90% of cancer-related deaths. Intravasation into the circulation is a critical step for
tumor cells to reach distant organs. We have recently observed a dramatic effect of the
adaptive immune system on vascular structures and metastasis. In the project, we
hypothesize that adaptive immune cells may prevent the cancer cells from entering into
the circulation through normalizing the tumor-associated vasculatures.
We utilized a p53-null murine mammary tumor model to test our hypothesis. To
determine the role of adaptive immune cells in intravasation, we transplanted tumor
cells into mammary gland of Balb/c mice (immunocompetent) and nude mice
(immunodeficient). Through quantifying circulating tumor cells (CTCs) by qPCR, we
found that the number of CTCs in Balb/c mice was around 100 times lower than that in
nude mice. To further investigate the relation between adaptive immune cells and
vascular normalization, we compared pericyte coverage of tumor-associated
vasculatures in Balb/c mice and nude mice, and found that pericyte coverage in tumors
of Balb/c mice was higher than that of nude mice. To directly test the function of T
lymphocytes in vascular normalization, we reconstituted T lymphocytes in tumor bearing
nude mice, and found that pericyte coverage of tumor-associated vasculatures
increased significantly in nude mice with T cell reconstituted.
This study indicates that adaptive immune cells may decrease vasculature
permeability through increasing pericyte coverage. As a consequence, could inhibit
intravasation and decrease metastasis frequency.
Contributors: Lin Tian1,3, Thomas Welte1, Xiang Zhang1,2.
1 Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX
3 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College
of Medicine, Houston, TX
335
KCNQ2 VARIANTS CAUSING EPILEPTIC ENCEPHALOPATHY DISPLAY VARIABLE
COMPLEXITY OF DISRUPTION OF LOCALIZATION AND FUNCTION AT THE AXON
INITIAL SEGMENT
Baouyen Tran
Advisor: Edward Cooper, M.D./Ph.D.-Department of Neurology
KCNQ2/3 are voltage-gated potassium channels underlying the M-current (IM)
regulating neuronal excitability. Genetic mutations in KCNQ2/3 may lead to mild disorders such as Benign Familial Neonatal Seizures (BFNS) or severe disorders such as
epileptic encephalopathy. Localization and concentration of KCNQ2/3 channels at the
axon initial segment (AIS) tightly regulates normal firing patterns. However, changes in
distribution of KCNQ2/3 channels throughout development may play a role in severity of
mutations of the KCNQ2 gene. In addition, mutations leading to epileptic encephalopathy may have different mechanisms that fail, which include gating, trafficking, or rapid
degradation, based upon their location in critical regions of the channel subunit. In order
to test for this, I performed immunofluorescence labeling in rodent tissue, cultured rat
hippocampal neurons, and perform biotinylation assay in CHO cells transfected with
mutant KCNQ2 in order to determine whether or not trafficking of the KCNQ2/3
channels are altered or abnormally degraded. immunofluorescence microscopy
performed on tissue sections from transgenic mice overexpressing the dominant
negative mutant G279S revealed an aberrant labeling pattern: KCNQ2 was completely
absent at the AIS and was retained at intracellular puncta in the soma and dendrites.
KCNQ3 was partially redistributed to these puncta. In pore mutations, surface biotinylation assays in CHO cells show no changes. However, a variant located within the
calmodulin binding region shows a decrease in total protein, leading us to believe the
KCNQ2 protein itself is unstable. Additionally, in co-immunoprecipation experiments,
calmodulin binding with the variant is decreased. Inversely, hippocampal neuron
cultures show a disruption of KCNQ2 and KCNQ3 proteins detected at AISs in pore
mutations, yet variants within the calmodulin binding region seem to target normally at
the AIS. Further studies determining the half-life of this KCNQ2 variant is necessary as
well as electrophysiological studies to determine whether or not activity is reduced.
Some mutations may act by preventing surface trafficking and AIS concentration. Since
such effects that may not be easily revealed through heterologous expression, further
development of in vivo models is warranted.
Contributors: Li, Li; Xu, Mingxuan; Cooper, Edward C.
336
ROLE OF RUNX1 AND P53 IN MEDIATING TGF 
-INDUCED MYOFIBROBLAST
PROLIFERATION AND DIFFERENTIATION
Linda Lien Tran
Advisor: David Rowley, Ph.D.-Department of Molecular & Cellular Biology
Background:
In prostate cancer, reactive stroma is observed to co-evolve with disease and be
indicative of rate of progression, rate to biochemical recurrence after prostatectomy,
and mortality. Previous xenograft studies in our lab involving the recombination of
carcinoma and stromal cells have shown that reactive stroma is tumor promoting.
Myofibroblasts are a key component of this microenvironment, yet little is known about
the mechanisms guiding their expansion and differentiation. p53 is a transcription
factor (TF) that has been widely studied for its role in the regulation of DNA damage
response. Modifications to this protein can determine whether a cell is fated to
apoptosis or cell cycle arrest. The Cooney lab and collaborators have implicated p53 in
the differentiation potential of mouse embryo fibroblasts (MEFs). p53-deficient MEFs
spontaneously differentiated into several different cell types. RUNX1 is a TF critical in
normal hematopoiesis and has been identified by the Rowley lab as a key TF in TGFe ta 1-mediated activation of human prostate mesenchymal stem cells (hpMSCs) to
myofibroblasts. This indicates a role for RUNX1 in stem cell biology and multipotency.
While p53 status appears to be important for differentiation commitment and resistance
to plasticity, RUNX1 seems to be essential for transit amplification of MSCs and
maintenance of their plasticity. RUNX1 is known to complex with p53 and affect
transcriptional activity of downstream target genes. Therefore RUNX1 and p53 may be
coordinating MSC expansion and myofibroblast differentiation. We hypothesize that as
RUNX1 levels decrease with myodifferentiation, p53 levels/activity will increase,
signaling the commitment of these cells to a specific lineage and removing them from
the multipotent pool. Following myofibroblast differentiation, RUNX1 and p53 protein
expression increase. Our studies suggest that RUNX1 and p53 do not physically
interact during myofibroblast proliferation/differentiation. Knockdown of RUNX1 with
siRNA does not appear to affect p53 levels or activity. Knockdown of p53 with siRNA
and CRISPR have shown that p53 is not necessary for myofibroblast differentiation.
Growth curves of these cell lines indicate that p53 is necessary for density-dependent
inhibition of growth. These data suggest that p53 may function to block return to stem
cell status during myofibroblast proliferation and differentiation. This may play a key
role in the abnormal expansion of reactive stroma.
Contributors: Tran, Linda; Wang, Hongran; Cooney, Austin; Rowley, David
337
YKI INTERACTS WITH THE JNK PATHWAY TO REGULATE EPIDERMAL WOUND
HEALING IN DROSOPHILA LARVAE
Chang-Ru Tsai
Advisor: Michael Galko, Ph.D.-Biochemistry and Molecular Biology
To cope with inevitable injury, organisms possess efficient wound healing
mechanisms to maintain tissue integrity and guard against infection. However, the
cellular and molecular details by which wound healing is accomplished remain poorly
defined. Drosophila melanogaster serves as a great model organism to study wound
healing because of the versatile genetic tools available and the simple anatomy of the
epidermal barrier. Upon larval epidermal wounding, epidermal cells around the wound
elongate and migrate in the absence of cell division to ultimately close the wound.
Using the wound closure assay we established, we found the Hippo downstream
transcriptional regulator, Yorkie (Yki) and its TEAD binding partner Scalloped (Sd) to be
required for epidermal wound healing. Intriguingly, unlike in other regenerative contexts,
Yki does not regulate the balance of mitosis or apoptosis in the healing larval epidermis.
Rather, it seems to regulate actin polymerization in the migrating wound-edge
epidermal cells. Moreover, with a series of genetic analyses, we found that Yki has a
strong genetic interaction with another wound closure signaling, the Jun N-terminal
Kinase (JNK) pathway. Our results suggest that there is a positive feedback loop
between these two pathways to facilitate proper epidermal wound healing.
Contributors: Tsai, Chang-Ru, Anderson, Aimee, Burra, Sirisha, Galko, Michael
338
THE ROLE OF G3BP1 ARGININE METHYLATION IN STRESS GRANULE
ASSEMBLY AND ANTIVIRAL FUNCTION
Wei-Chih Tsai
Advisor: Richard Lloyd, Ph.D.-Department of Molecular Virology & Microbiology
Stress granules (SG) are RNA granules formed when eukaryotic cells cope
with different external stresses, including virus infection. Stress signals are sensed by
four eIF2α kinases which can shutdown global translation initiation. RasGAP-SH3binding protein 1 (G3BP1)is a key nucleator of SG, targeting stalled mRNP/mRNA
complexes to drive SG assembly. Many viruses have evolved to prevent SG formation
via digesting or sequestering G3BP1 and other essential SG components. We have
shown that G3BP1 recruits PKR to SGs to activate PKR and innate immunity, and
reduces enterovirus titers. However, it is still unclear how G3PB1 is regulated in the
process of SG nucleation or innate immune activation. Type I protein arginine
methyltransferases (PRMT) are the family of enzymes in eukaryotic cells that catalyze
arginine methylation in the RGG domains of many RNA-binding proteins. G3BP1
contains a C-terminal RGG domain that contains five potential arginine methylation
sites. Deletion of the RGG domain abolishes SG assembly in stressed G3PB1 KO
mouse embryonic fibroblasts (MEF) and represses NFκB reporter activity. Site directed
mutagenesis of putative arginine methylation sites created an array of defects in SG
formation, and the number of SGs in cells correlated with NFκB reporter activity, and
gene expression of TNF-a and IFN-b. Mass spectrometry (MS) analysis confirmed four
methylation sites within the RGG domain. To our surprise, G3BP1 became less
methylated in the stressed condition. Endogenous G3BP1 in stressed conditions
contained reduced asymmetric and symmetric methylation in the RGG domain.
Inhibiting histone methyltransferases by Adox in HeLa cells increased SG formation. On
the other hand, inhibiting histone demethylase activity by N-Oxalylglycine (NOG) in cells
significantly decreased SG assembly. Taken together, these data suggest that arginine
demethylation of G3BP1 is a new SG regulation signal that can also modulate innate
immunity by controlling NFκB activation.
Contributors: Tsai, Wei-Chih; Lloyd, Richard E.
339
THE CELL-ADHESION GPCR BRAIN-SPECIFIC ANGIOGENESIS INHIBITOR 1
REGULATES DENDRITICSPINE MORPHOLOGY AND SYNAPTOGENESIS
Yen-Kuei Tu
Advisor: Kimberley Tolias, Ph.D.-Department of Neuroscience
Synapses are specialized sites that mediate communication between neurons.
Most excitatory synapses in the brain reside on actin-rich structures called dendritic
spines. The formation, regulation and maintenance of excitatory synapses are crucial
for normal cognitive function.
Recently, we have identified the adhesion G protein-coupled receptor (GPCR)
brain-specific angiogenesis inhibitor 1 (BAI1) as a key regulator of synapse
development. We show that BAI1 is highly localized to spines. Furthermore, knockdown
of BAI1 results in decreased spine and synapse density in both cultured hippocampal
neurons and cortical neurons from intact mouse brains. Synaptic loss caused by BAI1
knockdown can be rescued by full-length BAI1, but not by a BAI1 truncation mutant,
which fails to interact with the Par3/Tiam1 polarity complex. Tiam1 is a Rac1-guanine
nucleotide exchange factor (GEF) that promotes spine and synapse development by
inducing Rac1-dependent actin remodeling. Tiam1 is restricted to spines by the polarity
protein Par3, enabling for spatial control of Rac1 activation. We show that BAI1
regulates spine and synapse development by recruiting the Par3/Tiam1 complex to
spines, resulting in localized Rac activation and actin polymerization. Although these
findings elucidate how BAI1 signals inward to promote post-synaptic development, it is
unclear whether BAI1 also signals across the synapse to induce pre-synaptic
differentiation. Utilizing a COS7 cell-neuron co-culture system, we show that BAI1
increases pre-synaptic termini formation on the axons of cultured hippocampal neurons
that contact BAI1-expressing COS7 cells. These results indicate that BAI1 can induce
pre-synaptic as well as post-synaptic development. Our future investigations are twofold: (1) We have also shown that BAI1 interacts with important ligands that regulate
synaptic development such as Neuroligin-1 and the complement factor C1ql3.
Investigating the importance of N-terminal domains that interact with these ligands will
further elucidate mechanisms for BAI1's regulation of synaptogenesis. (2) Given that
the two other BAI family members are also highly expressed in the brain, affect neuron
morphology, and have been linked to neurological disease, we are investigating their
role as regulators of spine and synapse development. Results from our study should
help to elucidate the mechanisms that regulate excitatory synapse development, and
provide potential therapeutic targets for the treatment of neurological disease.
Contributors: Tu, Yen-Kuei; Duman, Joseph; Tolias, Kimberley
340
BIASED AGONISM THROUGH TOLL-LIKE RECEPTOR 4 AND CONTROL OF
INFLAMMATION
Hui-Ying Tung
The immune basis of allergic versus non-allergic diseases differs vastly,
however, toll like receptor (TLR) 4 is involved in both. While the canonical TLR4 ligand,
lipopolysaccharide (LPS), induces T helper (Th) 1 immune responses, our laboratory
has shown that expression of allergic airway disease requires fibrinogen cleavage
product (FCP) that signal through TLR4. We hypothesize that TLR4 exhibits biased
agonism in response to LPS and FCP liganding and aim to elucidate the distinct
signaling mechanisms activated by FCP through TLR4.
Reverse phase protein array (RPPA) of LPS versus FCP treated RAW 264.7
cells demonstrated a distinctive protein activation profile induced by FCP in comparison
to LPS. Western blot analyses further showed that LPS and FCP modulate NF-kB and
MAPKs through distinct signaling pathways. Moreover, FCP upregulated mucin
(MUC5AC), Arginase 1 and interleukin-13 receptor alpha (IL13Ra) gene expressions in
the lungs critical for expression of allergic asthma, as well as airway eosinophilia, which
are all regulated by the transcriptional factor, Signal transducer and activator of
transcription 6 (STAT6). Western blot results based on mouse bone marrow derived
macrophages (BMDMs) showed that STAT6 was clearly induced by FCP, but not LPS
through TLR4. Blocking of Janus kinase 1 (JAK1) by JAK1 specific inhibitor and siRNA
knockdown inhibited FCP -induced STAT6 phosphorylation, indicating that JAK1 was
required for FCP-induced STAT6 activation. In addition to initiating allergic airway
inflammation, FCP could elicit fungistasis in BMDMs and splenocytes. Nuclear factor,
NF-kB, was found to regulate FCP-induced anti-fungal immunity, independent of
STAT6.
These results indicate that LPS and FCP, both ligands for TLR4, modulate NFkB and MAPKs through distinct signaling pathways and these ligands differentially
activate STAT6 through TLR4. Collectively, TLR4 appears to exhibit biased agonism in
response to distinct ligands and it is the TLR4 response induced by FCPs that is
required for expression of allergic airway disease and antifungal immunity.
Contributors: Tung, Hui-Ying; Landers, Cameron; Lu, Wen; Yuping, Qian; Luz, Roberts; Corry,
David
341
KREBS CYCLE ENZYME ISOCITRATE DEHYDROGENASE 3 REGULATES
SYNAPTIC TRANSMISSION IN DROSOPHILA
Berrak Ugur
We performed an unbiased, forward genetic screen for essential genes on the
Drosophila X-chromosome that cause late developmental defects and
neurodegeneration. Through this screen, we isolated mutations in numerous nuclear
genes that encode mitochondrial proteins, including 4 different alleles of the fly homolog
of isocitrate dehydrogenase 3a (CG12233). The protein encoded by CG12233 (idh3a)
contains a mitochondrial localization signal and functions in the Krebs cycle to catalyze
the oxidative decarboxylation of isocitrate resulting in α-ketoglutarate and CO2.
Surprisingly, loss of idh3a causes severe synaptic transmission defects and a
progressive, light-independent neurodegeneration of fly photoreceptors. Mutant larval
neuromuscular junctions (NMJs) exhibit a loss of synaptic transmission and increased
mini frequency when stimulated in 0.5mM Ca+2. Since IDH3 is a key enzyme in Krebs
cycle, we hypothesize that altered levels of a metabolite contributes to the defects
observed in idh3a mutants. Indeed, we detected a significant decrease in the levels of
the IDH3a product, alpha-ketoglutarate (aKG) in mutant larvae. In addition, we found
that loss of isocitrate dehydrogenase causes an overall increase in almost all amino
acids. These results argue that IDH3a might function as a gatekeeper regulating amino
acid entry ino the Krebs cycle, thereby regulating metabolic homeostasis. To test
whether the reduced aKG levels affect synaptic transmission, we raised the mutant
larvae in aKG supplemented food. When stimulated in 1mM Ca+2, excitatory junction
potential was significantly higher than mutants grown with normal food, implying that
IDH3a is also important for Ca+2 regulation at synaptic terminals. Similarly, aKG
supplement rescues the synaptic transmission in photoreceptors but it does not rescue
the progressive, light-independent neurodegeneration in fly photoreceptors indicating a
separation of function at the synaptic terminal. To delineate the mechanism by which
loss of IDH3a causes loss of synaptic transmission we aim to perform genetic
interaction assay with various synaptic proteins. Furthermore, we aim to understand the
contribution of Ca+2 in idh3a mutants, via recording Ca+2 levels at the synaptic
terminal and synaptic mitochondria.
Contributors: Haelterman, Nele A; Jaiswal, Manish; Yamamoto, Shinya; Bellen, Hugo J.
342
THE ROLE OF LOCAL ASTROCYTES IN OLFACTORY BULB NEURONAL CIRCUIT
REFINEMENT AND PROCESSING
Kevin Ung
Synaptic plasticity provides extraordinary cellular adaptability for both neuronal
morphology and function. Although much has been learned about the general
phenomenology associated with synaptic plasticity, many of the molecular and genetic
mechanisms that contribute to plasticity, synaptic circuit formation, and behavior, are
not well understood. Previous studies have shown that synaptic plasticity can occur
through pre- or post-synaptic mechanisms. While typical experimental models focus on
neuronal effectors of plasticity, recent studies have demonstrated non-autonomous
contributions from astrocytes.
Astrocytes influence many aspects of neuronal development, ranging from
trophic support to neuromodulation, and synaptic stability. Moreover, defective
astrocytes have been associated with several conditions, such as Alzheimer’s disease,
schizophrenia, epilepsy, and tumorigenesis. Taking into account the known functional
roles of astrocytes along with our preliminary data, we are currently testing the
hypothesis that: local astrocyte populations play essential roles in synaptic plasticity
and circuit behavior in the olfactory bulb.
Towards this, we have used chemogenetics to selectively manipulate
astrocyte activity. Through electrophysiological recordings, we found that induced
astrocyte activity imparts an excitatory effect onto nearby neurons, and this activity
manipulation affects olfactory circuit processing and olfactory behavior. By monitoring
neuronal activity during olfactory behavior, we have shown that astrocytes contribute to
olfactory circuit processing by modulating neuronal responses to odorants.
Elucidating the molecular and cellular interactions between neurons and
astrocytes that influence postnatal synaptogenesis and circuit integration will enhance
our understanding of circuit formation in the developing, aging, and diseased brain.
Uncovering novel functional roles for astrocytes toward adult-born neurons may not
only better inform us of normal neural development and brain function, but also help
explain how defects in astrocytes may contribute to neurodegenerative diseases.
Contributors:
343
ROLE OF ATOH1-DEPENDENT NEURONS IN VENTILATORY CHEMORESPONSES
Meike Esther Van der Heijden
Autonomic control of breath depth and frequency are important to maintain
oxygen homeostasis upon a wide range of environmental and physiological changes.
Ventilatory chemo-responses are responsible for increasing breath depth and frequency
during changes in air gas concentrations such as hypoxia (low O2) or hypercapnia (high
CO2). Immature or abnormal chemo-responses in newborn babies are thought to be an
underlying cause for sudden infant death syndrome (SIDS). Chemo-responses are
regulated by chemo-responsive neurons in the hindbrain that are integrated in the
complex breathing circuitry. Normal development of a subset of glutamatergic neurons
populations in this circuitry is dependent on the transcription factor Atoh1 and loss of
Atoh1 leads to early neonatal death in mice due to respiratory failure. Previous studies
have shown that loss of Atoh1 from the retro-trapezoid nucleus (RTN) results in partial
lethality (50%). The surviving mice exhibit attenuated hypercapnic and exaggerated
hypoxic chemo-responses in surviving adult mice, indicating that neonatal survival
depends on multiple Atoh1-dependent populations and that early lethality might be due
to abnormal excitatory drive during chemo-challenges. We found that in addition to the
RTN, Atoh1-dependent neurons in the parabrachial/Kolliker Fuse nuclei (PB/KF) show
enhanced activity during chemo-challenges (hypoxia and hypercapnia). When Atoh1
was deleted from rhombomere1, in which the PB/KF resides, we did not observe
neonatal lethality but we discovered these mice show significantly attenuated
hypercapnic and abnormal hypoxic ventilatory chemo-responses. Interestingly, silencing
all Atoh1-dependent neurons in adult mice results only in mild attenuation of
hypercapnic ventilatory chemo-responses, but no changes in hypoxic responses.
Together these results suggest that two populations (the PB/KF and RTN) of Atoh1dependent neurons are necessary for the chemo-responses: both are essential for
hypercapnic responses but with a possible opposing role in modulating hypoxic chemoresponses. Future studies have to identify whether loss of Atoh1 from both these
populations results in neonatal lethality and whether silencing of these specific
populations indeed leads to opposing hypoxic ventilatory responses.
Contributors: Van der Heijden, Meike; Zoghbi, Huda
344
DEVELOPMENT OF A GENOMIC ASSEMBLY PIPELINE
Paul Jerrod Vandeventer
Advisor: Koen Venken, Ph.D.-Department of Biochemistry & Molecular Biology
To explore genome organization and functionality at the chromosome level, I am
developing novel genome engineering methods to generate and manipulate entire
chromosomes for multicellular eukaryotes. To demonstrate proof of principle, I am
focusing on the 1.2 Mbp coding region of the Drosophila 4th chromosome. This
chromosome is particularly interesting because small size, organization, and that many
of its 80 are essential and highly conserved. The experimental pipeline I am developing
can be broken down into three key steps: 1) Identification and upgrading of source
material, 2) Creation of retrieval vectors that will harbor the assemblies, and 3)
Developing a serial multipartite assembly process that includes verification and
validation of the DNA assemblies.
The source material consists of a combination of previously generated genomic
DNA libraries: Bacterial Artificial Chromosome (BAC) libraries used to determine the
reference genome sequence, and the P/ΦC31 artificial chromosome for manipulation
(P[acman]) libraries generated for complementation experiments after site-specific
integration. I generated genome-wide tiling-paths for both resources that will be used as
starting material for the assembly pipeline. Clones of these tiling paths will be modified
for downstream synthetic assembly through bacterial recombineering. Recombineering
will trim the clones to the bare minimum and concomitantly move in meganuclease
cutting sites for release of the DNA pieces so they can be assembled using
homologous recombination.
The retrieval vector is a custom shuttle plasmid consisting of elements needed
for maintenance in both bacteria and yeast, where homology arms for retrieval will be
incorporated.
The cloning pipeline itself is a serial assembly scheme that relies on
transformation-associated recombination (TAR) cloning in yeast. TAR cloning exploits
the high efficiency of homologous recombination to assemble large DNA fragments into
ever increasing molecules, ultimately leading to the regeneration of the 4th
chromosome, which will ultimately be tested for functionality in vivo.
Contributors: Vandeventer, Paul; Venken, Koen
345
MOLECULAR ROLE OF CYTOCHROME P450 1B1 IN HYPEROXIC LUNG INJURY
Alex Christopher Veith
Advisor: Bhagavatula Moorthy, Ph.D.-Department of Pediatrics
Supplemental oxygen (hyperoxia) is a life saving intervention provided to
individuals with respiratory insufficiency, such as patients with acute lung injury (ALI)
and acute respiratory distress syndrome (ARDS). ALI/ARDS poses a significant disease
burden in the United States, with high mortality rates and reduced quality of life in
survivors. While improvement in critical management of ALI/ARDS patients has
improved survival, there is no targeted therapy for the treatment and/or prevention of
ALI/ARDS. However, hyperoxia itself has been shown to exacerbate initial lung injury as
well as cause lung injury directly. Animal models of hyperoxic lung injury have shown
that reactive oxygen species (ROS) may be important in the pathogenesis of hyperoxic
lung injury but the molecular mechanisms underlying this have yet to be elucidated.
Previous studies have highlighted a potential role for the cytochrome P450 (CYP)
enzymes in hyperoxic lung injury. Our lab has shown that cytochrome P4501A1
(CYP1A1) and 1A2 (CYP1A2) are required to protect against hyperoxia indicated by
increased susceptibility to oxygen toxicity in Cyp1a1-KO and Cyp1a2-KO mice. It is
known that CYP1A1/1A2 as well as CYP1B1 are transcriptionally regulated by the
ligand activated transcription factor, the aryl hydrocarbon receptor (AHR), which has
also been implicated in hyperoxic lung injury. We have generated preliminary data
suggesting that CYP1B1 has a novel role in the development of hyperoxic lung injury.
Here we hypothesize that CYP1B1 is a crucial factor in the lung that promotes the
development of hyperoxic lung injury through increased ROS levels and modulation of
AHR-dependent transcription. We will characterize the response to hyperoxia in
Cyp1b1-KO mice during hyperoxia exposure and quantify markers of oxidative stress
and AHR-dependent transcription. Furthermore, we also aim to identify the cell type
within the lung, which expresses CYP1B1 and promotes hyperoxic lung injury. Finally,
we will identify metabolite differences enriched in Cyp1b1-KO mice during hyperoxia
exposure and their effect on hyperoxia susceptibility. These experiments will help us
understand the molecular mechanism by which CYP1B1 promotes hyperoxic lung
injury. This proposal presents an exciting opportunity to identify a factor that can be
manipulated to improve ALI/ARDS patient’s lives and quality of life.
Contributors: Veith, Alex C.; Bou Aram, Boura’a; Jiang, Weiwu; Wang, Lihua; Couroucli, Xanthi;
Moorthy, Bhagavatula
346
MANAGEMENT OF SUPERCOILING AS CAUSE OF AND ROLE FOR
REPLICATION-GENERATED COHESION IN E. COLI
Bryan Joseph Visser
Advisor: David Bates, Ph.D.-Department of Molecular & Human Genetics
Cohesion of replicated DNA allows for proper segregation to daughter cells
preventing aneuploidy that may cause serious genetic disorders. Cohesion is also
observed in E coli as a sliding window behind the replication fork, but the mechanism
that promotes it and what role it plays beyond promoting segregation is unclear. It is
known that cohesion is regulated positively by SeqA and negatively by topoisomerase
IV (Topo IV). SeqA binds hemimethylated GATCs behind the replication fork and is
important in the sequestration period between rounds of replication initiation and Topo
IV is responsible for the final decatenation reaction that allows for segregation of the
completely replicated chromosomes. Topo IV likely affects cohesion by removing
intersister coiling (precatenation) which physically prevented separation of the two DNA
molecules. Precatenanes are predicted to form when DNA polymerase rotates around
the DNA to relieve positive supercoiling ahead of the replication fork. Removal of
positive supercoiling is necessary for preventing polymerase stalling and fork collapse.
We have demonstrated that a topological barrier is capable of halting polymerase
hundreds of kilobases upstream in spite of wild type levels of DNA gyrase. This
indicates that DNA polymerase is generating more supercoiling during DNA replication
than gyrase is able to remove. I hypothesize that cohesion absorbs and sequesters
positive supercoiling to alleviate supercoiling generated by DNA polymerase to ensure
replication fork stability. To investigate this hypothesis I will complete the following aims:
1. Determine whether production of cohesion is compensation for excess DNA
polymerase-generated supercoiling 2. Determine whether cohesion affects fork stability
and subsequent DNA damage and repair. In completing these aims I will provide a
more complete model of the mechanism and purpose behind cohesion in E. coli. It is
very likely that premetaphase cohesion in eukaryotes operates under a very similar
mechanism as the one to be elucidated here indicating broad application for these
experiments.
Contributors: Joshi, Mohan; Barker, Anna; Bates, David
347
CAUSES OF THROMBOCYTOPENIA DURING DENGUE VIRUS INFECTION
Megan Brittany Vogt
Advisor: Rebecca Rico, M.P.H.-Department of Molecular Virology & Microbiology
Dengue virus is the most common cause of viral hemorrhagic fever in humans,
causing 50,000 deaths and $12 billion in economic losses each year. A common
clinical sign of dengue hemorrhagic fever is thrombocytopenia (platelet loss). Currently,
the cause of thrombocytopenia during dengue infection is unknown; however, possible
causes of thrombocytopenia include increased clearance of platelets in the blood
vessels or decreased production of platelets in the bone marrow. Because dengue
virus has been found in the bone marrow during human infection, we will investigate
whether dengue virus infects and suppresses megakaryocytes (platelet progenitor
cells). To answer this question, we will expose human megakaryocytes (derived ex vivo
from CD34+ stem cells isolated from human umbilical cord blood) to dengue virus and
determine whether dengue infects megakaryocytes using a quantitative reverse
transcription polymerase chain reaction assay specific for dengue virus RNA and an
enzyme-linked immunosorbent assay specific for a dengue virus non-structural protein
(NS1). We will also assess the differentiation and structure of infected megakaryocytes
using flow cytometry and immunofluorescence, respectively. Production of platelet-likeparticles by infected megakaryocytes will be quantified via flow cytometry. In addition,
we will investigate how dengue virus affects megakaryocyte populations in a mouse
model of dengue fever (humanized NOD/SCID/IL-2 gamma chain null mouse). Results
from these studies may lead to a model of thrombocytopenia causation in dengue
hemorrhagic fever and possibly other viral hemorrhagic fevers.
Contributors: Vogt, Megan; Rico-Hesse, Rebecca
348
GLANDULAR MORPHOGENESIS OF THE MOUSE UTERUS
Zer Vue
Advisor: Richard Behringer, -Molecular Genetics - M.D. Anderson
The focus of this study is on the development of the endometrial glands of the
mouse uterus. Endometrial glands secrete substances that are essential for uterine
receptivity to the embryo, implantation, conceptus survival, development and growth. In
models where uterine glands have been phenotypically knocked out, females are
infertile due to defects in implantation and early pregnancy loss, suggesting its essential
role for fertility. Adenogenesis is the formation of glands within the stroma of the uterus.
Uterine glands form after birth but before sexual maturity in the mouse. At P5, gland
formation initiates from the luminal epithelium. By P12, the glandular epithelium invades
the adjacent stroma. In domestic animals, glands are thought to be highly coiled and
branched. However, the morphology of mouse uterine glands is poorly understood
because it is based on two-dimensional histology. We are using various imaging
methods to determine the 3D structure of forming uterine glands and gland morphology
during the estrous cycle. By labeling the uterine epithelial cells with an antibody and/or
genetically with a conditional lacZ reporter (R26R-lacZ; Wnt7a-Cre), we have generated
3D models of uterine glands, using Optical Projection Tomography. We have shown
that SOX9 is expressed in the glandular epithelium in mouse and human uterus.
Analysis of Sox9-eGFP in the mouse uterus has provided insights into the structure of
developing glands and their distribution. Cumulatively from our reconstructions, we
conclude that glands are “buds” at P8 and become elongated tubes at P11. By P21,
these elongated tubes are curved, whereas in adults, glands are branched and coiled,
depending on the stage of the estrous cycle. To examine glands at single cell
resolution, we are using a conditional fluorescent reporter (Wnt7a-Cre; R26R-RG) to
quantify the number of cells in developing and mature glands. Our studies should
provide insights into the cellular mechanisms that result in the development of uterine
glands.
Contributors: Wang, Ying; Gonzalez, Gabriel; Stewart, Allison; Chen, Jichao; Behringer,
Richard R.
349
NEURONAL POPULATIONS IN MACAQUE PRIMARY VISUAL CORTEX
ENCODE UNCERTAINTY DURING PERCEPTUAL DECISIONS
Edgar Yasuhiro Walkder
Organisms typically base their perceptual decisions on noisy and ambiguous sensory
observations. There can be top-down sensory uncertainty due to ambiguity and bottomup sensory uncertainty due to noise in the perception system. In many tasks, optimal
performance requires the brain to represent and utilize, on every trial, knowledge about
the level of both bottom-up and top-down uncertainty. In earlier work, we introduced a
simple orientation classification task with controlled top-down and bottom-up sensory
uncertainty for which optimal performance requires the observer to utilize sensory
uncertainty on a trial-by-trial basis, and demonstrated that both humans and monkeys
do so. Here, our goal is to identify the neural substrates of this computation. The
theoretical framework of probabilistic population coding (PPC) postulates that the brain
decodes sensory uncertainty from a noisy pattern of population activity through a
“likelihood function” over the stimulus. This function represents the probability of the
observed pattern given each hypothesized stimulus value, and the width of this function
is a proxy of sensory uncertainty. We hypothesized that the width of the likelihood
function that can be decoded from trial-to-trial population activity in primary visual cortex
(V1) is informative about the animal’s decision. To test this hypothesis, we trained
macaque monkey on our classification task. We implanted a chronic multi-electrode
array in V1 to record the population activity while the monkey performed the
classification task. On each trial, we decoded from single-trial V1 population activity the
width of the likelihood function under a Poisson-like population coding model. The
monkey’s trial-by-trial classification decisions were better predicted by a Bayesian
model utilizing the width of the likelihood function than by a non-Bayesian model only
utilizing a point estimate of the stimulus orientation. We also tested the models on a
shuffled data where the widths of the likelihood functions were shuffled among trials
with identical stimulus condition, effectively removing trial-by-trial correlation between
the likelihood width and the monkey’s decision, while keeping the average correlation
between likelihood widths and stimulus orientations intact. We observed that Bayesian
model’s performance dropped significantly on the shuffled data when compared to the
fits on the original data, supporting our hypothesis that trial-by-trial variation in the
likelihood width is informative about the decision. This result provides population-level
physiological evidence in support of the PPC framework.
Contributors: Walker, Edgar Y.; Cotton, R. James; Ma, Wei Ji; Tolias, Andreas
350
DIFFERENT AFFINITIES OF ANKYRIN/SPECTRIN COMPLEXES IN THE
CLUSTERING OF SODIUM AND POTASSIUM CHANNELS AT NODES OF RANVIER
Chih-Chuan Wang
Myelination and the formation of nodes of Ranvier have been proposed to be an
evolutionarily critical advance allowing saltatory and rapid propagation of action
potentials in axons. The saltatory conduction of action potential depends on the nodal
clustering of Na+ and K+ channels. It has been proposed that AnkyrinG is of critical
importance in the clustering of Na+ and K+ channels. AnkyrinG interacts with NF-186,
βIV-spectrin, Na+ channels and K+ channels. NF-186 is the axonal receptor of glialderived extracellular matrix (ECM) and mediates ECM-based clustering of ion channels.
+
βIV-spectrin is the scaffold protein bridging the nodal protein complex, including Na
+
and K channels, to the actin cytoskeleton. However, we found that both AnkyrinG and
βIV-spectrin are dispensable for nodal clustering of Na+ channels. In AnkyrinG
conditional knockout mice and βIV-spectrin mutant mice, the nodal clustering of Na+
channels is still intact. We also found that the nodal clustering of Na+ channels is
compensated by AnkyrinR and βI-spectrin. Although it has been shown that Na+ and K+
channels share conserved domains that interact with Ankyrin, our results indicated that
AnkyrinG and βIV-spectrin are required for nodal clustering of K+ channels. The nodal
clustering of K+ channels cannot be compensated for by AnkyrinR and βI-spectrin in the
AnkyrinG conditional knockout mice and βIV-spectrin mutant mice.
Contributors: Wang, Chih-Chuan; Ho, Tammy Szu-Yu; Rasband, Matthew N.
351
SKELETAL MUSCLE CALCIUM RELEASE CHANNEL REGULATES ADAPTIVE
THERMOGENESIS IN BROWN ADIPOSE TISSUE
Hui Wang
Advisor: Susan Hamilton, Ph.D.-Department of Molecular Physiology & Biophysics
Lawrence Chan, D.Sc.-Department of Medicine
BACKGROUND: Adaptive thermogenesis (AT) is a finely regulated bioenergetic
process that dissipates energy as heat in response to environmental temperature or
diet. Dysregulation of AT precedes metabolic diseases and malignant hyperthermia
(MH), a life-threatening condition of systemic hypermetabolism triggered by
environmental or exercise induced heat. Preformed brown adipose tissue (BAT) and
skeletal muscle derived from the common myogenic progenitor lineage are the two
principle sites of AT. However, pathways regulating brown adipogenesis and the BATmediated thermogenesis in MH response remain to be investigated. Mouse with a
mutation (Y524S, YS) in the skeletal muscle calcium release channel, the ryanodine
receptor (RyR1) is a model for MH in humans, and displays high susceptibility to heatinduced sudden death. HYPOTHESIS: YS mutation in RyR1 upregulates brown
adipogenesis, which enhances BAT mediated thermogenesis and exacerbates the heat
sensitivity in YS mice. METHODS: Activity of BAT was detected by functional imaging
with integrated micro-PET/CT system, using the glucose analogue 18F-FDG as
contrast agent. After BAT activation by pre-exposing the mice at 4°C for 6 hours, mice
were subsequently heat challenged at 37°C and measured for hypermetabolism and
thermogenesis with indirect calorimetry. Preformed BAT and skeletal muscle common
progenitors were isolated from the stromal vascular fraction of interscapular BAT, which
were then differentiated in vitro for 3 days. The expression of genes regulating brown
adipogenesis and thermogenic function was assessed by quantitative real-time PCR.
RESULTS: The total 18F-FDG uptake in the interscapular region was significantly
increased in YS mice for more than 2-fold compare to control littermates (n=5, P<0.01),
suggesting the enhanced interscapular BAT activity. YS mice pre-exposed to cold
displayed exacerbated hypermetabolic response during heat challenge compare to
controls without cold pre-exposure (n=5, P<0.05) Surprisingly, preliminary results
showed that RyR1, which has been previously known to express predominantly in
skeletal muscle, was detected in BAT at the transcriptional level. Moreover, RyR1 was
found to be the major isoform of RyRs in common progenitor cells isolated from YS
mice, and the RyR1 expression increased for about 5-fold after differentiation in vitro. In
addition, the upregulation of essential genes for brown adipogenesis (Prdm16) and
thermogenesis (Ucp1) during differentiation was enhanced in YS progenitor cells
compare to that from the control littermates for about 10% and 60%, respectively.
CONCLUSION: Preliminary results suggest that YS mutation in RyR1 upregulates
brown adipogenesis, which contributes to the enhanced adaptive thermogenesis and
exacerbated heat sensitivity in mice.
Contributors: Wang, Hui; Lee, Chang Seok; Hanna, Amy; Xia, Yan; Dong, Keke; Gaber,
Waleed; Rendon, David; Sarkar, Poonam; Chan, Lawrence; Hamilton, Susan
352
REGULATION OF SHANK3 BY MAPK/ERK PATHWAY
Li Wang
SHANK3 haploinsufficiency causes Phelan-McDermid Syndrome (PMS) whereas
SHANK3 duplications lead to manic-like behavior, suggesting a proper dosage of
SHANK3 is critical for brain function. Previous work found that Shank3 is enriched yet
dynamic in postsynaptic density. It is important for cytoskeleton remodeling and activitydependent synaptic plasticity. However, mechanisms that regulate Shank3 function are
largely unknown. Phosphorylation is a common mechanism for controlling protein
stability and function. We explored Shank3 phosphorylation pattern and identified 41 in
vivo phosphorylated residues, suggesting a phosphorylation-dependent regulation.
To identify kinases that regulate Shank3 abundance, I made a DsRed-IRESEGFP-Shank3 transgenic cell line. The ratio of EGFP to DsRed serves as a read-out for
Shank3 levels controlling for transgene transcription efficiency. Using this system, I
performed a kinome-wide RNA interference (RNAi) screen, with each of the 638
kinases targeted by 3 siRNAs independently, to identify potential modifiers that change
EGFP/DsRed ratio. The screen revealed that multiple components of MAPK/ERK
pathway are involved in SHANK3 regulation. Blocking ERK pathway by either of two
highly selective MEK inhibitor increased Shank3, as well as its binding partners in
primary cortical neurons. Primary cultures treated with Bicuculline, a GABAA receptor
antagonist, decreased Shank3, which is partially blocked by ERK inhibition. These
observations suggest that ERK modulates activity-dependent Shank3 regulation. In
parallel, study of Shank3 interactome using immuno-precipitation (IP) followed by massspectrometry analysis identified ERK2 as one of the top kinase interactors of Shank3.
This protein-protein interaction is validated by both bimolecular fluorescence
complementation assay and in vivo IP-western blotting. In vitro kinase assay revealed
that ERK directly phosphorylates Shank3 at 3 specific serine residues. Phosphorylation
at these residues is regulated by neuronal activity, consistent with the effect of ERK on
activity-dependent Shank3 regulation.
Currently, we are exploring the effect of ERK phosphorylation on Shank3 stability
by analyzing phosphomutants of Shank3. We are also testing ERK as Shank3 modifier
in vivo using both genetic and pharmacological strategies. Our study will elucidate the
regulatory mechanism of Shank3 and may pinpoint potential therapeutic entry point
targeting Shank3 abundance for Shankopathies.
Contributors: Wang, Li; Han, Kihoon; Pang, Kaifang; Holder, Jimmy; Jain, Antrix; Jung, Sung;
Zoghbi, Huda
353
THE STOCHASTIC KINETICS OF E. COLI TRANSCRIPTION AT A SINGLE GENE
LOCUS
Mengyu Wang
Advisor: Ido Golding, Ph.D.-Department of Biochemistry & Molecular Biology
Gene regulation consists of a series of stochastic, single-molecule events,
resulting in substantial randomness in mRNA production between individual cells, and
even between the individual copies of the same gene within a single cell. To
characterize the stochastic kinetics of transcription in E. coli, we combine fluorescently
labelled DNA-binding proteins and single-molecule fluorescence in situ hybridization
(smFISH), to simultaneously detect a gene of interest and measure its transcriptional
activity, in individual bacteria. Our preliminary results, using the lactose promoter (Plac),
indicate that we can reliably detect the site of active transcription. They also indicate
that active transcription involves a change in the gene’s spatial position in the cell.
Under conditions of strong repression, we find that transcription is more likely to occur
close to the time of gene replication. Next, we will use mathematical modeling to map
the observed nascent and total mRNA copy-number statistics to the underlying
stochastic kinetics of transcription at a single gene locus. We will examine how this
stochastic kinetics is modulated by transcription factors that regulate gene expression,
and whether the activity of multiple copies of the same gene is correlated within the
single cell.
Contributors: Wang, Mengyu; Zhang, Jing; Sepulveda, Leonardo; Golding, Ido.
354
SYSTEMATIC CHARACTERIZATION OF MIRNA A-TO-I RNA EDITING HOTSPOTS
IN HUMAN CANCERS
Yumeng Wang
Advisor: Han Liang, Ph.D.-Bio informatics and Computational Biology
MicroRNAs (miRNAs) are small, non-coding RNAs that function in posttranscriptonal gene regulation by base-paring with its target mRNAs. It was estimated
that more than 60% of human protein-coding genes contain at least one conserved
miRNA binding site. Therefore, these ~22 nucleotides small molecules participates in
nearly all cellular pathway and pathological processes. miRNAs biogenesis is strictly
regulated temporally and spatially, and RNA editing is one of the modifications that
increase miRNA diversity by altering the sequence content. The most prevalent type of
miRNA editing in human is adenosines to inosines (A to I), which is catalyzed by
adenosine deaminase acting on RNA (ADAR) enzymes. miRNA editing may regulate
the processing of precursor miRNAs into mature miRNA, affect miRNA target
recognition, and has been implicated in many human diseases. Initial reports revealed
that miRNA editing events might be widespread in human based on low-throughput
experiments. Large-scale small RNA sequencing technology has the potential to
unravel the full extent of miRNA editing. However, the problem of high false discovery
rate in miRNA editing calling based on next-generation sequencing data has not been
resolved until recently. As a consequence, systematic analysis of miRNA editing events
is still in its infant stage, limiting to several individual cases and specific human tissues.
Very little was known about the regulation of edited miRNA in various tissue types and
human diseases. Therefore, considering the vital role of miRNA in post-transcriptional
modification, more and more attention was drawn to the metabolism and functional
impact of edited miRNA, especially disease-related editing events. We developed a
computational pipeline that carefully controls for false positive rate, and detect high
confident miRNA editing events. To deepen our understanding of post-transcriptional
regulation mediated by small RNA, integrative analysis cooperating various levels of
genomic data including mutation, gene expression, copy number variation, protein
expression, and patient clinic outcomes were performed. These results will provide
valuable insight into the functional effects of miRNA editing regulation during tumor
development, and can potentially identify edited miRNA as biomarker for diagnosis and
prognosis to improve cancer treatments.
Contributors: Wang, Yumeng; Xu, Xiaoyan; Yu, Shuangxin, Chen, Hu; Han, Leng, Yuan, Yuan,
Lu, Yiling, Mills Gordon; and Liang, Han.
355
MASTER REGULATORS OF DICTYOSTELIUM DEVELOPMENT
Amanda Nicole Webb
Dictyostelium has been used as a model organism since the isolation of
Dictyostelium discoideum by Kenneth Raper in 1933 and has since become a preferred
model among those interested in various processes including chemotaxis,
development, cell differentiation, and social evolution. Their particular appeal lies in the
fact that, upon starvation, these otherwise single-cellular amoebae aggregate and
develop into a complex multi-cellular structure. During development, the cells undergo
distinct stages that follow a strict timeline, making them an ideal model for systematic
developmental study. Despite significant genetic divergence, this is true of many
dictyostelid
species.
The genes involved in development tend to evolve much more rapidly than
vegetative growth genes, but the timing and structural changes of the developmental
program have remained intact across many different species over time. Two species in
particular, Dictyostelium discoideum and Dictyostelium purpureum, share similar
developmental timelines and morphological phenotypes, though their protein
sequences differ from each other as much as do humans’ from boney fish. This
developmental conservation is likely due to the fact that both species of Dictyostelium
have highly conserved transcriptomes, in which huge changes in gene expression
correspond to distinct stages in development. Such strong conservation suggests the
presence of crucial and stable regulatory mechanisms. What it is that provides this
regulation, however, is still a mystery.
Considering the powerful role that many play in gene regulation, as well as
the fact that they are often conserved, we hypothesize that the master regulators of
development are a particular set of transcription factors. By identifying these master
regulators and characterizing their roles during Dictyostelium development, we may
provide novel insights into the general principles governing eukaryotic development.
356
CD133 DOES NOT ENRICH FOR THE STEM CELL ACTIVITY IN VIVO IN ADULT
MURINE PROSTATES
Xing Wei
Advisor: Li Xin, Ph.D.-Department of Molecular & Cellular Biology
CD133 is widely used as a marker for stem/progenitor cells in many organ
systems. Previous studies using in vitro stem cell assays have suggested that the
CD133-expressing prostate basal cells may serve as the putative prostate stem cells.
However, the precise localization of the CD133-expressing cells and their contributions
to adult murine prostate homeostasis in vivo remain undetermined. We show that loss
of function of CD133 does not impair murine prostate morphogenesis, homeostasis and
regeneration, implying a dispensable role of CD133 for prostate stem cell function.
Using a CD133-CreERT2 model in conjunction with a fluorescent report line, we show
that CD133 is not only expressed in a fraction of prostate basal cells, but also in some
luminal cells and stromal cells. CD133+ basal cells possess higher in vitro sphereforming activities than CD133- basal cells. However, the in vivo lineage tracing study
reveals that the two cell populations possess the same regenerative capacity and
contribute equally to the maintenance of the basal cell lineage. Similarly, CD133+ and
CD133- luminal cells are functionally equivalent in maintaining the luminal cell lineage.
Collectively, our study demonstrates that CD133 is not a marker for mouse prostate
stem cells in vivo, and supports that facultative function of specific cell populations
induced by experimental conditions of in vitro assays may not contribute to the in vivo
biology substantially.
Contributors: Wei, Xing; Orjalo, Arturo; Xin, Li
357
CONSTITUTIVE EXPRESSION OF THE PROGESTERONE RECEPTOR B ISOFORM
RESULTS IN SEVERE SUBFERTILITY AND THE DEVELOPMENT OF POORLY
DIFFERENTIATED OVARIAN NEOPLASMS
Margeaux Wetendorf
Advisor: Francesco Demayo, Ph.D.-Department of Molecular & Cellular Biology
Ming-Jer Tsai, Ph.D.-Department of Molecular & Cellular Biology
The healthy ovary is responsible for the production of the steroid hormones, estrogen and
progesterone, which function via their cognate receptors, the estrogen receptor and progesterone
receptor (PGR). The appropriate balance of these steroids, hormone receptors, and hormone
receptor isoforms is critical for reproductive function. Correct levels of the PGR-A isoform are
necessary for ovulation and a successful pregnancy, while the PGR-B isoform is important for
mammary gland development. Dysregulation of these PGR isoforms or a pre-dominance of a
single PGR isoform is not only detrimental to reproduction but also underpins endometrial and
ovarian cancer susceptibility. To further understand the role of PGR isoform dysregulation in
hormone-responsive tissues, a conditional mouse model was generated to constitutively express
Pgr-B, using a PRcre driven lox-STOP-lox system. Through the constitutive expression of the
PGR-B isoform in PGR positive tissues, mice exhibited severe subfertility due to the inability of
embryos to implant and the failure of uterine decidualization. At 34 weeks of age, mice
developed large ovarian tumors identified as poorly differentiated neoplasms with a tumor
penetrance of 64%. To determine whether the neoplasms developed from the uterus or the ovary,
ovaries were removed from young mice and allowed to age. Ovariectomized PGR-B expressing
mice failed to develop tumors, confirming that tumorigenesis originates from the ovary or
oviduct. Preliminary studies indicate that the PGR antagonist, mifepristone, successfully reduces
tumor volume in PGR-B expressing mice. An RNA microarray was performed on ovarian tumor
tissue compared to wildtype age-matched healthy ovaries. Upon filtration by fold change and a pvalue of ≤0.05, 1199 and 1653 genes were found to be upregulated and downregulated
respectively. The major upregulated gene pathways represent cell proliferation and cell motility
pathways such as MAPK, p53, and AKT pathways. These data will increase our understanding of
the role that progesterone signaling plays in the progression of ovarian cancer and further the
discovery and development of new biomarkers and effective treatment options. This work was
supported by NIH Grants: R01HD042311, 5U54HD007495 (to FJD) and R01CA77530 (to JPL)
and NURSA grant: U19DK62434 (to MJT, SYT & FJD).
Contributors: Wetendorf, Margeaux; Wu, San-Pin; Creighton, Chad; Lanz, Rainer; Rosen,
Daniel; Tsai, Sophia; Tsai, Ming-Jer; Lydon, John; DeMayo, Francesco.
358
THE IDENTIFICATION OF CARBOXY-TERMINAL FRAMESHIFT MUTATIONS AND
THEIR ROLE IN HUMAN DISEASE
Janson James White
Advisor: James Lupski, M.D./Ph.D.-Department of Molecular & Human Genetics
Nonsense-mediated decay (NMD) is an RNA quality control process by which mutant
transcripts containing a premature termination codon (PTC) are actively degraded.
However, NMD is sometimes unable to recognize a PTC located at the portion of the
transcript encoding the C-terminus of the protein (last and portion of the penultimate
exon), allowing for the production of a mutant protein product with a large C-terminal
aberration. The notion that a proportion of variants escape NMD is important clinically,
as some genes only display pathogenesic phenotypes due to a dominant-negative or
gain-of-function mechanism and have been demonstrated to alter disease inheritance
patterns (e.g. recessive loss-of-function disease resulting from a dominant mutation)
and explain allelic affinity in genes with a known disease association. To investigate the
frequency of these C-terminal mutational events we examined our in-house cohort of
~5000 exomes consisting of a wide variety of phenotypes. We selected all frameshifting
variants located in the final exon plus 200bp upstream in the penultimate exon
according to the RefSeq transcripts. All candidates were filtered against exome data in
publicly available databases, including the NHBLI Exome Sequencing Project (ESP)
and the Atherosclerosis Risk in Communities Study (ARIC) database. Of the resulting
~2000 unique variants we manually curated those predicted to escape NMD. Our list of
variants includes genes where C-terminal truncation has previously demonstrated an
altered pathogenesis and the disease state is not a result of haploinsufficiency including
AHDC1, FBN1, DMD and DVL1. We have recently reported the variant alleles in DVL1
escape NMD and all share an identical PTC within the last exon that creates a mutant
DVL1 protein with a large atypical C-terminal peptide tail. This rare mutational
mechanism was reported as a common cause of Robinow syndrome likely due to a
gain-of-function and/or dominant-negative mechanism (PMID: 25817016). Similar to the
variants identified in DVL1, we have identified greater than 25 novel candidate variants
in multiple affected patients with unsolved exomes that present with overlapping
phenotypes, this includes cohorts of osteoporosis, Robinow syndrome, heterotaxy, and
various forms of intellectual disability. Our data show that variants that do not lead to
haploinsufficiency are important contributors to human disease and should be carefully
analyzed. We suggest that gain-of-function and dominant-negative mutations are
highly under-recognized in genomic analyses and likely result in a wide variety of
human disease.
Contributors: White, Janson; Coban-Akdemir, Zeynep; Jhangiani, Shalini; Baylor-Hopkins Center for
Mendelian Genomics; Boerwinkle, Eric; Gibbs, Richard A.; Lupski, James R.; Carvalho, Claudia M. B.
359
GENETIC SILENCING OF THE OLIVOCEREBELLARPATHWAY CAUSES
DYSTONIC MOVEMENTS
Joshua J White
Advisor: Roy Sillitoe, Ph.D.-Department of Pathology & Immunology
Dystonia is a severe neurological disorder that can cause painful muscle
contractions. Although several regions of the motor system are thought to be involved,
there has been a major shift in attention towards understanding how the cerebellum
contributes to the disease. Clearly, the Purkinje cells and their target cerebellar nuclei
are involved, but how specific synaptic and circuit malfunctions lead to dystonia are not
fully understood. Therefore, the goal of this study was to test whether manipulating
certain cerebellar synapses triggers dystonia. In order to accomplish this, we needed a
flexible animal model that enabled the targeted manipulation of selective synapses in
the cerebellar circuit, but without causing structural alterations, circuit degeneration, or
cell loss. Towards this, we devised an inducible model by using a conditional genetics
approach to target the olivo-cerebellar pathway, a connection that may be central to the
defects in humans and animal models with dystonia. With this model, we show using a
combination of high-resolution anatomy, behavioral paradigms, and in vivo awake
electrophysiology that loss of olivo-cerebellar synaptic neurotransmission induces
abnormal firing within the cerebellar circuit, which causes severe muscle cocontractions that produce overt dystonic movements. We also used pharmacologic and
deep brain stimulation techniques to overcome the dystonic phenotype by blocking the
abnormal output of the cerebellar nuclei. Altogether, our data show that loss of climbing
fiber induced complex spikes causes downstream changes in cerebellar circuit function
that lead to dystonic-like behaviors. We propose that a powerful therapeutic strategy for
human dystonia might be to directly repair cerebellar activity before it communicates
toxic signals to other regions.
Contributors: White, Joshua J & Sillitoe, Roy V
360
THE IMPACT OF FGFR4 ARG388 ON NEUROBLASTOMA INCIDENCE, OUTCOME,
AND RESPONSE TO TREATMENT
Sarah Brackley Whittle
Advisor: Peter Zage, M.D./Ph.D.-Department of Pediatrics
Children with high-risk, metastatic solid tumors have poor outcomes with frequent
recurrences, treatment resistance, and refractory and recurrent pediatric solid tumors respond
poorly to salvage therapy. Improved understanding of the mechanisms underlying the
pathogenesis, recurrence, and treatment resistance of pediatric solid tumors will identify novel
targets for future therapies.
Aberrant growth factor receptor expression and receptor tyrosine kinase signaling are
associated with the pathogenesis of many malignancies, and these kinases serve as targets for
a number of novel therapies. A germline polymorphism in the FGFR4 gene, resulting in the
expression of FGFR4 containing arginine at codon 388 (Arg388) rather than the more common
glycine (Gly388), has been shown to be associated with decreased survival rates, treatment
resistance, and more aggressive disease in a variety of malignancies.
We screened DNA from neuroblastoma tumor cells and 129 neuroblastoma patients for
the FGFR4 genotype using RFLP analysis. Allele frequencies were determined and compared
to the allele frequencies of an age, sex and race matched control population as well as a
representative control population as reported in HapMap (release 27, NCBI build 36).
Additionally, whole exome sequencing from the BASIC3 (Baylor Advancing Sequencing Into
Childhood Cancer Care) study was evaluated for 54 solid tumor patients and allele frequencies
were compared to those from the HapMap control population. To evaluate the degradation rate
of FGFR4, neuroblastoma tumor cells were starved in serum free media overnight then treated
with ligand (bFGF) to induce receptor endocytosis. Western blots using antibodies against
individual receptors were performed and relative expression levels were determined. We
treated a panel of established neuroblastoma tumor cell lines with increasing concentrations of
Ponatinib (a pan-FGFR inhibitor) and viability was determined using MTT assays. We used a
wound healing assay to determine cell migration with ponatinib. Cells were treated with 5uM
Ponatinib and western blots using antibodies for downstream targets of FGFR4 were
performed.
The frequency of the A allele in neuroblastoma patients was increased compared to the
matched control population, suggesting an association of the FGFR4 genotype with
neuroblastoma incidence (p= 0.014). Furthermore, logistic regression analyses suggested an
association between having the "A" allele and having neuroblastoma, with a 3.3-fold increase in
individuals with the AA genotype having neuroblastoma compared to those with the GG
genotype. Additionally, ligand-induced FGFR4 protein degradation occurs much more slowly
than that seen with EGFR, consistent with increased FGFR4 stability. All neuroblastoma cell
lines tested were sensitive to Ponatinib with IC50 values in the nanomolar to micromolar range.
Migration of neuroblastoma cells was inhibited by treatment with Ponatinib at 5μM
concentration. Phospho-MEK and Phospho-ERK were significantly decreased after treatment
with Ponatinib 5 uM.
Contributors: Whittle, Sarah MD; Du, Melissa; Zhang, Linna MD; Wang, Linghua; Woodfield,
Sarah PhD; Indiviglio, Sandra PhD; Okcu, Fatih MD; Parsons, Donald W MD PhD; Lupo, Philip
PhD; Scheurer, Michael PhD; Zage, Bean Andrew PhD, Peter MD PhD
361
TARGETING THE TUMOR VASCULATURE WITH TEM8-SPECIFIC ENGAGER T
CELLS
LaTerrica Chemise Williams
Xiao-Tong Song, Ph.D.-Department of Pathology & Immunology
Background: T-cell immunotherapy with antigen-specific T cells has shown
promise in preclinical models as well as early clinical studies. However, patients with
solid tumors often do not respond as well as patients with hematological malignancies.
This lack of efficacy for solid tumors is most likely due to several factors including the
inability for infused T cells to a) redirect resident T cells to tumor cells, and b) destroy
the vascular bed of solid tumors, which is critical for their malignant growth. We have
recently developed a strategy to overcome the first obstacle by generating T cells that
secrete bispecific T-cell engagers, which consist of two single chain variable fragments
specific for CD3 expressed on T cells and a tumor antigen. These so called engager
(ENG) T cells have the unique ability to redirect bystander T cells to tumor cells,
amplifying antitumor effects. The aim of this project is to compare two T-cell engagers
specific for tumor endothelial marker (TEM)8, and evaluate their anti-vasculature and
anti-tumor activity in preclinical tumor models.
Methods/Results: We generated two retroviral constructs encoding a TEM8specific engager consisting of the TEM8-specific single chain variable fragments AF344
or L2. CD3/CD28-activated T cells were transduced with RD114-pseudotyped retroviral
particles to generate TEM8-specific engager T cells and expression was confirmed by
FACS analysis. To evaluate the functionality of the TEM8-ENG T cells we used TEM8negative cell lines (LAN1 and 293T) and genetically modified both to express murine
TEM8 (293T.mTEM8 and LAN1.mTEM8). TEM8-ENG T cells recognized target cells in
an antigen-dependent fashion as judged by their ability to secrete pro-inflammatory
cytokines (IFN- a nd IL-2) in coculture assays, and kill TEM8-positive target cells.
Importantly, TEM8-ENG T cells readily recognized mTEM8-positive target cells, which
will allow us to evaluate the safety and efficacy of TEM8-ENG T cells in a xenograft
tumor model using LAN1.
Conclusion: We have constructed two TEM8-specific T-cell engagers and have
shown that T cells expressing these engager molecules recognize and kill mTEM8positive target cells. Animal studies are in progress to determine their safety and
efficacy. Targeting the tumor vasculature with TEM8-ENG T cells may improve current
T-cell immunotherapies for solid tumors.
Contributors: Kalra, Mamta; Du, Wa; Phung, Thuy; Rowley, David; Gottschalk, Stephen
362
METEOR: CREATION AND APPLICATION OF A LITERATURE-BASED NETWORK
FOR PRECISION MEDICINE
Stephen Joseph Wilson
Advisor:Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Objectives: As highlighted by the Precision Medicine Initiative of the Executive Branch,
precision medicine holds the promise of dramatically changing how healthcare is
administered with the goal of greatly improved health outcomes. The basic principle
underlying precision medicine is that disease is not uniform and that the genetic and
lifestyle factors of a patient need to inform treatment. However, to be effective,
precision medicine requires reliable gene, drug, and disease information. Because of
this, there is a significant need for biological data discovery and aggregation.
Methods: Towards that end, we have created a novel biological network that
aggregates data from millions of PubMed articles in the literature. By using MeSH terms
as a summary of articles in PubMed, we created the MeSH Term Objective Reasoning
(MeTeOR) network that represents relationships between genes, drugs, and diseases.
Results: Comparisons to current knowledge from publically available databases
support MeTeOR as an accurate source of information, and when predictions were
made on time-stamped versions of the network using non-negative matrix factorization,
we accurately predicted gene-gene and gene-disease relationships that were later
discovered. MeTeOR also improves accessibility to the literature with direct references
on every association. When combined with community detection and network
connectivity, MeTeOR offers prioritized biological context to any gene or set of genes.
Conclusions: Overall, MeTeOR provides high-quality information about genes, drugs,
and diseases to rationally guide researchers to precision medicine and pathway level
discoveries.
Contributors: Wilson, Stephen; Wilkins, Angela
HIPPOCAMPAL REPLAY CORRELATES WITH MEMORY RETRIEVAL IN
INHIBITORY AVOIDANCE TASK
363
Chun-Ting Wu
Advisor: Daoyun Ji, Ph.D.-Department of Molecular & Cellular Biology
The hippocampus is intricately involved in the initial encoding and subsequent
retrieval of episodic memories. Experiments involving recording the activity of
ensembles of individual rodent hippocampal neurons have linked a particular pattern of
activity – the expression of the place cell map – to the initial encoding process.
However, how this neural ensemble expresses memory retrieval remains unclear.
Recent data report that sequences of place cell activity exhibited while animals explore
are subsequently reactivated in quiescent periods in a time compressed fashion. Such
"awake replay" has been hypothesized as a neural substrate of memory retrieval.
However, evidence to support the hypothesis that replay corresponds to the actual
experience of remembering is limited, mainly because the reward-based tasks used in
the past do not have a clear behavioral report of that memory retrieval has taken place.
Here, after rats acquired fear memory by receiving mild foot shocks at a specific zone
(shock zone) of a linear track, we analyzed hippocampal place cell activity when the
animals were placed back to the track and displayed memory retrieval behavior:
approaching and turning away from the shock zone. We found that before the animals
turned away and avoid the shock zone, the place cells associated with the shock zone
were reactivated. This reactivation occurred with awake replay of the place cell
sequences representing the path ending around the shock zone. The result reveals the
role of hippocampal place cells in contextual fear memory and provides strong evidence
for the function of awake replay in memory retrieval. By demonstrating that
hippocampal replay is a substrate for memory retrieval, we lay the foundation for
therapeutic interventions that might target these neural patterns, either to dampen
episodic memories, i.e., in PTSD, or to enhance them in cases of memory impairment.
Contributors: Kemere, Caleb; Ji, Daoyun;
364
HOLLIDAY-JUNCTION TRAP REVEALS RECOMBINATION CAUSES AND A
JUNCTION-GUARDIAN ROLE OF RECQ HELICASE
Jun Xia
DNA repair by homologous recombination (HR) is critical to cell survival, but is also
genome destabilizing, provoking cancer, human diseases, and aging. HR is used
universally for repair of spontaneous DNA damage, and yet the usual initiating events
and relative importance (frequency) of HR repair to cellular maintenance are not known.
Here we present engineered fluorescent proteins that trap Holliday junctions (HJs), a
central DNA intermediate in most HR, and use the HJ trap to elucidate underlying
instigators/origins of spontaneous HR in a proliferating Escherichia coli-cell model
system. The HJ trap binds and labels HJs as HR-protein-dependent fluorescent foci in
cells proportionately to enzyme-induced double-strand breaks (DSBs), and at their sites
of repair, shown by chromatin immunoprecipitation. Microfluidic imaging and genetic
experiments demonstrate that spontaneous HJ-HR events are correlated with DNA
replications, implicating replication as a main driver, although at rates lower than
predicted. Unexpectedly, most spontaneous HR foci do not originate from DSBs but
rather from non-DSB DNA damage. Moreover, we find that RecQ, ortholog of human
cancer-prevention proteins BLM, WRN, RECQ4, RECQ1 and RECQ5, both promotes
spontaneous HJ formation, and, when HJs increase during RecA overproduction, RecQ
promotes HJ reduction. This dual action shows a novel junction-guardian role for RecQ
as a main modulator of HJ levels during spontaneous DNA-damage repair. Most tumors
have overproduced RAD51 (RecA ortholog) and may also have abnormally high HJ
levels. We propose that one or more human RecQ orthologs may act similarly, both as
primary promoters of spontaneous-repair HJs and primary agents of their removal when
excessive in tumors.
Contributors: Xia, Jun; Chen, Li-Tzu; Ma, Chien-Hui; Mei, Qian; Lin, Hsin-Yu; Hamilton, Holly M;
Magnan, David; Xi, Shen; Hastings, PJ; Jayaram, Makkuni; Rosenberg, Susan M
365
MOLECULAR AND GENETIC STUDIES OF ATOH1 REGULATION
Wei Xie
Atonal homolog 1 (Atoh1) is a proneural basic helix loop helix (bHLH)
transcription factor that is crucial for the proper development of many cell populations.
These cell populations are both neuronal – cerebellar granule neurons, hindbrain
neurons, spinal cord interneurons – and non-neuronal – inner ear hair cells, intestinal
secretory cells, and Merkel cells. To get a better understanding of Atoh1 function, we
investigated phosphorylation as a potential regulatory mechanism. Based on
evolutionary conservation, serine 193 (S193) emerged as a potentially interesting
phosphorylation site. In vitro studies in our lab demonstrated that S193 phosphorylation
affects Atoh1 transcriptional activity. Follow up studies showed that the DNA binding
properties of the S193 phosphomutants were altered. In sum, our results pointed to a
pivotal role of S193 phosphorylation on Atoh1 function.
To study the effect of S193 phosphorylation in vivo, we generated a phosphonull (S193A) knock-in mouse. We performed histology and behavioral studies to
characterize mice that were homozygous for the mutant allele (Atoh1S193A/S193A),
and mice with the mutant allele over a null background (Atoh1S193A/lacZ).
Surprisingly, Atoh1S193A/S193A did not show any morphological defects or motor
coordination defects. However, Atoh1S193A/lacZ mice showed a motor coordination
deficit on the rotating rod as well as inner ear hair cell loss. Our findings suggest that
the S193A mutation is a very mild hypomorph.
Overall, our results indicate that the inner ear hair cells are one of the Atoh1dependent cell populations that are more susceptible to loss of function. Our data also
suggest that phosphorylation at S193 functions as an “off” switch, resulting in inhibition
of Atoh1 function. Preventing phosphorylation at this site, however, does not seem to
result in overt effects on Atoh1 function, suggesting that this phosphorylation site is just
one of many regulatory signals for Atoh1.
Contributors: Xie, Wei; Jen, Hsin-I; Groves, Andrew; Klisch, Tiemo; Zoghbi, Huda
366
THE CONTRIBUTION OF MUTATIONS IN SYNDROME-CAUSING GENES TO NONSYNDROMIC RETINITIS PIGMENTOSA
Mingchu Xu
Retinitis pigmentosa (RP) is a rare inherited retina

2015 graduate student symposium

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