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F l o r i d a a t l a n t i c U n i v e r s i t y
May 17, 2013Research Day
Dedication
This book is dedicated to the Schmidt Family Foundation for its
generosity and vision of a biomedical science program
at Florida Atlantic University.
Charles E. Schmidt
Charles E. Schmidt College of Medicine
We enjoy research affiliations with leading organizations including the Scripps Research Institute, the Max
Planck Florida Institute for Neuroscience, the Torrey Pines Institute for Molecular Studies, and the Vaccine
Gene Therapy Institute of Florida. These affiliations provide additional opportunities of collaborations and
research training for our students.
Among the research highlights for our College this fiscal year 2012‐13 include NIH grants for Huntington’s
disease and breast cancer metastasis, the identification of a unique mechanism in bacteria that has the
potential to serve as a target for developing new antibiotics for diseases such as AIDS and soft tissue
infections, a unique collaboration with Georgia Aquarium to conduct the first study of key immune cells
(dendritic cells) of Bottlenose dolphins and their response to environmental stressors, and a first of its kind
collaboration with Boca Raton Regional Hospital aimed at predicting breast cancer behavior and
developing therapies that will block the metastatic process. Our faculty have joined forces with industry
partners to further develop and test the effectiveness of INTERACT (Interventions to Reduce Acute Care
Transfers) and provide recommendations to our federal government. Our faculty members have published
the results of their research in scientific journals that include The Lancet, American Journal of Medicine, the
Journal of Biological Chemistry, the Journal of Psychiatric Research, the Journal of the American Geriatric Society,
Molecular Neurobiology, and numerous others.
During Research Day 2013, we will find examples of innovative research that is taking place throughout the
College. The Dean’s Office is committed to promoting the research efforts of our faculty and students. In the
coming year, I look forward to continuing work with faculty and leadership to increase opportunities for
funded research and discovery at the Charles E. Schmidt College of Medicine.
John W. Newcomer, M.D.
Vice Dean for Research and Graduate Programs
A Message from the Vice Dean for Research
and Graduate Programs
Our shared goal in the Charles E. Schmidt College of Medicine is to conduct
basic, translational and clinical research that increases the understanding of
human biology, and particulary the treatment and prevention of human
disease. The ultimate goal of our biomedical research is to advance global
health and the health of our local community in the process. Research in the
College is focused in the areas of cardiovascular disease and stroke, cancer,
neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease,
genetic eye diseases, macular degeneration and cataracts, autoimmune
diseases, malaria, HIV/AIDS, and healthy aging.
Research Day Agenda
Charles E. Schmidt College of Medicine
Lobby and Room 126
Noon Posters will be displayed in the lobby
Noon to Registration and Lunch (Lobby and Room 126)
to 12:30 p.m.
12:30 p.m. Welcoming Remarks John W. Newcomer, M.D.
Vice Dean for Research and Graduate
Programs
12:40 p.m. Improving Breast Cancer Survival: Vijaya Iragavarapu‐Charyulu, Ph.D.
Prevention of Metastasis Associate Professor of
Biomedical Science
1:10 p.m. A Test and Treat Pilot Study to Joanna Drowos, D.O., M.P.H., M.B.A.
Combat Lymphatic Filiriasis in Director, Community and Preventive
Palm Beach County Medicine Clerkship and Assistant
Professor of Clinical Biomedical
Science
1:40 p.m. Student Poster Presentations
and Competition (Lobby)
3:00 p.m. Announcement of Poster Winner John W. Newcomer, M.D.
Confocal image for the Research Day book cover provided by Dr. Vijaya Iragavarapu‐Charyulu
Research
Day
Posters
Posters
1. Intracellular trafficking of an androgen receptor splice variant, AR8 Ciny John and Michael L. Lu
2. Pulmonary inflammation associated with chitinase‐3‐like‐1 molecule (CHI3L1) expression accelerates breast cancer metastasis to the lung
Stephania Libreros, Ramon Garcia‐Areas, Roberto Carrio and Vijaya Iragavarapu‐Charyulu
3. Determination of levels and expression of IL‐1β, IL‐4, TNF‐α, IL‐10, and IFN‐γ in the lacrimal glands of a predisposed mouse model of Sjögren’s Syndrome (SS): the influence of sex
hormones and genetic background in the pathogenesis of SS
Stefanie P.C. Czerwinski, Safinaz Mostafa and Ana Maria Azzarolo
4. Fast Glutamate Uptake by EAAT2 Prevents Glutamate Depletion in Rod Photoreceptors L.A. Purpura, H. Ripps and W. Shen
5. Structure‐energy study of interactions between Membrane‐type matrix metalloproteinase 1 and Tissue inhibitors of metalloproteinase
Haiyin Zou, Tim Logue, Ying Wu, Michelle Lizotte‐Waniewski and Keith Brew
6. Ribosomal RNA damage and degradation under oxidative stress Min Liu and Zhongwei Li
7. Effects of Green Tea Catechins on Diastolic Function in Mice with Restrictive Cardiomyopathy Lei Zhang, Pierre‐Yves Jean‐Charles, Changlong Nan and Xupei Huang
8. Annotation of neurodevelopmental regulators in dopaminergic and GABAergic neurons for links to ischemia‐hypoxia response
Kim McKain, Maryanne Joseph and Rainald Schmidt‐Kastner
9. Cholera toxin induces a shift from inactive to active COX‐2 in alveolar macrophages activated by Mycobacterium bovis BCG
Mari Kogiso, Tsutomo Shinohara, C. Kathleen Dorey and Yoshimi Shibata
10. Semaphorin7A is up‐regulated in human breast tumors and promotes tumor growth and metastasis in a mouse breast cancer model
R. Garcia‐Areas, S. Libreros, S. Amat, S. Durat, K. Schilling, E. Wojcikiewicz and
V. Iragavarapu‐Charyulu
11. TNF‐alpha‐ mediated modulation of mammary epithelial cell biophysical properties promote cell adhesion to collagen type I
Samrat Dutta, Justyna Jaczewska and Ewa P. Wojcikiewicz
12. Investigation of cell stiffness and cytoskeletal remodeling in response to inflammatory mediators using atomic force microscopy
Sherylne Magny and Ewa P. Wojcikiewicz
13. Interprofessional Education: Evaluation of a Pilot Program Mario Jacomino, Joseph Ouslander, Lindsey Henson, Mira Sarsekeyeva and Jo Ann Bamdas
14. Whole Transcriptome Analysis of Chicken Eye Lens Development and Cellular Differentiation Pathways Reveals Key Autophagy and Mitophagy Genes Required for Cell Differentiation
Daniel Chauss, Lisa Brennan, J. Fielding Hejtmancik, Sue Menko, Subhasaree Basu, Ales
Cvekl and Marc Kantorow
15. Delaying the Progression of Driving Impairment in Individuals with Mild Alzheimer’s Disease P. Holland, R. Tappen, L. Fisher and A.L. Curtis
16. The Impact of DaTscan on the Diagnosis and Management of Movement Disorders: A Retrospective Study
Kimberly D. Seifert and Jonathan I. Wiener
17. Thermodynamics analysis of substrate binding to a Bacteroides ovatus family 6 glycosyltransferase
Brittany Stinson, K. Ravi Acharya, Percy Tumbale, Michelle Lizotte‐Waniewski and Keith
Brew
18. Chronic variable stress‐induced dendritic plasticity and associated changes in brain‐derived neurotrophic factor in the hippocampus and the basolateral amygdala in the novelty‐seeking
phenotype: Implications for depressive‐ and anxiety‐like behaviors
Ozge Oztan, Cigdem Aydin, Fareen Hossain and Ceylan Isgor
19. Does High‐Density Lipoprotein Influence the Development of Vein Graft Disease After Coronary Artery Bypass Graft Surgery?: Analysis from the CASCADE Randomized Trial
Katie Jerzewski and Alexander Kulik
20. The Safety of Ketorolac After Cardiac Surgery: Is the Black Box Warning Justified? Lisa Oliveri, Katie Jerzewski, James J. Morris and Alexander Kulik
21. Reliability of the Diagnosis of Peruneural Invasion as a Prognostic Indicator in Colorectal Cancer
S. Millette, D. Allende, E. Silva, A. Maya, N. Sengul, F. Potenti, S. Wexner and M. Berho
22. The Impact of Histopathological Features in Survival of Stage I‐II Colorectal Cancer Patients? A. Maya, S. Wexner, H. Chong, F. Potenti, S. Millette, D. Allende and M. Berho
23. Improving the immunogenicity of vaccines through TLR agonists Mahyar Nouri‐Shirazi and Elisabeth Guinet
Abstracts
ABSTRACT TITLE: Intracellular trafficking of an androgen receptor splice variant, AR8
AUTHORS: Ciny John and Michael L. Lu
DEPARTMENT: Biomedical Sciences
BACKGROUND: The classical model of the intracellular trafficking of the ligand‐stimulated androgen
receptor (AR) involves receptor dimerization upon ligand binding, dissociation from chaperones and
interactions with nuclear membrane importins and Ran‐GTPases in order to gain access to nucleus. In the
nucleus, AR forms complexes with transcriptional co‐regulators and binds to target sequences. However,
the molecular mechanism AR signaling between the cytoplasm and the membrane remains undetermined.
In current study, we determined the intracellular trafficking of a newly identified AR isoform, AR8. AR8 is
a splice variant of the original full‐length AR and is shown to be up‐regulated in castration‐resistant
prostate cancer cells. The unique C‐terminal amino acid sequence immediately following the N‐terminal
domain (NTD) of a conventional AR makes AR8 structurally different from other AR variants. Two cysteine
residues within this sequence were identified to be palmitoylated, and is determined to be responsible for
the localization of AR8 to plasma membrane.
OBJECTIVES: It was previously established that AR8 predominantly localizes on the plasma membrane.
In this study, we wanted to elucidate a possible mechanism of intracellular trafficking between the
membrane and cytoplasm and predict potential functions of AR8.
METHODS: In order to investigate the intracellular trafficking of AR8, we used a mCherry fluorescent
protein tagged to AR8 and confocal microscopy, as well as time‐lapse confocal imaging, to determine
localization and migration of AR8 and markers for the components of the endocytic pathway.
RESULTS: Using mCherry‐AR8 and confocal microscopy, we confirmed the plasma membrane localization
of AR8, as previously described, and determined that AR8 co‐localized with a Golgi marker, GM‐130, as
well as GFP tagged galactosyltransferase (GalT), a golgi‐targeting sequence (GalT‐GFP; green fluorescent
protein) at Golgi compartments. Additionally, we found that mCherry‐AR8 co‐localized with EGFR‐GFP
(epidermal growth factor receptor‐GFP) on the plasma membrane within the raft domain. Using time‐lapse
confocal microscopy, we observed that AR8 co‐migrated with EGFR in a retrograde fashion and associated
with endosomal compartments upon EGF (5ng/ml) stimulation, which later fused with lysosomes shown by
co‐localizations of mCherry‐AR8 with GFP‐LAMP‐1.
CONCLUSIONS: Our observations propose a potential intracellular trafficking pathway associated with
AR8 and are consistent with the original report that EGF stimulation promotes the interactions between
AR8 and EGFR. AR8 localizes specifically to the lipid raft domain as shown by the co‐localization of AR8
with caveolin‐1, co‐migrates with EGFR in a retrograde manner as seen in the time‐lapse experiment, and
AR8 trafficking also involves endosome and lysosome transport. This co‐localization also suggests the
possibility that AR8 also migrates from the plasma membrane through receptor‐independent pathways.
Additionally, AR8 also co‐localizes with Golgi markers, suggesting potential trafficking to and from the
Golgi apparatus.
REFERENCES: Yang, X., Guo, Z., Sun, F., Li, W., Alfano, A., Shimelis, H., Qiu, Y. (2011). Novel membrane‐
associated androgen receptor splice variant potentiates proliferative and survival responses in prostate
cancer cells. J Biol Chem, 286(41), 36152‐36160. doi: 10.1074/jbc.M111.265124
ABSTRACT TITLE: Pulmonary inflammation associated with chitinase‐3‐like‐1 molecule (CHI3L1)
expression accelerates breast cancer metastasis to the lung
AUTHORS: Stephania Libreros1, Ramon Garcia‐Areas1, Roberto Carrio2 and Vijaya Iragavarapu‐
Charyulu1. Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL.
DEPARTMENT: Biomedical Science
Metastasis of a primary tumor to distant organs is the principal cause of mortality in patients with breast
cancer. Recently, it was reported that asthmatic patients have a greater propensity to develop distant
metastasis to the lung. Using a ragweed pulmonary inflammation model, we found that asthmatic mice
implanted with 4T1 mammary tumors have a 5‐fold increase in formation of metastatic foci in their lungs
compared to non‐asthmatic 4T1 mammary tumor‐bearing mice. Further, asthmatic tumor‐bearing mice
showed accelerated tumor growth and shorter disease‐free survival. We have previously reported that
Chitinase‐3 like‐1 protein (CHI3L1) expression is increased in mammary tumor‐bearing mice and that it
induces production of prometastatic mediators, CCL2, CXCL2 and MMP‐9 by macrophages. Asthmatic
patients have been reported to exhibit elevated levels of CHI3L1 in their lungs and circulation while in
breast cancer patients these levels are further elevated. We found that the levels of CHI3L1 are higher in
circulation and lungs of asthmatic mammary tumor‐bearers compared to mice with either asthma or tumor
alone. It is unknown if exposure to high levels of CHI3L1 previous to tumorigenesis accelerates metastasis.
We hypothesize that CHI3L1‐induced pulmonary inflammation generates the proper environment for
recruiting circulating breast cancer cells as well as proinflammatory leukocytes (M2 macrophages and
myeloid derived suppressors), resulting in increased rate of metastasis to the lung. Treatment of 4T1
asthmatic mammary tumor‐bearing mice with chitin microparticles, the ligand of CHI3L1, resulted in
decreased levels of metastasis to the lung. These studies provide an understanding of the role of CHI3L1
and existing inflammation in accelerating metastasis, the major cause of death in breast cancer patients.
ABSTRACT TITLE: Determination of levels and expression of IL‐1β, IL‐4, TNF‐α, IL‐10, and IFN‐γ in the
lacrimal glands of a predisposed mouse model of Sjögren’s Syndrome (SS): the influence of sex hormones
and genetic background in the pathogenesis of SS
AUTHORS: Stefanie P.C. Czerwinski, Safinaz Mostafa, Ana Maria Azzarolo
DEPARTMENT: Integrated Medical Science
BACKGROUND: Sjögren’s Syndrome (SS) is a chronic, inflammatory autoimmune disease affecting mostly
the exocrine cells of lacrimal and salivary glands, leading to diminished secretory function and resulting in
keratoconjunctivitis sicca (dry eye disease) and/or stomatitis sicca (dry mouth disease). Nevertheless, the
exact etiology and mechanisms of SS remain unknown. SS has been diagnosed predominantly in post‐
menopausal women with the female to male ratio reaching 9:1, suggesting a role of ovarian sex hormones in
the pathogenesis of SS. However, not all postmenopausal women develop SS, indicating the contribution of
other factors such as a genetic background to the onset of SS. SS has also been characterized by the
upregulation of several pro‐inflammatory cytokines in the lacrimal gland. However, the source and time
course of the cytokine increase remain to be elucidated. We have been studying the disease progression
using ovariectomized (OVX) NOD.B10.H2b mice, which provide a model of menopause and genetic
predisposition to SS. We have previously shown that lymphocytic infiltration followed by acinar cell
apoptosis in the lacrimal gland significantly increased at 3, 7, 21 and 30 days after OVX, and this infiltration
and apoptosis was prevented by physiological doses of 17β estradiol (E2) or dihydrotestosterone (DHT).
OBJECTIVES: In this study, we investigated the time course of changes in the levels and expression of
cytokines IL‐1β, IL‐4, TNF‐α, IL‐10 and IFN‐γ in the lacrimal glands of NOD.B10.H2b mice following OVX.
We also investigated whether treatment with E2 or DHT prevented the changes in cytokine levels and
expression found after OVX.
METHODS: Six weeks old NOD.B10.H2b and C57BL/10 mice were sham operated, OVX, OVX+E2 or
OVX+DHT for 3, 7, 21 and 30 days. At the end of each experimental time period, lacrimal glands were
collected and processed for RNA analysis by rt‐qPCR and protein assays by ELISA to evaluate cytokine
expression and concentrations of IL‐1β, IL‐4, TNF‐α, IL‐10 and IFN‐γ.
RESULTS: A significant increase in the expression of IL‐1β, TNF‐α, and IL‐4 in the lacrimal glands of
NOD.B10.H2b mice was seen at 3, 7, 21, and 30 days after OVX as compared to sham operated animals,
while a significant increase in the expression of IL‐10 was seen only at 7, 21, and 30 days after OVX. A
significant but much smaller increase in expression of IL‐1β and IL‐4 was seen at only 7, 21, and 30 days
after OVX in the lacrimal glands of C57BL/10 mice. No significant changes were observed for TNF‐α and IL‐
10 at any of the experimental times. The cytokine levels of IL‐1β, TNF‐α, and IL‐4 were significantly
increased at 3, 7, 21, and 30 days after OVX in the lacrimal glands of NOD.B10.H2b mice as compared to
sham operated animals. Significant increase in levels of IL‐10 was seen only at 21 and 30 days after OVX.
No significant differences were observed in the levels of IL‐1β, TNF‐α, IL‐4, and IL‐10 for all treatment
groups at all four experimental times in the lacrimal glands of C57BL/10 mice. Treatment with physiological
doses of E2 or DHT at time of OVX prevented the increase in cytokine levels and expression in both strains.
Both expression and levels of IFN‐γ remained undetected in both mouse strains for all experimental groups
and times.
CONCLUSIONS: The results of this study suggest ovarian sex hormones and genetic background seem to
be necessary for the pathogenesis of SS. Furthermore, this study suggests that cytokines are key players in
the etiology of SS. It has been previously reported that IL‐1 and TNF‐ may be involved in different
pathways leading to the clinical manifestations of SS, including the activation of the transcription factor NF‐
B. NF‐B has been reported to be involved in the transcription of various inflammatory and pro‐apoptotic
genes. Therefore, IL‐1 and/or TNF‐ mediated NF‐B activation could cause apoptosis and a persistent inflammatory reaction in the lacrimal glands of SS patients. Furthermore, the specific time frame of up‐
regulation of each cytokine is crucial to the understanding of the exact disease mechanism. For instance, this
study showed a delayed increase in IL‐10, which is a reportedly anti‐inflammatory cytokine known to
inhibit the Th1 response. This observation suggests that IL‐10 could be responsible for a switch in the
immune response from an early Th1 to a later Th2 in the progression of SS. Overall, this study further
unravels possible mechanisms for SS manifestations, allowing for potential therapeutic advancements in
treatment of this disease, including cytokine‐directed therapies.
REFERENCE: Mostafa S, Seamon V, Azzarolo AM, (2012). Influence of sex hormones and genetic
predisposition in Sjögrenʹs syndrome: a new clue to the immunopathogenesis of dry eye disease. Exp Eye
Res. 96(1):88‐97.
ABSTRACT TITLE: Fast Glutamate Uptake by EAAT2 Prevents Glutamate Depletion in Rod
Photoreceptors
AUTHORS: L.A. Purpura1; H. Ripps2; W. Shen1 1 College of Medicine, Florida Atlantic University, Boca
Raton, FL, United States. 2 Ophthalmology and Visual Science, University of Illinois College of Medicine,
Chicago, IL, United States.
DEPARTMENT: Biomedical Science
BACKGROUND: The rapid reuptake of extracellular glutamate by high‐affinity excitatory amino acid
transporters (EAATs) is critical for continued glutamate release in photoreceptors. As of now, five subtypes
of EAATs, (EAAT1 to EAAT5) have been cloned and localized in both neurons and glial cells of the CNS. In
salamander, the five EAATs have been identified, and their molecular properties and distributions have
been described [1,2]. sEAAT2A and sEAAT5 are expressed in salamander photoreceptors, and biophysical
studies have shown that the transporters display two separate conductance when activated (i) a coupled
Na+,H+,K+‐dependent conductance, which is necessary for the transporter to bind and translocate glutamate,
and (ii) a stoichiometrically uncoupled Cl‐ conductance, gated by Na+ and glutamate. Although a large
EAAT‐mediated Cl‐ conductance was first recorded from glutamatergic retinal neurons, i.e., photoreceptors
and bipolar cells[3,4], the role of the membrane transporter at the synapse between photoreceptors and
second‐order neurons was largely unknown.
OBJECTIVES: Previously, we demonstrated that EAAT2A (excitatory amino acid transporter 2A) is
expressed predominantly in the photoreceptor terminals, and showed that its function is important in
shaping the cone‐mediated light‐offset response in bipolar cells. In this study we examined how EAAT2A
prevents glutamate depletion in rods, and the contribution of EAAT2A to synaptic plasticity in the distal
retina.
METHODS: Whole cell patch‐clamp recordings were obtained from rod photoreceptors in tiger
salamander retinal slices. Single and multiple depolarizing pulses were applied to trigger glutamate release
from the photoreceptors. After release, EEAT2A serves to restore glutamate in the receptor terminals, and
the synaptic EAAT currents were recorded in rods with electrodes containing a high intracellular
concentration of CsNO3. DHKA, a specific inhibitor of the EAAT2 subtype, was used to block the synaptic
EAAT2A currents.
RESULTS: We find that DHKA blocks 80% of the EAAT2A‐mediated current in the rod terminals, an
indication that the transporter effectively takes up glutamate in dark. However, DHKA only blocked 35‐
40% of the transporter current in cones, and the DHKA‐ insensitive currents were blocked by TBOA, a
broad‐spectrum EAAT inhibitor,. EAAT2A activation in rods was tightly controlled by glutamate levels in
the synaptic cleft, whereas the kinetics of the transporter was not affected by glutamate levels. After
applying a 2ms depolarizing pre‐pulse to trigger a rapid release of glutamate from rod terminals, we found
that EAAT2A activates in less that a millisecond. Our results also show that internal Ca2+ levels, protons,
and the cell’s ECl can affect the action of EAAT2A in rod terminals.
CONCLUSIONS: Our study reveals a role for EAAT2A in preventing glutamate depletion at the rod
photoreceptor terminals. The fast reuptake of glutamate by EAAT2A may help to sustain glutamate release
from rods in darkness.
REFERENCES:
1. Eliasof S, Arriza JL, Leighton BH, Kavanaugh MP, Amara SG (1998) Excitatory Amino acid transporters
of the salamander retina: identification, Localization and functions. J Neuroscie. 18(2):698‐712.
2. Eliasof S, Arriza JL, Leighton BH, Amara SG & Kavanaugh MP (1998) Localization and function of five
glutamate transporters cloned from the salamander retina. Vision Res 38, 1443‐1454.
3. Arriza JL, Eliasof S, Kavanaugh MP & Amara SG (1997). Excitatory amino acid transporter 5, a retinal
glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U SA 94, 4155‐4160.
4. Grant GB & Werblin FS (1996). A glutamate‐elicited chloride current with transporter ‐like properties in
rod photoreceptors of the tiger salamander. Vis Neurosci 13, 135‐144.
5. Rowan MJ, Ripps H & Shen W (2010) Fast glutamate uptake via EAAT2 shapes the cone‐mediated light
offset response in bipolar cells. J Physiol., 588:3943‐56.
ABSTRACT TITLE: Structure‐Energy study of interactions between Membrane‐ type matrix
metalloproteinase 1 and Tissue inhibitors of metalloproteinase
AUTHORS: Haiyin Zou, Tim Logue, Ying Wu, Michelle Lizotte‐Waniewski and Keith Brew
DEPARTMENT: Biomedical Science
BACKGROUND: The matrix metalloproteinases (MMPs) play key roles in many biological processes,
such as wound healing, embryo implantation, bone remodeling, and organogenesis. The activities of MMPs
are regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).
Uncontrolled matrix degradation occurs when the balance between TIMPs and active MMPs is disrupted,
resulting in serious disease processes such as cancer cell metastasis, arthritis and chronic tissue ulceration.
Thus, the engineering of TIMPs to produce highly selective and efficacious inhibitors of individual MMPs
has potential for developing novel disease treatments. A goal of our research is to understand the
biophysical and structural basis of TIMP/MMP interactions to facilitate the design of MMP‐specific TIMP
variants with high binding affinity and specificity (1).
OBJECTIVES: TIMP‐1 and its inhibitory domain, N‐TIMP‐1 are weak inhibitors of MT1‐ MMP, a key
enzyme in the pericellular degradation of the ECM. In previous studies, a variant of N‐TIMP‐1, T98L
was identified (2) that has a higher affinity for MT1‐MMP. The objective is to determine the
thermodynamic profiles of the interactions of N‐TIMP‐2, N‐TIMP‐1 wild‐type and its mutant T98L
with MT1‐MMP to understand how structural differences change the binding affinity and the
specificity of TIMP/MMP interactions.
METHODS: Human MT1‐MMP, N‐TIMP‐2 and ‐1 and the N‐TIMP‐1 T98L mutant were expressed in
E. coli BL21(DE3) cells as inclusion bodies and were folded and purified as previously described (2, 3).
Inhibition constants (Ki values) with MMPs were measured by fluorimetric assays (2). Solutions of N‐
TIMPs and MMPs were extensively dialyzed against various buffers and degassed. N‐TIMPs (12‐20
μM) were titrated with an MMP (120‐300 μM) at different temperatures and in buffers with different
enthalpies of ionization using a MicroCal VP‐ITC microcalorimeter. The ITC data were analyzed to
elucidate the thermodynamic profiles for the interactions of MT1‐MMP with N‐TIMP‐2, N‐TIMP‐1
wild‐type and its T98L mutant. ITC requires relatively large quantities of purified proteins, but is
capable of measuring the changes in enthalpy (ΔH) and free energy (ΔG) of binding from which the
entropy (ΔS) can be calculated. Also, the heat capacity change, ΔCp (the temperature dependence of
ΔH) and ionization changes associated with the interactions can be measured. ΔCp can be used to
estimate the contribution of solvent ΔS arising from changes in the exposure of hydrophobic groups to
the ΔS of binding. With information about the structures of experimentally determined and modeled
complexes, it is possible to correlate the structures of interaction sites with thermodynamic parameters
(3).
RESULTS: The interactions of MT1‐MMP with N‐TIMP‐2, N‐TIMP‐1 (wild‐type) and T98L mutant
have large positive enthalpy changes (9.9±0.04; 8.9±0.5; 14.7±0.02 kcal mol‐1) compensated by favorable
entropy change. ΔHobs for all these interactions shows a strong negative dependence on temperature (ΔCp)
suggesting that a large fraction of the ΔSof binding arises from the hydrophobic effect. The associations of
MT1‐MMP with N‐TIMP‐ 1 (WT and T98L mutant) have slightly larger (more negative) heat capacity
changes (ΔCp) than the interaction with N‐TIMP‐2 (‐0.39±0.01, ‐0.58±0.004 vs ‐0.29±0.01 kcal mol‐1).
Compared with N‐TIMP‐1 (WT), the interaction of the T98L mutant with MT1‐MMP has a modified
thermodynamic profile, that includes a larger (unfavorable) enthalpy change (8.9±0.5 vs 14.7±0.02 kcal mol‐
1) and larger (favorable) entropy change, as well as an increased hydrophobic contribution to the binding
together with a large estimated increase in conformational entropy.
CONCLUSIONS: Our current results show that the hydrophobic contributions to the interactions of
MT1‐MMP with N‐TIMP‐2, N‐TIMP‐1 wild‐type and mutant T98L accounts for the most of the
entropic contribution to the Gibbs free energy of binding. The interaction of N‐TIMP‐2 with MT1‐MMP
has a smaller (negative) ΔCp, smaller entropy change and more positive (unfavorable) ΔH than that for
the N‐TIMP1/MT1‐MMP interaction. These suggest that there may be a large conformational change in
the formation of N‐TIMP‐1/MT1‐ MMP complex but only minor changes in N‐TIMP‐2/MT‐MMP
complex that gives rise to the positive ΔH values for the interactions. The higher binding affinity of
T98L for MT1‐ MMP compared with that of wild‐type N‐TIMP‐1 wild‐type appears to be linked to a
greater increase in conformational dynamics during the formation of complex. The interactions of both
N‐TIMP‐2/MT1‐MMP and N‐TIMP‐1 T98L/MT1‐MMP do not result in the uptake or release of protons,
but the association of N‐TIMP‐1 (wild‐type) with MT1‐MMP is accompanied by release of a proton.
The difference between wild‐type N‐TIMP‐1 and the T98L mutant seem likely to arise from different
conformational changes in that occur when these variants interact with MT1‐MMP.
This work was supported by grant RO1 AR40994 from NIAMS, NIH.
REFERENCES:
1. Brew, K and Nagase H, (2010). The tissue inhibitors of metalloproteinases (TIMPs): Anancient family
with structural and functional diversity. Biochim Biophys Acta 1803(1): 55–71.
2. Hamze, A B, Wei, S., Bahudhanapati, H. Kota, S., Acharya, K.R. and
Brew. K (2007). Constraining specificity in the N‐domain of tissue inhibitor of metalloproteinases‐1 ;
gelatinase‐selective inhibitors: {JOURNAL, VOLUME???] 1905‐ 1913.
3. Wu, Y., Wei, S., Van Doren, S.R., and Brew, K. (2011) Entropy increases from different sources support
the high‐affinity binding of the N‐terminal inhibitory domains of tissue inhibitors of metalloproteinases (N‐
TIMPs) to the catalytic domains of matrix metalloproteinases (MMPs) ‐1 and‐3. J. Biol. Chem. 286, 16891‐
16899.
ABSTRACT TITLE: Ribosomal RNA damage and degradation under oxidative stress
AUTHORS: Min Liu*, Zhongwei Li
(*Currently at Tsinghua University)
DEPARTMENT: Biomedical Science
BACKGROUND: RNA is now well recognized as a major target of chemical damage such as those created
by free radicals and other reactive species that are increased under oxidative stress. Recent compelling
evidence suggests that RNA damage causes detrimental effect on protein products and cell viability.
Ribosomal RNA (rRNA) accounts for the majority of cellular RNA in any living organism, and is
presumably a major target of oxidative damage. We have previously shown that rRNA, although highly
structured and associated with ribosomal proteins under normal conditions, is highly oxidized when cells
are challenged by an oxidant. We hypothesize that rRNA oxidation abolish its function in protein synthesis,
and that cells control the quality of rRNA by degradation under oxidative stress conditions. Although
rRNA degradation has been previously reported under other conditions, we are interested in elucidating
degradation mechanisms that may be employed specifically under oxidative stress.
OBJECTIVES: To understand the nature of rRNA oxidation and the mechanism of rRNA degradation
caused by oxidative stress.
METHODS: RNA oxidation level was determined by HPLC analysis of 8‐hydroxyguanosine. Oxidative
stress was created by addition of hydrogen peroxide or other oxidants. RNA fragmentation was detected by
Northern blotting.
RESULTS: We have been testing these hypotheses using Escherichia coli (E. coli) cell cultures. We have
shown that RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic
acid damages in are present in RNA as shown by the high levels of 8‐oxo‐G, an oxidized form of guanine.
Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither
RNA structure nor association with proteins protects RNA from oxidation. When E. coli cultures were
treated with hydrogen peroxide (H2O2), 8‐oxo‐G forms at higher levels in ribosomal RNA than in non‐
ribosomal RNA species. The preferential formation of 8‐oxo‐G in ribosomal RNA is related to the high
order structure of the RNA since oxidation produces more 8‐oxo‐G in native RNA than in denatured RNA,
and in a RNA:DNA duplex than in single‐stranded RNA of the same sequence. H2O2‐induced 8‐
oxo‐G in ribosomes is removed specifically depending on the activities of polynucleotide phosphorylase
(PNPase) and RNase R, two 3’ exoribonucleases capable of degrading structured RNA. H2O2–treatment of
E. coli cultures also causes rRNA degradation in a dosage dependent manner. In cells lacking the RNA‐
degradation exoribonucleases, RNase R, PNPase, and/or RNase II, rRNA fragments accumulated to a high
level upon H2O2‐treatment. The pattern of the rRNA fragments suggested a specific rRNA degradation
pathway that is initiated by endonucleolytic cleavages of 16S and 23S rRNA in the intact ribosomes or
subunits of ribosomes, followed by the degradation of the fragments exonucleolytically. Surprisingly, none
of the known specific endoribonucleases, RNase E, G, or P, is involved in the initial cleavages of 16S rRNA.
CONCLUSIONS:
1. RNA is oxidized to higher levels than DNA under both normal and oxidative stress conditions,
which suggests that RNA damage may contribute to increased cell death under oxidative stress.
2. RNA structure and association with proteins do not protect RNA from being oxidized.
3. Our results demonstrate a major role for RNA degradation in controlling oxidized RNA.
4. We have identified activities that may work in specific pathways for selectively degrading damaged
RNA. These activities may play pivotal role in controlling oxidized RNA and protecting cells under
oxidative stress.
REFERENCES:
1. Li Z, Wu J and DeLeo CJ. (2006) RNA damage and surveillance under oxidative stress. IUBMB Life.
58: 581‐588.
2. Liu M, Gong X, Alluri RK, Wu J, Sablo T and Li Z. (2012) Characterization of RNA damage under
oxidative stress in Escherichia coli. Biol. Chem. 393:123‐132.
Acknowledgement‐This work was supported by NIH grant S06 GM073621 and R15 GM097693 to Z.L. M.L.
was supported by FAU Integrated Biology Ph.D. program.
ABSTRACT TITLE: Effects of Green Tea Catechins on Diastolic Function in Mice with Restrictive
Cardiomyopathy
AUTHORS: Lei Zhang, Pierre‐Yves Jean‐Charles, Changlong Nan and Xupei Huang
DEPARTMENT: Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida
Atlantic University, Boca Raton, FL
BACKGROUND: Cardiomyopathy refers to heart muscle disorders that cause various cardiac
dysfunctions. Among the major cardiomyopathies, restrictive cardiomyopathy (RCM) is associated with a
severe prognosis often leading to heart failure and sudden death. Cardiac dysfunction in RCM is
characterized by damaged diastolic function resulting in a reduced blood filling of the ventricles during
diastole. Understanding the mechanisms behind diastolic dysfunction and RCM will have a significant
impact on the prevention and treatment of cardiomyopathies and heart failure, since up to 40% of heart
failure patients present diastolic dysfunction. Our laboratory have previously generated RCM transgenic
mice that manifest a phenotype similar to that observed in RCM patients i.e. enlarged atria, ventricles
presenting normal morphology but increased stiffness and impaired cardiac relaxation (Du et al, 2006;
2008). We also found that increased myofibril sensitivity to Ca2+ is a key mechanism behind the impaired
relaxation in RCM (Li et al, 2010). Compounds with Ca2+ desensitizing abilities suitable for therapeutic use
are rare. Catechins which are biologically active polyphenols from green tea, have been recently reported to
possess some Ca2+ desensitizing properties and various protective effects against cardiovascular diseases
(Robertson et al, 2009; Tadano et al, 2010). In the present study, we determined the effects of (‐)‐
epigallocatechin gallate (EGCg), a most biologically active component in catechins, on cardiac function in
transgenic RCM mice.
OBJECTIVES: This study aimed at exploring the effect of catechins, on cardiac function in RCM mice to test
the hypothesis that desensitization of myofibrils to Ca2+ is useful in correcting the impaired relaxation and
diastolic dysfunction in RCM. The therapeutic potential of Ca2+ desensitizers for cardiomyopathies with
diastolic dysfunction will be determined from our experiments.
METHODS: RCM cTnI TG mice (cTnI193His) expressing cTnI R193H in the heart were generated in our
laboratory (Du et al, 2006) and the transgenic colonies are maintained in the Animal Care facilities at Florida
Atlantic University. For cell‐based assays, mouse cardiomyocytes were freshly isolated from WT and
cTnI193His transgenic mice. The mechanical properties and calcium transients of single cardiomyocytes were
determined using an IonOptix Myocam system (IonOptix Inc., Milton, MA). Fura‐2 was used as calcium
indicator (Li et al, 2010). The tested cells were exposed to various concentrations of EGCg for the
measurement of sarcomere contractility and Ca2+ dynamics. For in vivo experiments, 2 month‐old male
R193H transgenic and WT mice were treated with (‐)‐epigallocatechin gallate (EGCg, i.p. injection) for three
months. Animals injected with vehicles were used as control. Cardiac functions were measured in vivo on
mice using a Vevo 770 High‐Resolution echocardiography (VisualSonics, Toronto, ON, Canada) as
described (Du et al, 2006, 2008; Li et al, 2010; Jean‐Charles et al, 2011; Chen et al,2012).
RESULTS: Untreated transgenic cardiomyocytes presented prolonged sarcomeric relaxation and delayed
Ca2+ decay compared to WT. EGCg treatment improved the sarcomere contractility in the diseased animals
in a dose dependent manner. The delay in Ca2+ decay kinetics in the RCM cardiomyocytes was restored as
well following the treatment. The systolic parameters of the cardiomyocytes were not affected by the drug.
Cardiac function measurement with echocardiography revealed that EGCg was able to improve cardiac
function in the transgenic animals. Left ventricular end diastolic dimension (LVEDD) was significantly
increased in RCM mice after treatment and the isovolumetric relaxation time (IVRT) significantly reduced
when compared with untreated RCM mice. These changes in diastolic parameters confirm the beneficial
effect of the compound on diastolic function in vivo. The ejection fraction and fractional shortening in WT
and RCM mice with or without treatment of EGCg did not present significant difference.
CONCLUSIONS: The Ca2+ desensitizing molecule, (‐)‐epigallocatechin gallate (EGCg), can reverse the
impaired relaxation and delayed Ca2+ decay in RCM cardiac myocytes by restoring the myofilament Ca2+
sensitivity. EGCg improves diastolic function in RCM mice without damaging the systolic function. The
restored diastolic function was manifested by the correction of the prolonged IVRT in the RCM animals and
the increased of the left ventricular end diastolic dimension in RCM mice following the treatment. The Ca2+
desensitizing properties of the green tea extract, EGCg, are beneficial for RCM and provide a therapeutic
alternative for diastolic dysfunction due to increased myofilament Ca2+ sensitivity.
REFERENCES:
Du J, C Zhang, J Liu, C Sidky, XP Huang. Arch Biochem Biophys 456:143‐150, 2006.
Du J, et al. Am J Physiol Heart Circ Physiol 294:H2604‐H2613, 2008.
Robertson IM, MX Li, BD Sykes. J Biol Chem 284:23012‐23023, 2009.
Tadano N, et al. Bri J Pharmacology 161:1034‐1043, 2010.
Li Y, et al. J Mol Cell Cardiol 49:402‐411, 2010.
Jean‐Charles P, Y Li, C Nan, XP Huang. J Geriatr Cardiol 8:168‐183, 2011.
Chen G, et al. Biochem Res Internat doi:10.1155/2012/715197, 2012
ABSTRACT TITLE: Annotation of neurodevelopmental regulators in dopaminergic and GABAergic
neurons for links to ischemia‐hypoxia response
AUTHORS: Kim McKain, Maryanne Joseph, Rainald Schmidt‐Kastner
DEPARTMENT: Integrated Medical Science
BACKGROUND: A leading concept for the pathogenesis of schizophrenia (SCZ) is based on gene –
environment interactions during neurodevelopment. In previous in silico work, genes associated with the
risk of SCZ were examined for links to ischemia‐hypoxia responses and vascular factors, and a significant
overlap was demonstrated (1, 2). Our analysis has stimulated clinical studies of gene x environment
interactions in SCZ (3). In preparation for translational research, we are now developing strategies to create
gene databases related to a) known pathophysiological mechanisms for SCZ, b) regulation of
neurodevelopment, and c) ischemia‐hypoxia responses in the brain. Dysfunction of the dopaminergic (DA)
system in SCZ has been confirmed by recent PET studies of dopamine receptors, and abnormalities of
GABAergic cortical interneurons have been described in post‐mortem tissue.
OBJECTIVES: The aim of this study is to define possible interactions between neurodevelopmental
mechanisms for DA and GABAergic neurons and ischemia‐hypoxia responses. Using literature‐mining, we
generated databases for genes/proteins broadly related to the development of the DA midbrain neurons or
GABAergic telencephalic interneurons, and then looked for an overlap with our own databases for
ischemia‐hypoxia responses.
METHODS: Lists of genes related to regulation of DA and GABAergic neurogenesis were generated via
literature mining (PubMed, Web of Knowledge) and by compilation of gene expression profiling studies.
Genes expressed during development of DA and GABAergic were annotated using the ischemia‐hypoxia
response (IHR) gene database. The overlap was tested using the chi‐square test (two‐sided, p<0.025).
Selected data sets were examined for enrichment of pathways using DAVID Bioinformatics.
RESULTS: N=94 genes were retrieved as related to the development of DA neurons and 23% matched with
the IHR list (p=0.015). LHX2, NR4A2/NURR1 and RGS4 stand out as regulated genes in DA neuron
development. N=416 genes were retrieved for the development of GABAergic cortical interneurons and
22% overlapped with the IHR list (p=0.0001). CXCR4, DLX1 and GRM5 are of particular interest for
developing GABAergic neurons.
CONCLUSIONS: Literature for neural stem cells has indicated a positive interaction between low oxygen
levels and generation of DA neurons, and our analysis provides putative genes involved in this interaction.
The strong match between genes for GABAergic interneurons and response to hypoxia could reflect the
metabolic stress during tangential migration from the ganglionic eminence to the cortex. Both the DA
neurons and GABAergic neurons may react to hypoxic conditions during neurodevelopment which could
program long‐lasting changes that in turn increase the risk of SCZ.
REFERENCES:
(1) Schmidt‐Kastner, R., van Os, J., Steinbusch, H.W.M., Schmitz, C. Gene regulation by hypoxia and the
neurodevelopmental origin of schizophrenia. Schizophrenia Res. 84: 253‐271, 2006
(2) Schmidt‐Kastner, R., van Os, J., Esquivel, G., Steinbusch, H.W.M., Rutten, B.P.F. An environmental
analysis of genes associated with schizophrenia: hypoxia and vascular factors as interacting elements in the
neurodevelopmental model. Mol. Psychiatry 17 (12): 1194‐1205, 2012 (Advance Online Publication, Jan. 31,
2012)
(3) Nicodemus, K.K., Marenco, S., Batten, A.J., Vakkalanka, R., Egan, M.F., Straub, R.E.,
Weinberger, D.R. Serious obstetric complications interact with hypoxia‐regulated/vascular‐expression
genes to influence schizophrenia risk. Mol. Psychiatry 13(9): 873‐877, 2008
ABSTRACT TITLE: Cholera toxin induces a shift from inactive to active COX‐2 in alveolar macrophages
activated by Mycobacterium bovis BCG
AUTHORS: Mari Kogiso, Tsutomu Shinohara, C. Kathleen Dorey and Yoshimi Shibata
College of Medicine, Florida Atlantic University, Boca Raton, FL, Virginia Tech Carilion School of Medicine,
Roanoke, VA
BACKGROUND: The development of effective mucosal vaccines has been hindered by the lack of useful
adjuvants and by our limited knowledge of their modes of action (1). The cyclooxygenase (COX) product
prostaglandin E2 (PGE2), pharmacologically targeted by non‐steroidal anti‐inflammatory drugs, is
commonly considered a potent pro‐inflammatory mediator; PGE2 shifts T cells to Th2, Th17 and regulatory
T cell responses and shifts macrophages to alternatively activated macrophages (M2) in autoimmune
diseases, cancer and other chronic inflammatory diseases (2). In contrast, PGE2 in the lungs has complex
pro‐ and anti‐ inflammatory roles modulating not only the immune‐inflammatory responses, but also
mucosal protection from inflammatory injuries and tissue repair processes (3‐5). More specifically, lung
PGE2 is reported to play paradoxical roles including up‐regulation of apoptotic death of bactericidal
macrophages, thereby inhibiting replication of intracellular Mycobacterium tuberculosis (6), and inhibition of
Th2 differentiation and allergic inflammation (5, 7). This complexity is at least in part explained by multiple
pulmonary mucosal and inflammatory cells differentially expressing four distinct PGE2 receptors, termed E‐
prostanoid 1 – 4, which are pharmacologically targeted for chronic inflammatory diseases. However,
despite recognition of the indispensable roles of constitutive COX‐1 and inducible COX‐2 for PGE2‐
mediated mucosal inflammation, there is still insufficient information regarding the activities of these two
rate‐limiting enzymes for PGE2 release during the course of mucosal vaccination. Our previous studies (8, 9)
indicate that intranasal BCG develops bactericidal alveolar macrophages (M1), but both□ COX‐1 and COX‐
2 are dissociated from the nuclear envelop (NE), accumulated in aggregates in the endoplasmic reticulum
(ER), and catalytically inactive; these COX‐2+ macrophages release no PGE2 (8).
OBJECTIVES: This study tested the hypothesis that intranasal administration of BCG with cholera toxin
(CT), a mucosal vaccine component, would shift the inactive/NE‐dissociated COX‐1/COX‐2 to active/NE‐
associated forms.
METHODS: Groups of mice (6 mice/group) were given 50 μl of saline containing 500 μg of M. bovis BCG 1
μg of CT, 500 μg of BCG and 1 μg of CT, or saline (controls) intranasally at 0 h, and samples were collected
at 24 h. Alveolar macrophages were fixed, permeabilized, and incubated in blocking buffer consisting of
10% FBS overnight at 4ºC prior to incubation with anti‐COX‐2 or anti‐COX‐1 antibody overnight at 4°C.
Subsequently, cells were washed with PBS and incubated with Alexa Fluor 488 goat anti‐rabbit IgG for
COX‐1 and COX‐2 for 1 h at 22°C. Nuclei and BCG were stained with propidium iodide. Cells were
examined with a laser scanning confocal microscope (LSM700, ZEISS).
RESULTS: The results showed increased PGE2 release in the lungs and NE‐associated COX‐2 in the
majority of COX‐2+ macrophages. These COX‐2+ macrophages were the primary source of PGE2 release in
the lungs, since there was only slight enhancement of NE‐associated COX‐1, and no change in COX‐1/COX‐
2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the
effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro
macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX‐2‐
mediated PGE2 release, but macrophages in vivo exhibited less activation of NE‐associated COX‐2.
CONCLUSIONS: Our results indicate that including CT in the intranasal BCG vaccination enhances COX‐
2‐mediated PGE2 release by alveolar macrophages, and further suggest that the effect of CT in vivo is
mediated by other lung cells. The full article has been published in 2013 (10).
REFERENCES:
1. Negri, D. R., D. Pinto, S. Vendetti, M. Patrizio, M. Sanchez, A. Riccomi, P. Ruggiero, G. Del Giudice,
and M. T. De Magistris. 2009. Cholera toxin and Escherichia coli heat‐labile enterotoxin, but not their
nontoxic counterparts, improve the antigen‐presenting cell function of human B lymphocytes. Infect
Immun 77:1924‐1935.
2. Kalinski, P. 2012. Regulation of immune responses by prostaglandin E2. J Immunol 188:21‐28.
3. Morsy, M. A., Y. Isohama, and T. Miyata. 2001. Prostaglandin E(2) increases surfactant secretion via
the EP(1) receptor in rat alveolar type II cells. Eur J Pharmacol 426:21‐24.
4. Savla, U., H. J. Appel, P. H. Sporn, and C. M. Waters. 2001. Prostaglandin E(2) regulates wound
closure in airway epithelium. Am J Physiol Lung Cell Mol Physiol 280:L421‐431.
5. Vancheri, C., C. Mastruzzo, M. A. Sortino, and N. Crimi. 2004. The lung as a privileged site for the
beneficial actions of PGE2. Trends Immunol 25:40‐46.
6. Chen, M., M. Divangahi, H. Gan, D. S. Shin, S. Hong, D. M. Lee, C. N. Serhan, S. M. Behar, and H. G.
Remold. 2008. Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and
LXA4 in the induction of macrophage death. J Exp Med 205:2791‐2801.
7. Martin, J. G., M. Suzuki, K. Maghni, R. Pantano, D. Ramos‐Barbon, D. Ihaku, F. Nantel, D. Denis, Q.
Hamid, and W. S. Powell. 2002. The immunomodulatory actions of prostaglandin E2 on allergic
airway responses in the rat. J Immunol 169:3963‐3969.
8. Shinohara, T., T. Pantuso, S. Shinohara, M. Kogiso, Q. N. Myrvik, R. A. Henriksen, and Y. Shibata.
2009. Persistent inactivation of macrophage cyclooxygenase‐2 in mycobacterial pulmonary
inflammation. Am J Respir Cell Mol Biol 41:146‐154.
9. Kogiso, M., T. Shinohara, C. K. Dorey, and Y. Shibata. 2012. Role of PPARgamma in COX‐2
Activation in Mycobacterial Pulmonary Inflammation. Inflammation 35:1685‐1695.
10. Kogiso, M., T. Shinohara, C. K. Dorey, and Y. Shibata. 2013. Cholera Toxin Induces a Shift from
Inactive to Active Cyclooxygenase 2 in Alveolar Macrophages Activated by Mycobacterium bovis
BCG. Infect Immun 81:373‐380.
Additional Project.
11. C‐J Huang, KN Beasley, EO Acevedo, RL Franco, TL Jones, DC Mari, and Y Shibata, Chitin enhances
obese inflammation ex vivo, Submitted for publication.
12. Kamba A, Y Shibata, E Mizoguchi, Potential roles of chitin in mucosal inflammation, in preparation.
ABSTRACT TITLE: Semaphorin7A is up‐regulated in human breast tumors and promotes tumor growth
and metastasis in a mouse breast cancer model
AUTHORS: R. Garcia‐Areas1, S. Libreros1, S. Amat1, S. Durat1, K. Schilling2 E. Wojcikiewicz1, V.
Iragavarapu‐Charyulu1. 1Florida Atlantic University, Schmidt College of Medicine, Boca Raton, USA. 2Boca
Raton Regional Hospital, Boca Raton, USA.
Semaphorins are a family of proteins that were originally discovered to play a role in neurogenesis and are
now found to have important physiological roles outside of the nervous system. Moreover, semaphorins
have also been found to be dysregulated during tumorigenesis. Although most vertebrate semaphorins
have been linked to various cancers, no published studies to date have shown a role for the novel
Semaphorin7A (Sema7A) in tumorigenesis. Our laboratory has discovered that Sema7A is highly expressed
in tumor tissues of breast cancer patients and is weakly expressed in normal breast epithelia. To determine
a functional role for Sema7A in breast tumors, we have used shRNA technology to silence Sema7A in the
highly metastatic 4T1 mammary tumor cells. To assay the in vivo effects of tumor‐derived Sema7A, we
implanted BALB/c mice with 4T1 control cells or 4T1 Sema7A knock‐down cells. Mice bearing Sema7A
knock‐down cells showed a significant decrease in tumor growth and metastasis, while having enhanced
survival. Using pathway focused gene arrays, we found that knocking down Sema7A reduced the
metastatic and mesenchymal phenotype of the 4T1 mammary tumor cells. We further analyzed 4T1
Sema7A knock‐down cells using Atomic Force Microscopy. We found that decreasing Sema7a increased cell
stiffness in 4T1mammary tumor cells, which may also be contribute to their decrease in metastatic potential.
Using a combination of clinical studies and animal models we have uncovered a novel role for Sema7A in
breast cancer. Sema7A could prove to be a novel therapeutic target to limit tumor growth and metastasis
not only for breast cancer but for other solid tumors as well.
ABSTRACT TITLE: TNF‐alpha‐ mediated modulation of mammary epithelial cell biophysical properties
promote cell adhesion to collagen type I
AUTHORS: Samrat Dutta, Justyna Jaczewska, and Ewa P. Wojcikiewicz
DEPARTMENT: Biomedical Science
BACKGROUND: Studies have shown that cancerous cells are less stiff as compared to normal cells (1,2).
However, the mechanisms through which their biophysical properties are altered have not been fully
elucidated. Tumor necrosis factor alpha (TNF‐alpha) is a widely studied inflammatory cytokine involved
in apoptosis, cell survival, inflammation and tumor angiogenesis. It has also been shown to promote cell
migration and cytoskeletal rearrangement.
OBJECTIVES: We hypothesize that TNF‐alpha promotes prometastatic changes in normal mammary
epithelial cells by altering their biophysical properties. These changes facilitate adhesive interactions with
the extracellular matrix (ECM) and promote cell migration and metastasis.
METHODS: MCF‐10A cells were sustained in a 1:1 mixture of Dulbeccoʹs Modified Eagleʹs Medium and
Hamʹs F‐12 (DMEM/F‐12) that were supplemented with 5% horse serum, epidermal growth factor (EGF) 20
NG/ML, hydrocortisone (0.5 g/ml), cholera toxin (100 ng/ml), insulin (10 μg/ml) and
penicillin/streptomycin. The MFP‐3D Bio atomic force microscope (Asylum Research, Santa Barbara, CA)
was used to measure the cell stiffness of the mammary epithelial cell line. The obtained force‐indentation
curves were fitted to the Young’s modulus. In addition, AFM single cell force spectroscopy was carried out
on collagen type I (3). All measurements were performed using living cells in liquid at 37 deg. C. Data was
analyzed using the Igor Pro analysis platform.
RESULTS: Preliminary results suggest that 24‐hour TNF‐alpha treatment promotes a less stiff mammary
epithelial cell phenotype. Cell stiffness was reduced by ~80% following treatment. TNF‐alpha treatment
also enhanced adhesion of MCF10A to collagen type I, the chief component of the mammary extracellular
matrix.
CONCLUSIONS: Taken together, our results implicate that decreased cell stiffness most likely mediated
by cytoskeletal remodeling facilitates cell spreading on collagen. This allows for the formation of more
adhesive contacts and therefore enhanced cell adhesion.
REFERENCES:
1. Lekka M, Laidler P, Gil D, Lekki J, Stachura Z, Hrynkiewicz AZ. Elasticity of normal and cancerous human
bladder cells studied by scanning force microscopy. Eur Biophys J 1999;28(4):312‐316.
2. Cross, S., Y. Jin, et al. (2007). ʺNanomechanical analysis of cells from cancer patients.ʺ Nat Nanotechnol
2(12): 780‐783.
3. Wojcikiewicz, E., R. Koenen, et al. (2009). ʺLFA‐1 binding destabilizes the JAM‐A homophilic interaction
during leukocyte transmigration.ʺ Biophys J 96(1): 285‐293.
ABSTRACT TITLE: Investigation of cell stiffness and cytoskeletal remodeling in response to inflammatory
mediators using atomic force microscopy
AUTHORS: Sherlyne Magny and Ewa P. Wojcikiewicz
DEPARTMENT: Biomedical Science
BACKGROUND: Atomic force microscopy (AFM) is a novel technology with emerging potential for cancer
detection based on cell stiffness measurements. Studies have shown that cancerous cells were recognized to
be less stiff than normal epithelial cells (1,2). However, the mechanisms through which their biophysical
properties are altered have not been fully elucidated.
OBJECTIVES: In this study, we investigated the role of transforming growth factor‐β (TGF‐β) as a potential
mediator involved in altering the biophysical properties such as cell stiffness of mammary epithelial cells
(MCF10A). AFM was used to obtain cell stiffness measurements. We hypothesized that TGF‐β will
promote decreased cell stiffness through the disruption of f‐actin using the AFM.
METHODS: MCF‐10A cells were sustained in a 1:1 mixture of Dulbeccoʹs Modified Eagleʹs Medium and
Hamʹs F‐12 (DMEM/F‐12) that were supplemented with 5% horse serum, epidermal growth factor (EGF) 20
NG/ML, hydrocortisone (0.5 g/ml), cholera toxin (100 ng/ml), insulin (10 μg/ml) and
penicillin/streptomycin. The MFP‐3D Bio atomic force microscope (Asylum Research, Santa Barbara, CA)
was used to measure the cell stiffness of the mammary epithelial cell line. The obtained force‐indentation
curves were fitted to the Young’s modulus. All measurements were carried out on living cells in liquid at
37 deg. C. Data was analyzed using the Igor Pro analysis platform.
RESULTS: We determined that disrupting f‐actin of MCF10A using cytocholasin D decreased measured
cell stiffness. However, disrupting microtubules using colchicine did not alter cell stiffness. Additionally,
our AFM measurements revealed that MCF10A treatment with TGF‐β reduced the measured cell stiffness
more than 3‐fold, down to the level measured for MDA‐MB‐231 cancer cells in our previous studies.
CONCLUSIONS: TGF‐β promotes metastatic progression in part by disrupting the f‐actin cytoskeleton that
likely leads to the reduced stiffness measured by AFM. This study will make a contribution towards
unraveling the mechanism through which a normal cell’s biophysical properties are modulated during
prometastatic progression.
REFERENCES:
4. Lekka M, Laidler P, Gil D, Lekki J, Stachura Z, Hrynkiewicz AZ. Elasticity of normal and cancerous human
bladder cells studied by scanning force microscopy. Eur Biophys J 1999;28(4):312‐316.
5. Cross, S., Y. Jin, et al. (2007). ʺNanomechanical analysis of cells from cancer patients.ʺ Nat Nanotechnol
2(12): 780‐783.
ABSTRACT TITLE: Interprofessional Education: Evaluation of a Pilot Program
AUTHORS: Mario Jacomino, Joseph Ouslander, Lindsey Henson, Mira Sarsekeyeva, Jo Ann Bamdas
DEPARTMENT: Integrated Medical Science
BACKGROUND: The Interprofessional Education (IPE) program was created to help students from the
Florida Atlantic University’s (FAU) Colleges of Medicine, Nursing, and School of Social Work learn and
work collaboratively. The program is led by a steering committee and faculty subcommittees. The IPE
program had two components: IPE development sessions (IPEDS) and interdisciplinary team visits. The
first component included three sessions with small group case discussion facilitated by faculty from each
college following recommended “Core Competencies for IPE.” The second component of the program
included four student IP team visits with Senior Aging and Geriatrics Educator (SAGE) Mentors at a local
retirement community (Abbey Delray and Abbey Delray South). The second year of the program was
revised based on feedback from faculty and students.
OBJECTIVES: The objectives were to: 1) Develop an IPE program based on the Interprofessional Education
Collaborative “Core Competencies for Interprofessional Collaborative Practice;”2) Create and implement
evaluation methods to assess the IPE program; 3) Conduct collaborative interprofessional activities
concentrating on a geriatric population; 4) Disseminate the lessons learned from the IPE pilot program.
METHODS: A combination of pre‐test and post‐test questionnaires was distributed for all students during
the three IPEDS: 1) introduction to roles and responsibilities, care, and collaboration; 2) communication and
teamwork; and 3) health care policy and quality. Likert scale questions and open‐ended questions were
included to obtain quantitative and qualitative data that would complement each other. At each IPED, case
studies were created by subcommittee faculty teams and facilitated in 9‐10 small groups of students. Based
on feedback, in the second year the groups were divided into smaller groups of 3‐5 students. Also, an IRB‐
approved survey for students and faculty facilitators was drafted to gather data from this component of the
program. For the SAGE Mentor program, the College of Medicine created an IRB‐approved survey with
Likert‐scale and open‐ended questions. This survey is administered to medical students and SAGE mentors
at the end of each year of the SAGE mentor visits program.
RESULTS: In the first year, a total of 64 medical students, 48 social work students, and 46 nursing students
(n=158) participated in the IPE pilot program. At the end of the first year, 155 students remained. A total of
fifteen faculty members were involved in one or more of the IPEDS. In the second year, a total of 61
medical students, 75 nursing students, and 66 social work students (n= 202) participated in the IPE
program. The response rate for the pre/post‐test survey of the IPED sessions varied from 85% to 95%. In
the first year, the results revealed that the majority of students felt that their expectations were met, core
competencies were known by them or presented to them during the sessions, and that the facilitators
helped guide the case discussion. The statistics indicated that almost all of the average scores for each
group of students and for all the students together improved slightly from pre to post test. Two out of the
three sessions (communication and health care policy and quality) were helpful for their professional
learning. The SAGE mentor survey was administered to the medical students and mentors in the first year.
A total of 63 out of 64 medical students and 36 out of 64 SAGE mentors at two facilities responded to the
survey. The SAGE Mentor program was deemed successful by the students and stated that “the best part
was working with my SAGE Mentor.” Students and mentors were most challenged by scheduling.
However, the challenges were outweighed by the amount of communication and learning that took place
during the SAGE Mentor visits.
DISCUSSION AND CONCLUSION: An IPE program was successfully developed, implemented, and
assessed at FAU. While year 1 of the program was centered on developing new pre and post‐test surveys,
year 2 was dedicated to gathering data and modifying instruments to better reflect input. Year 3 will be a
critical year in this longitudinal study to transfer what we have learned.
In this unique pilot program at FAU the majority of students found that two out of the three IPEDS were
beneficial for their professional learning. The collaborative interprofessional activities concentrating on a
geriatric population were very well received by students and mentors. Students and mentors were most
challenged by scheduling of the SAGE visits, but this has been outweighed by the amount of learning
taking place during the visits. Important lessons were learned to refine the content and logistics of the
program for the coming years. After two full years of this program with few monetary resources, the
program has been successful. In order to continue developing the program, several recommendations are
being made to the Interprofessional Education Steering Committee and the Administration at FAU.
REFERENCES
Interprofessional Education Collaborative Expert Panel. (2011). Core competencies for interprofessional
collaborative practice: Report of an expert panel. Washington, D.C.: Interprofessional Education Collaborative.
http://www.aacn.nche.edu/education‐resources/ipecreport.pdf
Acknowledgements: This Interprofessional Education pilot program has been supported in part by the Retirement
Research Foundation.
ABSTRACT TITLE: Whole Transcriptome Analysis of Chicken Eye Lens Development and Cellular
Differentiation Pathways Reveals Key Autophagy and Mitophagy Genes Required for Cell Differentiation
AUTHORS: Daniel Chauss (presenter), Lisa Brennan, Fielding Hejtmancik(1), Sue Menko (2), Subharsee
Basu(2), Ales Cvekl(3), Marc Kantorow
DEPARTMENT: BIOMED with (1) NIH; (2) Thomas Jefferson University, (3) Einstein University
BACKGROUND: The pathways that operate to initiate lens differentiation and degrade lens organelles
must be identified to reveal those genes important for prevention of cataract formation. Here 16,000 genes
were analyzed in the chick model of development and the pathways identified.
OBJECTIVES: To identify genes whose disruption is a factor in eye lens cataract formation‐a leading cause
of world blindness.
METHODS: Whole genome RNA sequencing analysis, Western Analysis, Spatial gene expression imaging.
Bioinformatic analysis, Gene‐silencing, Retroviral transgenic characterization.
RESULTS: Here 16,000 genes were analyzed in the chick model of development and the pathways
identified. At least 800 genes play key roles in autophagy, mitophagy and other pathways essential for lens
development and differentiation.
CONCLUSIONS: Autophagy and mitophagy pathways are essential for lens development and
differentiation and their disruption as a consequence of inherited mutation or environmental damage likely
plays a major role in cataract formation.
REFERENCES: Brennan et al., Molecular Vision 2013
ABSTRACT TITLE: Delaying the Progression of Driving Impairment in Individuals with Mild
Alzheimer’s Disease
AUTHORS: P. Holland, R. Tappen, L. Fisher and A.L. Curtis
DEPARTMENT: Integrated Medical Science and Christine E. Lynn College of Nursing, Florida Atlantic
University
OBJECTIVES: Evaluate the effects of 20 mg/day memantine on the inhibition of the progression of driving
impairment in 60 subjects with mild Alzheimer’s disease as measured by actual driving and cognitive
performance based on a battery of neuropsychological assessments testing abilities necessary for driving
(executive functioning, visuospatial abilities, attention, orientation and tests designed to measure driving
ability in older adults).
METHODS: A sample of 60 otherwise healthy men and women 60 years and older with mild (Mini Mental
State Exam‐MMSE ≥ 23) Alzheimerʹs Disease was randomized at a 1:1 ratio in a double‐blind, placebo
controlled, 12 month trial of memantine titrated to a target dose of 20mg/day verses placebo. Subjects with
poor vision and depression were excluded. Driving ability was measured by actual driving (on the road
driving test) and cognitive performance on a battery of neuropsychological assessments testing abilities
necessary for driving (executive functioning, visuospacial abilities, attention, orientation and tests designed
to measure driving ability in older adults). The primary outcome measure is the number of subjects in each
group who are able to pass the DriveABLE test at month 12 (endpoint). The secondary outcome measures
are the change from baseline to endpoint on driving related measures. Outcome measures (scores from
DriveABLE‐On Road Test and cognitive assessments) were acquired at baseline, 6‐months, and 12 months.
Cognitive assessments included ADAS‐Cog, Trail Making Tests A & B, Rey‐Osterrieth Complex Figure
Test, Useful Field of Vision, and the Motor Free Visual Perception Test ‐ Visual Closure Subtest.
RESULTS: 43 subjects were randomized. Preliminary analysis showed efficacy for memantine
delaying progression of driving impairment. At 12 months 100% of the treatment group either stayed
the same or improved their driving ability, while only 75% of the placebo group did the same or better
(p=.04).
CONCLUSION: In this preliminary analysis, addition of memantine to the drug regimen appears to have
efficacy in delaying driving impairment in subjects with mild Alzheimerʹs disease.
ABSTRACT TITLE: The impact of DaTscan on the diagnosis and management of movement disorders: A
retrospective study
AUTHORS: Kimberly D. Seifert and Jonathan I. Wiener
Florida Atlantic University Charles E. Schmidt College of Medicine1, Boca Radiology Group, Boca Raton
Regional Hospital2
The diagnosis of Parkinson’s disease remains a challenge in patients who have abnormal symptoms or
show a lack of response to medication. The imaging technique, DaTscan, can be used to visualize
dopamine degeneration in the nigro‐striatum, which is associated with Parkinsonian Syndrome. We
examined the use of the DaTscan in diagnosis, confidence in diagnosis, and clinical
management. Methods: Physicians of 125 patients were contacted to fill out a brief survey about changes
in diagnosis, confidence of diagnosis, and clinical management after assessment with the
DaTscan. Results: There was an overall increase in confidence of diagnosis with the results of the
DaTscan. Physicians also stated that the DaTscan impacted their diagnosis in 68% of the patients, as well as
an impact in the clinical management of 58% of the patients. Conclusion: The DaTsan can be used as a tool
to help diagnose Parkinsonian Syndrome in patients with unclear symptoms.
ABSTRACT TITLE: Thermodynamics analysis of substrate binding to a Bacteroides ovatus family
glycosyltransferase
AUTHORS: Brittany Stinson, K. Ravi Acharya, Percy Tumbale, Michelle Lizotte‐Waniewski
and Keith Brew
DEPARTMENT: Biomedical Science
BACKGROUND: Family 6 Glycosyltransferases (GT6) catalyze steps in the biosynthesis of complex
glycans that are important for pathogenicity, cellular recognition, and immunity. The immune system is
able to distinguish between pathogenic and mutualistic organisms partly by recognizing foreign glycans on
the cell surface. Some bacteria decorate their cell surfaces with glycans that mimic those of their hosts, to
avoid attack by the host immune system. We have identified a GT6 from a bacterial resident of the human
intestine, Bacteroides ovatus, that catalyzes the synthesis of glycans similar to human blood group A.
Although this enzyme, BoGT6a, is homologous with the enzyme that produces human blood group A and
catalyzes a similar reaction, it differs from mammalian GT6s in not requiring divalent metal ions for
activity. This is linked to the change of an Asp‐X‐Asp (DxD) sequence motif found in many GTs that
interacts with a Mn2+cofactor required for catalytic activity to Asn‐x‐Asn. α3GT from bovines contains a D‐
X‐D motif as well as an elongated N‐terminal region in comparison to BoGT6a. We are investigating
structure‐function relationships in BoGT6a to try and understand the basis of glycosyltransferase metal
dependence and its implications for the catalytic mechanism.
OBJECTIVES: Our goal is to determine the thermodynamic profiles for the binding of substrates to
BoGT6a and α3GT using Isothermal Titration Calorimetry (ITC). In collaborative studies we will use X‐ray
crystallography to determine the structure of the enzymes in the free state and in complexes with donor
and acceptor substrates.
METHODS: His‐tagged forms of BoGT6a and its E192Q mutant were expressed in soluble forms in E. coli
BL21(DE3) cells and purified using Ni‐NTA columns. The BoGT6a E192Q mutant was selected because this
mutation greatly lowers catalytic activity but does not eliminate substrate binding. Studies using wild type
forms of BoGT6a and α3GT were also performed for comparison. ITC was used to generate thermodynamic
profiles for BoGT6a E192Q substrate binding. For ITC studies BoGT6a was dialyzed into a buffer containing
20 mM HEPES, 1 mM DTT, and 0.2 M NaCl at pH 7.5. UDP, UDP‐Galactose (UDP‐Gal), UDP‐N‐
Acetylgalactosamine (UDP‐GalNAc), and UDP‐Glucose (UDP‐Glc) at 3 mM concentrations in the same
buffer were titrated into the enzyme (28 X 10 μL injections). The enthalpy (ΔH), entropy (ΔS), Gibbs free
energy (ΔG), and association constants (Ka) were measured for enzyme‐ ligand complex formation.
Titrations were conducted in different buffers (PIPES, MES, HEPES, MOPS, BES, and ACES) with different
enthalpies of ionization to determine ionization changes. Heat capacity changes (ΔCp) were determined by
measurements of ΔH at different temperatures: 291 K, 297 K, 300 K, and 303 K. X‐ray crystallographic
studies of BoGT6a E192Q complexes with donor and acceptor substrates were conducted by Dr. K.R.
Acharya and his group at Bath University, UK.
RESULTS: ITC shows that BoGT6a binding to its preferred substrate (UDP‐GalNAc) is similar to that of a
homologous mammalian enzyme, bovine α3GT, for binding to its preferred substrate (UDP‐Gal) despite the
absence of a divalent metal ion in BoGT6a studies. The Ka values for BoGT6a binding to UDP‐GalNAc and
α3GT +Mn2+ binding to UDP‐Gal are 18 x 10‐3 and 16.7 x 10‐3 respectively verifying the metal independence
of BoGT6a. A comparison of ΔH values of BoGT6a binding to UDP‐GalNAc (‐16.37 kcal/mol), UDP‐Gal (‐
14.75 kcal/mol), UDP‐Glc (‐13.68 kcal/mol), and UDP (‐13.00 kcal/mol) revealed that UDP‐GalNAc, the
preferred donor substrate for BoGT6a, has the highest ‐ΔH of binding. Binding to UDP‐Glc is least
thermodynamically favored. UDP‐GalNAc and UDP binding to BoGT6a have similar Ka values. However,
the entropies for these interactions are very different. ‐T ΔS for these interactions are 10.55 kcal/mol and 7.3
kcal/mol respectively. The temperature‐dependence of ΔH of binding revealed that the presence of a sugar
attached to the UDP of Leloir substrates makes ΔCp more negative indicating an increased hydrophobic
effect. ΔCp values for UDP, UDP‐GalNAc, and UDP Gal are ‐105 cal*K/mol, ‐704 cal*K/mol, and ‐1511
cal*K/mol respectively. ITC studies in different buffers revealed a slope of ‐0.96 when plotting observed
enthalpy changes against the buffer enthalpies of ionization for BoGT6a binding to UDP‐GalNAc. This
indicates that one proton is released to buffer when UDP‐GalNAc binds to BoGT6a.
CONCLUSIONS: ITC studies indicate that UDP‐GalNAc, the preferred donor substrate for BoGT6a, has a
greater affinity, and highest (negative) ΔH for binding to the enzyme than other substrates. UDP binds to
BoGT6a E192Q with a Ka value that is similar to UDP‐GalNAc but has a significantly less negative TΔS.
However, UDP interacts with BoGT6a wt with a lower Ka. This indicates that the UDP component of the
substrate is directly identified by the enzyme but may not cause a conformational change similar to that of
the preferred substrate. Measurements of heat capacity changes show that UDP‐GalNAc binding to BoGT6a
is linked to a greater hydrophobic effect than UDP binding. UDP‐GalNAc interactions with BoGT6a cause a
proton to be released. ITC studies have shown that Ka values for 2’fucosyllactose binding to BoGT6a
increased from 8.0 x 102 to 1.3 x 104 in the presence of UDP. This suggests that, although substrates can bind
randomly, there may be a preferred order of donor and acceptor substrates.
ABSTRACT TITLE: Chronic variable stress‐induced dendritic plasticity and associated changes in brain‐
derived neurotrophic factor in the hippocampus and the basolateral amygdala in the novelty‐seeking
phenotype: Implications for depressive‐ and anxiety‐like behaviors
AUTHORS: Ozge Oztan, Cigdem Aydin, Fareen Hossain and Ceylan Isgor
DEPARTMENT: Biomedical Science
Experimentally naive rats show variance in their locomotor reactivity to novelty, some displaying higher
(HR) while others displaying lower (LR) reactivity, associated with vulnerability to stress. The novelty‐
seeking phenotype (the LRHR phenotype) is proposed as an antecedent to development of stress hyper
responsiveness. We employed chronic (16 sessions), variable physical stress (CVP; restraint, cold, or ether)
and chronic variable social stress (CVS; isolation, novelty, or crowding) exposure or control handling to
LRHR rats during the peripubertal‐juvenile period (postnatal days 28‐44) to determine the effects of chronic
variable stress on dendritic plasticity underlying enhanced stress‐induced anxiety in the basolateral
amygdala (BLA) and differential regulation of depressive‐like behavior in the hippocampus of the juvenile
LRHR rats. Our previously published work showed opposite regulation of depressive‐ and social anxiety‐
like behaviors following chronic variable stress in the HR phenotype that may be mediated by increased
levels of BDNF in the hippocampus and in the BLA respectively. In addition, increased depressive‐ and
social anxiety‐like behaviors were observed following CVP in the LR rats that may be mediated by
decreased levels of BDNF in the hippocampus and increased levels of BDNF in the BLA
respectively. Concurrent with findings on regulation of hippocampal BDNF, our results showed a
significant increase in total mossy fibre volume, particularly in suprapyramidal mossy fibres (SP‐MF)
terminal field size in HRs and a significant decrease in SP‐MF terminal field size in LRs following CVP. In
the present study, changes in spine density in the BLA and dendritic processes of the hippocampal CA3
neurons that are affected by CVP and CVS were determined using Golgi‐Cox impregnation method and 3‐
D reconstruction of neurons following chronic variable stress in LRHR rats. CVP resulted in increased spine
density in the BLA spiny pyramidal neurons and decreased dendritic length and spine density in the apical
dendritic arbors of CA3 neurons in juvenile LRs, in line with increased BDNF levels in the BLA and
decreased BDNF levels in the hippocampus respectively. In contrast, both stress regimens caused increased
spinogenesis in the BLA and hypertrophy of the apical CA3 dendritic arbors in juvenile HRs, in line with
increased BDNF levels both in the BLA and the hippocampus. These findings implicate a phenotype and
stress specific remodeling of ultrastructure in the hippocampus and the amygdala.
Grant Support: NIH DA023675
ABSTRACT TITLE: Does High‐Density Lipoprotein Influence the Development of Vein Graft Disease
After Coronary Bypass Graft Surgery?: Analysis from the CASCADE Randomized Trial
AUTHORS: Katie Jerzewski and Alexander Kulik
DEPARTMENT: Integrated Medical Science
BACKGROUND: Low levels of high‐density lipoprotein (HDL) purportedly increase the risk after
coronary bypass surgery. This may relate to the development of saphenous vein graft (SVG) disease early
postoperatively, but this premise has never been evaluated in the context of a prospective trial.
METHODS: The CASCADE Trial was a multi‐center study of 113 patients evaluating the use of
postoperative clopidogrel. Patients received standard lipid management after surgery (96% statins). At 12
months, angiography and intravascular ultrasound was performed to assess SVG occlusion and intimal
hyperplasia, respectively. In this exploratory analysis, we evaluated the influence of HDL levels on the
development of SVG diseases at 12 months, using the established cut‐off of <40 mg/dL suggesting increased
risk.
RESULTS: While HDL levels increased over the time‐period of the trial (P<0.0001), 51.1% of patients had
HDL levels <40 mg/dL 12 months after surgery. Slightly more SVG occlusions occurred amongst patients
with HDL levels <40 mg/dL (6.8%), compared to patients with HDL levels >40 mg/dL (4.0%, P=0.5). With
multivariate adjustment, HDL levels <40 mg/dL was associated with a trend towards more SVG occlusions
(odds ratio 3.2; P=0.12). Lower HDL level was also associated with more intimal hyperplasia on
ultrasound at 12 months (P=0.10). Patients who had HDL levels >60 mg/dL had the least amount of intimal
hyperplasia, significantly less than the remainder of the cohort (P=0.01).
CONCLUSIONS: Within this population, lower HDL levels were associated with trends towards more
graft occlusions and more vein intimal hyperplasia. Modulation of postoperative HDL levels may represent
a valuable future strategy for the reduction of SVG disease.
ABSTRACT TITLE: The Safety of Ketorolac After Cardiac Surgery: Is the Black Box Warning Justified?
AUTHORS: Lisa Oliveri, Katie Jerzewski, James J. Morris and Alexander Kulik
DEPARTMENT: Integrated Medical Science
BACKGROUND: After cardiovascular safety concerns were identified with several non‐steroidal anti‐
inflammatory drugs (NSAIDs), the United States Food and Drug Administration (FDA) issued a black box
warning recommending against the use of ketorolac, an intravenous NSAID, following cardiac surgery.
The goal of this study was to assess the safety of ketorolac in the management of early postoperative pain
after cardiac operations.
METHODS: A retrospective chart review was performed at a single center to identify all patients who
received ketorolac for postoperative analgesia within 72 hours after cardiac surgery to limit postoperative
narcotic use. Perioperative data was collected, focusing on preoperative comorbidities, types of surgery,
and postoperative ketorolac dosing. Postoperative adverse events were recorded, including bleeding,
cardiovascular, and renal complications.
RESULTS: Between September 2006 and July 2012, 488 patients received at least one dose of ketorolac for
pain management after cardiac surgery (78.9% coronary bypass, 16.6% valve). Patients treated with
ketorolac were often young men (mean age 67.2 years, 70.3% male) with near normal preoperative renal
function (mean preoperative creatinine: 1.05 mg/dL). Ketorolac was administered on average 8.7 hours
after surgery (mean doses: 3.1; mean cumulative dose 81.7 mg). Eight patients (1.6%) required reopening
for bleeding after ketorolac administration, and 6 patients (1.2%) developed gastrointestinal bleeding.
While the mean postoperative increase in creatinine was 0.20 mg/dL, 2 patients (0.4%) developed acute
renal failure requiring dialysis. Two patients (0.4%) had a perioperative myocardial infarction, 4 patients
(0.8%) had a perioperative stroke or transient ischemic attack, and there were 2 perioperative deaths (0.4%).
CONCLUSION: Ketorolac appears safe for use as a postoperative analgesic when administered selectively
following cardiac operations. With a low risk of complications associated with its administration, our data
call into question the need for a black box warning recommending against the use of ketorolac in the
postoperative setting after cardiac surgery.
ASBTRACT TITLE: Reliability of the diagnosis of perineural invasion as a prognostic indicator in
colorectal cancer
AUTHORS: S. Millette, D. Allende, E. Silva, A. Maya, N. Sengul, F. Potenti, S. Wexner and M. Berho
DEPARTMENT: Integrated Medical Science and Cleveland Clinic Florida
BACKGROUND: Perineural invasion (PNI) has emerged as an adverse prognostic marker in colorectal
cancer, however its applicability may be limited due to variable inter‐observer variability.
AIM: To assess the impact of nerve stains (S100) in the recognition of PNI, to evaluate inter‐observer
variability (IOV) and to correlate PNI with other markers of prognosis.
METHODS: After IRB approval, patients with colorectal cancer who underwent surgical treatment from
1/2002 to 12/2009, were identified from a prospective database. Only cases in which clinical/surgical
information and slides/blocks were available were included. Clinical and histological parameters were
obtained by chart review. IOV in the detection of PNI by both H&E and S100 stained slides was assessed by
two gastrointestinal pathologists. Time required to read the H&E and S100 slides and the number of
positive slides for PNI were recorded.
RESULTS: 139 colorectal patients (mean 64 years, 67% female) were included in the study (colon: 46,
rectum: 93), with a mean follow‐up of 50.8 months. PNI was noted in 44% (pathologist A) and 52%
(pathologist B) of the cases on H&E; and 63% and 58% on S100, respectively. The time to diagnosis went
from 3.2 minutes (H&E) to 1.1 minutes (S100) for pathologist A and from 2.4 minutes (H&E) to 1.8 minutes
(S100) for pathologist B. IOV showed good levels of agreement for both H&E (k=0.75) and S100 (k=0.72).
Overall recurrence was 17%, mean time to recurrence was 36 months. PNI by both H&E and S100 was an
independent predictor of mortality (OR 3.39, p<0.05) after controlling for factors such as budding, node
status, and vascular invasion.
CONCLUSIONS: PNI can be effectively identified on routine H&E stains with good interobserver
variability. It is also an independent predictor of mortality. Even though S100 stain does not significantly
impact sensitivity or specificity, its use significantly reduced time to diagnosis (p=0.05).
ABSTRACT TITLE: The impact of histopathological features in survival of Stage I‐II Colorectal Cancer
Patients?
AUTHORS: A. Maya, S. Wexner, H. Chong, F. Potenti, S. Millette, D. Allende and M. Berho
DEPARTMENT: Integrated Medical Science and Cleveland Clinic Florida
BACKGROUND: A small but significant group of patients with node negative colorectal cancer (Stage I‐II)
will develop recurrent disease and may therefore benefit from adjuvant therapy. Determination of
clinicopathological variables that could predict an adverse prognosis is essential to plan treatment.
AIM: To identify histopathological parameters associated with poor outcome in patients with Stage I – II
colorectal cancer.
METHODS: After IRB approval, all the patients with Stage I‐II colorectal cancer who underwent surgical
treatment with curative intent and negative margins, from 12/2002‐1/2010, were identified from a
prospective database. Demographics, operative and tumor variables were obtained by chart review.
Patients with concomitant diseases or adjuvant treatment were excluded. Pathological variables were
abstracted from pathology reports as well as review of actual slides.
RESULTS: 193 patients (median 69 years, 42% male) underwent surgical treatment for colon (72.1%) and
rectal (27.9%) cancer. The median follow‐up in days was 1520.6 (SD: 681.7). Surgical procedures were as
follows: Right colectomy: 36.8%, low anterior resection: 24.9%, left colectomy: 19.2%, proctocolectomy:
10.4%, subtotal colectomy: 5.7% and APR: 2.1%. The median number of LN dissected was 29.6. Deceased
and alive groups were well matched for demographics, ASA score, surgical procedures, number of lymph
nodes dissected and comorbidities. 54 patients died during the follow up period. Analysis of the different
tumor variables revealed that only perineural invasion (PNI) was significantly associated with the deceased
group (p < 0.01). Vascular and perineural invasion were each associated with a <12 lymph nodes dissected
(p<0.001). All other histopathological variables were not correlated with prognosis. Lower number of lymph
nodes dissected was noted in the deceased group, however this difference did not reach statistical
significance, most likely related to the high median of lymph nodes found overall (29.6).
ABSTRACT TITLE: Improving the immunogenicity of vaccines through TLR agonists
AUTHORS: Mahyar Nouri‐Shirazi and Elisabeth Guinet
DEPARTMENT: Integrated Medical Science
BACKGROUND: Vaccines are one of the most successful means of preventing communicable disease
spread to human by directly protecting vaccinated individuals and indirectly preventing circulation of
infectious agents to individuals (newborns, elderly, pregnant women, HIV/AIDS, and
immunocompromised) who are not eligible for vaccines through its herd effect. In addition, vaccines ease
financial strain from illness‐related hospitalization and increase workforce productivity due to fewer sick
days. Today, one of the priorities of vaccine program agencies (WHO, NIH, CDC) is to put a global halt to
infectious diseases by developing new and improved vaccines that are safe and effective in all target
populations. However, the reduced protection seen in smokers compared to nonsmokers after vaccination
(1‐3) has been one of the major hurdles to this effort and could have dire consequences for public health
particularly during epidemic season, pandemic outbreak, or biological warfare. The current research
addresses this public health concern by identifying the means to optimize the efficacy of current and future
vaccines in vaccinated individuals.
OBJECTIVES: Our in vitro and in vivo work (4‐8) revealed that nicotine‐induced defects in the
differentiation and the biological activities of dendritic cells (DCs) diminishes the development of antigen‐
specific effector memory Th1 cells and antibody production, leading to poor host response to an otherwise
protective and therapeutic vaccine (5). We also reported that the defects observed in DCs are reversible and
IFN‐ known to be produced by NK cells, is an absolute requirement in this process. Hence, the objective
was to test whether an adjustment of vaccine formulation with immunological adjuvants, TLR agonists,
would restore the interactions between DC and NK cells and allow vaccines to also work optimally in
nicotine‐exposed host.
METHODS: Nicotine exposure model: The osmotic pump mode was used to increase the accuracy of
delivery, and served as a constant source for delivery of nicotine for up to 6 weeks. Transgenic mouse model:
We utilized the ovalbumin (OVA) transgenic mouse models (DO11.10) to evaluate both humoral and cell‐
mediated immune responses to a protein‐based vaccine formulated with TLR agonists. The TCR transgenic
mice, DO11.10 (BALB/c background), contain rearranged TCR and TCR genes in their germline DNA
encoding TCR specific for the ovalbumin peptide fragment (OVA 323‐339) bound to I‐Ad class II MHC
molecules. Disease model: We used the EG7‐OVA animal model of cancer to test both prophylactic and
therapeutic vaccines formulated with TLR agonists in nicotine‐exposed mice. The well‐established OVA‐
EG.7 immunogenic tumor cells (ATCC) expressing the gene for the known experimental antigen OVA are
mouse thymoma EL4 cells stably transfected with the complementary DNA of chicken ovalbumin (OVA)
and thus express OVA epitopes as a unique antigen. Flow cytometry: We used the flow cytometry to assess
the phenotype and functional characteristics of DCs and T cells in response to vaccination.
RESULTS: The key observations made were: 1) None of the TLR agonists could directly induce IFN‐ production by NK cells and the presence of nicotine had no effect. The candidate agonist, however, was the
only one capable of synergizing with exogenous IL‐12 to trigger adequate IFN‐ production by nicNKs. 2)
It was also the only agonist capable of triggering similar production of the key Th1 cytokine IL‐12 and IFN‐
by Flt3‐L expanded nicDCs as compared to control DCs. 3) Importantly, in contrast to TLR4 agonist LPS,
the candidate agonist was able to act on both nicDCs and nicNKs and restored nicDC:nicNK cross‐talk, as
evidenced by significant amounts of IFN‐ and IL‐12 produced during co‐cultures. 4) Immunization with
soluble OVA and LPS, induced clonal expansion of adoptively transferred OVA‐specific T cells (detected by
CD4+KJ+ monoclonal antibodies) in the spleen and lymph nodes of both control and nicotine‐exposed mice
(0.4 to 2.32% and 2.11% respectively). 5) Nicotine exposure prior to immunization, however, significantly
reduced the frequency of OVA‐specific IFN‐‐secreting effector Th1 cells (15.9% vs. 7.7%). 6) Immunization
with soluble OVA plus the candidate agonist induced clonal expansion of adoptively transferred OVA‐
specific CD4+KJ+ T cells, and promoted the differentiation of OVA‐specific IFN‐‐secreting effector Th1 cells in the spleen and lymph nodes of both control and nicotine‐exposed mice (14.1% vs. 14.7%).
CONCLUSIONS: Our research has revealed the physiological changes triggered by nicotine on host
immunity and opened the possibility of developing a vaccine formulation that works optimally in all target
populations. The lab is currently investigating into the mechanisms by which the candidate TLR agonist
exerts its unique adjuvant effects in nicotine‐exposed hosts.
REFERENCES:
1. J. F. Finklea et al., Am Rev Respir Dis 104, 368 (Sep, 1971).
2. J. S. MacKenzie, I. H. MacKenzie, P. G. Holt, J Hyg (Lond) 77, 409 (Dec, 1976).
3. A. P. Winter, E. A. Follett, J. McIntyre, J. Stewart, I. S. Symington, Vaccine 12, 771 (Jul, 1994).
4. E. Guinet, K. Yoshida, M. Nouri‐Shirazi, Immunol Lett 95, 45 (Aug 15, 2004).
5. M. Nouri‐Shirazi, E. Guinet, J Immunol (Jan 25).
6. M. Nouri‐Shirazi, E. Guinet, Immunology 109, 365 (Jul, 2003).
7. M. Nouri‐Shirazi, E. Guinet, Immunol Lett 103, 167 (Mar 15, 2006).
8. M. Nouri‐Shirazi, R. Tinajero, E. Guinet, Immunol Lett 109, 155 (Apr 15, 2007).
ABSTRACT TITLE: Inhibition of HIV‐1 replication by expression of the cellular splicing factors hnRNPA1
and SF2
AUTHORS: Jacques Kepler Jean‐Philippe, Sean Paz and Massimo Caputi
DEPARTMENT: Biomedical Science
BACKGROUND: Expression of the HIV‐1 genome is regulated by the interaction of several cellular and
viral factors with a number of viral sequences. The viral transactivator Tat and several cellular co‐factors
mediate transcription of the integrated viral genome via binding to the Trans‐activation response element
(TAR) to generate a single transcript. This viral pre‐mRNA is alternatively spliced into more than 40
mRNAs that code for the 9 viral genes. Regulation of viral splicing relies on the interaction of cellular
factors, namely members of the arginine‐serine rich (SR) and heterogeneous nuclear ribonucleoproteins
(hnRNP) protein families, with multiple viral sequences. We have previously characterized the activity on
viral splicing of several members of the hnRNP and SR protein families and shown that expression of a
number of these cellular factors can down‐regulate viral expression by deregulating proper processing of
the viral mRNA.
RESULTS: We wanted to determine if factors regulating the processing of the viral RNA might affect also
the regulation of viral transcription. To this aim we co‐expressed the cellular splicing factor SF2 with a
series of reporter minigenes under the control of the viral promoter LTR. We observed that over‐expression
of SF2 counteracts the activity of the viral transcriptional trans‐activator Tat. SF2 activity is independent of
the transcript generated by the LTR promoter. Thus, the inhibitory activity exerted by SF2 on Tat is not
dependent on SF2 splicing activity exerted on the viral mRNA. The results obtained by utilizing reporter
minigenes were confirmed by a series of assays that utilized full‐length pro‐viral HIV‐1 clones. We are now
in the process of dissecting the molecular mechanism underlying SF2 inhibition of Tat transcriptional
activity. To this end we are utilizing a genomic RNA sequencing (RNA‐Seq) approach coupled to chromatin
immunoprecipitation (ChIP) assays.
A second goal of our research was to deliver hnRNPA1 to infected CD4+ T cells. We have previously shown
that the cellular protein hnRNPA1 down‐regulates HIV replication in stable cell lines by inhibiting the
processing of Tat‐specific viral mRNAs. The lower level of Tat induces a decrease of transcription from the
viral LTR promoter. Although stable cell lines are useful models they do not faithfully reproduce the
complexity of CD4+ T cells, the main cellular type targeted by the HIV‐1 virus within the human host. We
have compared several transduction techniques to deliver hnRNP A1 expression vectors to T cells purified
from healthy donors and shown that electroporation is the most efficient method, achieving over 60% of
transfection efficiency. We are now analyzing the effects of hnRNPA1 over‐expression on HIV‐1 replication
in CD4+ T cells utilizing a combined RNA‐Seq and quantitative PCR approach. We have also isolated
truncated variants of the hnRNPA1 protein that efficiently down‐regulate viral replication. Given the
difficulty to deliver and expression vector to primary T cells we have produced a recombinant hnRNPA1
protein conjugated with a cell penetrating peptide (CPP) and the GFP protein in bacteria. Preliminary data
show that the recombinant hnRNPA1‐CPP‐GFP protein can reduce viral replication when added to the cell
culture media of infected cells.
CONCLUSIONS: The final goal of our research is the understanding of the mechanisms regulating the
replication of the HIV‐1 virus and the development of therapeutics to resolve the viral infection. To this end
we have studied the role of cellular proteins in the transcription and processing of the viral mRNAs. Our
recent findings show that the expression of key cellular proteins can severely reduce viral replication by
reducing viral transcription and the proper processing of the viral mRNA.
REFERENCES:
Massimo Caputi (2011) The regulation of HIV‐1 mRNA biogenesis. RNA Processing. InTech. 79‐100.
Joseph A. Jablonski, Antonio A. Amelio, Mauro Giacca and Massimo Caputi. (2010) The transcriptional
transactivator Tat regulates viral splicing. Nucleic Acids Res. 38(4), 1249‐60
Joseph A. Jablonski and Massimo Caputi. (2009). Role of cellular RNA processing factors in human
immunodeficiency virus type 1 mRNA metabolism, replication, and infectivity. J. Virology 83, 981‐992.
NOTES
NOTES
7 7 7 G l a d e s R o a d , B l d G . 7 1 , B o c a R a t o n , F l 3 3 4 3 1 • m e d . F a u . e d u