kamal a. shair central research science laboratory 5th
TRANSCRIPT
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Kamal A. Shair Central Research Science Laboratory
5th Research Conference
Friday, April 8, 2016
0099am to 0099 pm
Hostler Auditorium
"AUB 150 Research Conference"
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Talks Abstracts
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T1. Improvement of ketoprofen degradation in oxidative persulfate medium
using thermal and chemical activation processes.
Maya Amasha, Antoine Ghauch
American University of Beirut, Faculty of Arts and Sciences, Department of Chemistry
Activated persulfate (PS) chemistry has become a growing field of interest in terms of its
wide application in the remediation of heavily-contaminated effluents. Its powerful effect in this
field has triggered interested researchers to develop several techniques for PS activation into sulfate
radicals (SRs), the main active species in the contaminant destruction process. The aim of our
project is to utilize and study the influence of combined activation techniques of PS on the oxidative
degradation of the anti-inflammatory drug Ketoprofen (KTP). PS activation can take many forms,
of which we mainly focus on thermal activation, chemical activation as well as a combination of
both for synergistic effect. . Thermal activation experiments were conducted in 20 mL vials at pH
7 at four different temperatures (40, 50, 60, and 70◦C). KTP (7.25 μM) and PS (1.0 mM) KI were
monitored by HPLC-MS and by spectrophotometric KI complexation, respectively. The results
showed that KTP disappearance was pseudo- first order with determined activation energy of about
145 kJ.mol-1. Introducing iron scrap (1 mg of iFe) into the same reaction system yielded an
activation energy of 60 kJ.mol-1. The latter experiments however were not maintained at pH≈7 since
it was noticed that the presence of a phosphate buffer complexed the ferrous iron (Fe2+) and thus
suppressed the PS chemical activation process. Open system experiments were also conducted
through which the reaction systems were kept exposed to air and the degradation results of KTP
were investigated in comparison to the previous sets of experiments done in close systems. Varying
the concentration of PS in the medium from 1.0 mM to 0.75, 0.5, 0.25 and 0.1 mM was also
examined. In short, a comparative study showing the effect of varying the mentioned parameters
on KTP degradation will be presented.
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T2. Functional Interaction between Apolipophorins and Complement
Regulate the Mosquito Immune Response to Systemic Infections
Layla Kamareddine, Johnny Nakhleh, Mike A. Osta
Department of Biology, American University of Beirut, Beirut, Lebanon
In the malaria vector Anopheles gambiae, the complement-like attack mediated by the
thioester-containing protein 1 (TEP1) was shown to be a hallmark effector response against
Plasmodium ookinetes invading the midgut epithelium and against systemic infections with bacteria
and fungi. Due to its key role in mosquito immunity, TEP1 is tightly regulated at the transcriptional
and activation levels. The basal levels of TEP1 were initially shown to be controlled by the Rel1
and Rel2 signaling pathways. Enhancing TEP1 expression by silencing the negative regulators of
these pathways increased mosquito resistance to Plasmodium infections. A more recent report
revealed a significant role for the c-Jun N-terminal kinase (JNK) pathway in controlling the basal
expression levels of TEP1 in hemocytes, suggesting that multiple transcription factors act in concert
to control TEP1 promoter activity. At the activation level, full-length TEP1 is cleaved in the
hemolymph by an unknown protease into active TEP1 cut, which is stabilized by a complex of two
leucine-rich immune proteins, APL1C and LRIM. The APL1C/LRIM1 complex controls the
precocious activation of TEP1 in the hemolymph and is required for its binding to Plasmodium
ookinetes. It was shown later that nitration of ookinetes by HPX2 (heme peroxidase 2) and NOX5
(NADPH oxidase 5) during the traversal of midgut epithelial cells directs TEP1 to their surfaces.
HPX2 is also required for TEP1 binding to apoptotic sperm cells in mosquito males, suggesting that
nitration is probably a prerequisite for TEP1 binding to all its target cells. Following activation,
TEP1 accumulation on microbial surfaces is also controlled by two noncatalytic clip domain serine
proteases (CLIPs), SPCLIP1 and CLIPA2, which act as positive and negative regulators of that
process, respectively, suggesting the presence of a convertase-like activity analogous to that in
mammalian complement. Silencing LRIM1 in naive mosquitoes triggered the loss of TEP1cut ,
SPCLIP1 and CLIPA2 from the hemolymph, demonstrating the tight association of these CLIPs
with TEP1 activity. CLIPA2 has a particular RNAi phenotype characterized by increased mosquito
resistance to infections with Plasmodium, bacteria and fungi, in addition to a reduction in egg
laying.
Here, CLIPA2 coimmunoprecipitation from the hemolymph of Beauveria bassiana -
infected mosquitoes followed by mass spectrometry and functional genetic analysis led to the
identification of the Apolipophorin-II/I gene, encoding the two lipid carrier proteins Apo-I and II,
as a novel negative regulator of TEP1-mediated immune response during mosquito systemic
infections. Apo-II/I exhibits a similar RNAi phenotype as CLIPA2 in mosquito bioassays
characterized by increased resistance to B. bassiana and Escherichia coli infections. We provide
evidence that this enhanced resistance to systemic infections is TEP1 dependent. Interestingly,
silencing Apo-II/I but not CLIPA2 upregulated the expression of TEP1 following systemic
infections with E. coli and B. bassiana in a c-Jun N-terminal kinase pathway-dependent manner.
Our results suggest that mosquito Apo-II/I plays an important immune regulatory role during
systemic infections and provide novel insight into the functional interplay between lipid metabolism
and immune gene regulation.
We would like to acknowledge the KAS CRSL for providing access to the fluorescent microscope,
real time machine and nanodrop.
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T3. Through A Gendered Lens Women ISIS Recruits in the Media
Maream-Jena Halim Nabut
American University of Beirut, Sociology, Anthropology, and Media Studies
At a time when ISIS was making daily headlines all over the world, a particularly intriguing
phenomenon had appeared in the media: the recruitment of women by ISIS social media users. In
this study, I analyzed the coverage of this phenomenon as it had been treated by two major
networks- CNN and Al-Arabiya. In assessing this news coverage, I utilized critical media and social
constructionism theories. Meanwhile, content and discourse analysis were used for my
methodology. I intended to discovering how both CNN/U.S. and Al-Arabiya covered the news of
women who had been joining ISIS lately, in order to see how framing, representation, and
symbolization of women took place in media, as well as the extremist groups' consequences on
media's news. My findings suggested that females joining ISIS were constructed as deviant through
CNN/U.S. and Al-Arabiya's use of language. Women were represented in a feminine way (e.g.
emotional and powerless), taking secondary and stereotypically roles and were represented
according to their appearance. Nonetheless, media stereotyped women involved in extremism by
framing their stories around nationality and ethnicity.
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T4. Immobilized Polyoxometalates onto Highly Porous Aerogels for
Heterogeneous Catalysis
Christina Jalkh, Christelle Ghazaly, and Houssam El-Rassy
Department of Chemistry, American University of Beirut
Aerogels are dried gels exhibiting very high relative pore volume and surface area. They are
synthesized by low-temperature traditional sol-gel chemistry followed by supercritical drying. The
catalytic activity of Polyoxometalates (POMs), known to be green and efficient catalysts, was
previously investigated in homogeneous catalysis where high activities were reported. In this work
we immobilize various POMs onto amorphous inorganic solid networks such as titania aerogels
prior to their investigation in heterogeneous catalysis. The structural, textural, and morphologic
characterization of these materials was performed before and after immobilization of POMs.
Investigating the catalytic activity of these clusters in homogeneous and heterogeneous catalysis
for the biodiesel production from corn oil is done. Various conditions were studied to illustrate the
catalytic behavior in various media and allowed us to evaluate the efficiency of the catalyst in
heterogeneous conditions.
Acknowledgment:
The authors acknowledge the K. Shair CRSL where GC, AAS, SEM and TGA-FTIR analyses were
performed.
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T5. Electrospinning Nanotechnology: From mathematical model validation
to practical waterproof breathable electrospun
Fouad Junior Maksoud , Nagham Ismail, Nesreen Ghaddar, Kamel Abou Ghali, Ali Tehrani-
Bagha
From Gecko’s aptitude to walk on glass to water droplets rolling off lotus leaf, the secret to
these incredible abilities lies in the nano-structure found on them, thus the importance of Nanoscale
science and technology carried out on the 10-9 meters and applied to produce nano-sized materials
with significantly improved properties of novel structures via electrospinning nanotechnology
which has been increased rapidly in textile manufacturing through fabricating very thin fibers
capable of forming a highly porous mesh characterized by a large surface-to-volume ratio that
improves protection with better comfort.
Conventional protective clothing system subjects workers to heat stress because it impedes the
transmission of water vapor and reduces clothing air ventilation. Current available technology for
breathable fabrics is based on laminated membrane or coating, usually made of ePTFE (expanded
Polytetrafluoroethylene, also known as Teflon®) or PU (Polyurethane). Its main disadvantage is its
high price (e.g. Gortex TM, eVent TM). Then again electrospinning nanotechnology offers cheaper
solution for breathable fabric manufacturing.
In this project, the waterproof and breathability performance of produced electrospun nano-sized
mesh will be studied. Mathematical models will be validated for the electrospinning process to
predict and optimize the effect of the various physical parameters involved in the electrospinning
technology (polymer material and configuration of the electrospinning device) on the produced fiber
diameter, mesh porosity and thickness to obtain the required mesh protection and comfort
properties.
The approach of using electrospun nanofibers for producing an affordable waterproof breathable
fabric in the proposed work may set an example of technical advance to serve the need of sustainable
development.
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T6. Deep Brain Stimulation-Induced Neurogenesis is Gender-Independent
Farah Chamaa 1, Wafaa Sweidan 1, Ziad Nahas 2, Nayef Saade 1, Wassim Abou-Kheir 1
1: Department of Anatomy, Cell Biology 2: Department of Psychiatry
and Physiological Sciences Faculty of Medicine
Faculty of Medicine American University of Beirut
American University of Beirut
Background: Deep brain stimulation (DBS) provides substantial clinical benefits for a variety of
movement disorders and lately emerged as a potential treatment for cognitive and mood disorders.
Regulation of adult hippocampal neurogenesis may play a chief role in mediating DBS effects.
Nevertheless, there exist significant sex differences in the regulation of neurogenesis influenced
mainly by gonadal hormone exposure.
Objective: To investigate the hippocampal neurogenic differences between male and female awake
and unrestrained rats in response to unilateral anteromedial thalamic nucleus (AMN) stimulation.
Methods: Four groups of adult Sprague-Dawley male and female rats received unilateral stimulation
(n=6 each) or sham surgery of electrode implantation with no current delivery (n=4 each) in the
right AMN; A naïve group of males and females (n=4 each) was also included. Rats received 4
injections (50mg/Kg/injection) of 5’-bromo-2’-deoxyuridine (BrdU) 3 days post-surgery and were
euthanized 24h later. The fractionator method was used together with confocal immunofluorescent
analysis to probe for BrdU-, GFAP- and NeuN-positive cells in the dentate gyrus (DG).
Results: Focal neurogenesis was induced in the ipsilateral DG after AMN stimulation. Stimulation-
induced effects were gender-independent and translated into a 76% increase in proliferation of
neural stem/progenitor cells.
Conclusions: Despite the difference in basal neurogenic level between male and female rats, the
current study demonstrates that DBS elicits gender-independent neurogenic effects that might have
implications in the treatment of cognitive and behavioral disorders irrespective of sex of patients.
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T7. Detection of Furfural and 5-hydroxymethyl Furfural in the aerosol of
electronic cigarettes
Sarah Soussy, MS,†,§ Rima Baalbaki, MS,†,§ Ahmad EL-Hellani, PhD,†,§, Rola Salman, MS,‡,§
Alan Shihadeh, ScD,‡,§ and Najat A. Saliba, PhD†,§,*
† American University of Beirut, Lebanon, Chemistry Department, Faculty of Arts and Sciences.
‡ American University of Beirut, Lebanon, Mechanical Engineering Department, Faculty of
Engineering and Architecture
§ Center for the Study of Tobacco Products, Virginia Commonwealth University, Richmond,
Virginia
Funding source: Research was supported by the National Institute on Drug Abuse of the National
Institutes of Health under Award Number P50DA036105 and the Center for Tobacco Products of
the U.S. Food and Drug Administration.
Keywords: Electronic cigarette (ECIG), sweet flavored e-liquids, furan compounds (furfural and
5-Hydroxymethyl furfural (HMF))
Descriptive Statement:
Electronic Cigarette (ECIG) is designed to deliver nicotine, flavorings, water and humectants like
propylene glycol (PG) and/or glycerol (VG) in the form of aerosols. Unlike conventional cigarette,
ECIG uses a heating coil to heat the e-liquid in the absence of combustion. However; the thermal
degradation still delivers different toxicants depending on the chemical composition of the e-liquid.
The aim of this study is to discuss the degradation of sugar additives in ECIG.
Introduction: The diversity of sweet flavorings has been immensely attributed to the use of ECIG
among youth. The composition of sweet e-liquids includes the basic sugar components: glucose,
sucrose and sorbitol which can lead to furans upon heating. In this study the aerosol generated from
prepared sweet e-liquids were assessed for the formation of furan compounds under variable battery
outputs and puff durations.
Methods
Liquids of varying concentrations were prepared by adding aqueous solutions of glucose, sucrose
or sorbitol to a 70/30 PG/VG solution. Aerosols were generated using a commercially available
ECIG operating at 4 and 10 Watts, using puff durations of 4 and 8 s. Aerosols were trapped on filter
pads and extracted and purified using solid phase extraction (SPE) technique. Quantification of
furfural and 5-Hydroxymethyl furfural (HMF) was achieved using a novel gas
chromatography/mass spectroscopy (GC/MS) technique.
Results Furfural and HMF were found in aerosols under all conditions. Furan emissions increased
with sweetener concentration, electric power, and puff duration, and for some conditions the per-
puff yields exceeded values previously reported for combustible cigarettes.
Conclusion The addition of sweeteners to ECIG liquids likely increases ECIG user exposure to
furans. The obtained results call into question the safety of flavors in e-liquids.
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T8. Enhanced Neurogenesis in the Adult Olfactory Bulb is Transient and
Functional in Inducible Rb-Knockout Mice
Saad Omais1, Renaud Vandenbosch2, Sawsan Al-Lafi1 and Noël Ghanem1*
1Department of Biology, American University of Beirut, Beirut, Lebanon 2 GIGA-Neurosciences, University of Liège, Liège, Belgium
Acknowledgements: University Research Board (URB), Lebanese National Council for Scientific
Research (LNCSR), KAS Central Research Science Laboratory.
Keywords: neurogenesis, Rb, olfactory bulb, knock-out mice, survival, olfaction, plasticity
Descriptive Statement: This study investigates the fate and functional impact of an increased
supply of adult-born olfactory bulb neurons following the loss of the Retinoblastoma protein (Rb)
in the brain.
Introduction: Newborn neurons are continuously generated in the adult mammalian brain from
neural stem cells (NSCs) residing in the subventricular zone (SVZ) lining the lateral ventricles and
the subgranular zone (SGZ) in the hippocampus. SVZ-aNSCs generate fast-dividing progenitors
which in turn give rise to neuroblasts that migrate to the olfactory bulb (OB) where they differentiate
into GABAergic interneurons. We have shown recently that Rb controls progenitor proliferation in
the SVZ and the rate of adult neurogenesis whereby loss of Rb triggered enhanced neurogenesis in
the OB at 28 days post-Rb deletion. Despite this, the number of Rb-null newborn neurons gradually
declined to match control levels 3 months later. We have investigated here whether Rb plays a role
in the long-term survival of adult-born neurons and the functional outcomes associated with
enhanced neurogenesis in the OB.
Methods: We induced a temporal deletion of Rb in aNSCs/progenitors using NestinCreERT2;
Rosa26YFP; Rbflox/flox mice, and, retroviral-mediated Cre delivery in the lateral ventricles. We
assessed cell death and neuronal birthdating by immunohistochemistry against active-caspase 3 and
Bromodeoxyuridine (BrdU), respectively. We also performed odor-reward associative learning
tasks and habituation/dishabituation tests following dichlobenil-induced damage to the olfactory
system.
Results: Compared with heterozygous controls, we detected a significant increase in cell death
inside the OB in the absence of Rb in both our transgenic model and the viral-inducible KO (iKO)
model with a peak at 60 days post-deletion in the former model. Moreover, the number of newborn
neurons (YFP+;BrdU+) was severely reduced after loss of Rb. In parallel, the integration of excess
newborn neurons was dependent on the difficulty of olfactory associative task: conditional Rb-null
mice thus performed worse compared to controls when the task involved discrimination between
dissimilar odors but were slightly better discriminating between similar odors. Finally, the rate of
perceptual recovery was similar between groups four weeks after dichlobenil treatment. We are
further looking at the effect of dichlobenil induced damage on olfactory associative tasks in Rb iKO
mice.
Conclusion: Rb is required for the long-term neuronal survival of adult-born OB neurons. The
transient increase in OB neurogenesis observed in the absence of Rb seems to be functionally
significant in difficult olfactory discrimination tasks but not after indirect lesion to OB circuit. This
study holds a promising therapeutic potential for neurodegeneration.
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T9. Tbx5 the missing culprit gene in thalidomide toxicity
Athar Khalil1, Nehmé El-Hachem1, Fadi Bitar1,2, and Georges Nemer1
Departments of 1Biochemistry and Molecular Genetics, and 2Pediatrics and Adolescent Medicine,
American University of Beirut, Beirut, Lebanon
Congenital heart disease (CHD) is the leading cause of death in the first year of life affecting
approximately 1% of life births. Only 13 % of all CHD cases are thought to be inherited, and the
rest are sporadic in nature. Mutations in few genes encoding cardiac-enriched proteins have been
only linked to few cases of CHD whereas most of the cases are still with no know etiologies. In
addition, some CHD are caused by environmental factors and teratogens like thalidomide.
Thalidomide is a sedative drug that was used by pregnant women for morning sickness but was
removed from the market in 1961 because it caused severe malformations in newborns similar to
the Holt-Oram Syndrome (HOS) patients, which is characterized by severe cardiac and limb
malformations. Previous studies showed that the mRNA levels of TBX5, the culprit gene in HOS
were reduced in wing buds of chicken embryos after exposure to Thalidomide.
We aimed at investigating the effect of Thalidomide on TBX5 expression and function in vitro in
order to establish its causative role in cardiac and limb malformation. By using electric mobility
shift assay (EMSA) we showed that thalidomide decreased the binding affinity between TBX5
protein and a consensus sequence of T-box up to 40%. While thalidomide didn’t affect the cellular
localization or the protein stability of TBX5 as indicated by immuno-fluorescence and western blot
respectively. Suppressed expression activity of vascular endothelial growth factor (VEGF) and
atrial natriuretic factor (ANF) promoter was obtained in the presence of thalidomide assessed by
luciferase assay. While thalidomide was neither able to suppress the interaction of TBX5 with
GATA4 presented by VEGF promoter expression, nor affected this interaction on the protein level
as shown by co-immunoprecipitation assay. Indeed in silico assessment using the Autodock-Vina
software showed that Thalidomide binds readily to TBX5 mainly through three amino acids R81,
R81, and K226 all implicated in DNA binding., and ClusPro algorithm did show that TBX5
interacts with GATA4 via different amino acids in the T-Box domain than the ones implicated in
DNA binding. Moreover Thalidomide inhibited drastically the physical interaction of TBX5 with
HAND2 while the in silico docking experiments revealed that the same amino acids involved in the
interaction of TBX5 with the DNA are also involved in its binding to HAND2.
Our results were the first to show a direct effect of thalidomide on Tbx5 which affected the
heart and limb.
Funding source: Medical Practice Plan, University Research Board- AUB/ CEDRE
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Posters Abstracts
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P1. Towards an Ultrasensitive Detection of Traumatic Brain Injury
Biomarkers
Samir Abou Shaheen 1, Firas Kobaissy, 2 and Pierre Karam1 1Department of Chemistry, Faculty of Arts and Sciences,
2Department of Biochemistry and Molecular Genetics, Faculty of Medicine
American University of Beirut, Beirut, Lebanon
[email protected] [email protected]
Funding source: Farouk Jabre Biomedical Research Grant
Introduction: The call for an early and consistent detection of traumatic brain injury is escalating
as the number of victims, caused mainly by motor vehicle use, is dramatically increasing. Therefore,
having an ultrasensitive testing device for protein biomarkers will facilitate a more routine clinical
assessment for molecules that are signs of the disease. As such, we aim at developing a biosensor
platform based on electrochemical and fluorescent methods that would allow us to detect trace
amount of biomarkers specific for TBI.
Methods: The electrochemical scheme includes fabrication of nanostructured gold micro-
electrodes using amperiometry, functionalization of antibodies specific for TBI biomarkers and
building of a sandwiched assay of antigen-antibodies coupled with silica nanoparticles. The sensing
strategy depends on monitoring the current at each step using differential pulse voltammetry.
Whereas, the fluorescent scheme includes the use of gold-coated polystyrene beads,
functionalization of antibodies specific for TBI and building of a sandwiched assay of antigen-
antibodies coupled with labeled liposomes. The sensing strategy depends on monitoring the
fluorescence shift using flow cytometry.
Results: We succeeded in getting high sensitivities of antigen concentration (ng/ml) and low signal-
to-noise ratios.
Conclusion: We have reached an advanced stage in the development of an ultrasensitive approach
for the detection of traumatic brain injury.
Acknowledgment: The Flow Cytometry
measurements and SEM imaging were done in Kamal A. Shair Central Research Science
Laboratory (KAS CRSL).
Figure 2.Differential Pulse Voltammogram
showing the signal decrease after each
incubation
Figure 1. SEM images of
nanostructured-gold
microelctrode
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P2. Exploring Arylpyrenes in Blue Organic Light-Emitting Diodes
Tarek H. El Assaad and Bilal R. Kaafarani
Department of Chemistry, American University of Beirut, Beirut 1107-2020, Lebanon,
[email protected], [email protected]
The functionalization of pyrene through Suzuki coupling reaction to form tetrarylpyrenes is
explored. The detailed photophysical, electrochemical, and thermal properties of a series of nine
tetrarylpyrenes is presented. Furthermore, the X-ray structures and packing of six of these
tetrarylpyrenes are discussed. The application of these compounds in single-layer geometry devices
is outlined.
The authors would like to acknowledge the KAS CRSL for providing part of the equipment needed
for this work including NMR, UV-VIS and a spectrofluorometer.
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P3. Modulation of Liposomes Properties by Rhamnolipids: Curcumin as
Membrane Probe to Study Phase Transition Temperature
Zeinab Moussa and Digambara Patra*
Department of Chemistry, American University of Beirut, Beirut, Lebanon
Curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a polyphenol
present in tumeric, a spice extensively used food preservative, coloring agent and Asian traditional
medicine. Broad scale of curcumin as anti-oxidant, anti-carcinogenic, anti-mutagenic and anti-
inflammatory agent makes it predominantly interesting for the advancement of pharmaceutical
compounds. However, poor water solubility of curcumin creates its in vivo administration a very
complicated assignment. Coincidentally over the last years modern drug carriers, like liposome,
have demonstrated successful for delivery of lipophilic molecules. The physiochemical properties
of liposome and their interaction with drug/guest molecules are extremely crucial because it
describes the pharmacokinetic behavior and pharmacological response of the system. In this study
we have explored interaction of Rhamnolipids with phospholipids membrane implicating curcumin
as a fluorescent probe. Lipid membranes are known to exist in different phase depending on the
temperature. The effect of Rhamnolipids on the phase transition temperature of phospholipids from
gel to sol phase was determined by measuring the fluorescence intensity of curcumin. The impact
of RLs on the permeability and fluidity of the phospholipid membranes was investigated by
studying the quenching of curcumin by CPB and comparing it with pyrene, a well-established
probe.
Keywords: Curcumin; Phospholipids; Rhamnolipids; Liposomes.
Acknowledgement
Financial support provided by American University of Beirut, Lebanon through URB, Kamal A.
Shair Research Fund as well as Kamal A. Shair Central Research Science Laboratory (KAS CRSL)
facility to carry out this work is greatly acknowledged.
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P4. Tuning Selectivity and Sensitivity of Fluorescence Probe by
Nanocapsules for Non-Enzymatic Determination of Cholesterol
Mazhar Chebl and Digambara Patra*
Department of Chemistry, American University of Beirut, Beirut, Lebanon
Modern cholesterol determination are based on enzymatic method that requires fixing
cholesterol esterase on the surface of an electrode and measuring the oxidation or reduction current
of hydrogen peroxide generated, but this method is limited by the ascorbic and uric acid that can
interfere. Curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a
polyphenol present in turmeric, works as an anti-oxidant, anti-carcinogenic, anti-mutagenic and
anti-inflammatory agent. Other applications of curcumin include its use as a fluorescent molecule
to study membrane phase transiTon, bio-sensing and a reducing agent. In this study, curcumin is
integrated to a chitosan-silica aggregate, here referred as nanocapsules, to enhance selective
estimation of cholestrol. The nanosensor explicitly enhances analytical specificity of cholesterol
estimation without using any enzymatic reaction. The analytical selectivity in the presence of other
foreign substances such as ascorbic acid, uric acid, etc. has been tested. Even interference from
metal ions, which are well known fluorescence quencher for curcumin could be avoided because
association of chitosan oligosaccharide lactate blocks the keto-enol (or diketo) group of curcumin
that is responsible of metal ion binding. Moreover this method has shown a broad dynamic range
with a limit of detection and quantification similar to that reported in literature.
Keywords: Curcumin; Nanosensor; Cholesterol; Chitosan; Fluorescence
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P5. Study of the combined roles of p53 and Rb in the control of adult
neurogenesis in vitro
SALIBA Afaf1, AL LAFI Sawsan1, Jaafar Carine1, Raya Saab2 and GHANEM Noël1*
1Department of Biology, American University of Beirut, Beirut, Lebanon 2Department of Pediatric and Adolescent Medicine, AUBMC, Beirut, Lebanon
Funding source: Kamal Shair (KS) grant
Keywords: p53, Rb, adult neurogenesis, neural stem cells, neurosphere, proliferation,
differentiation.
Descriptive Statement: This study investigates the combined roles of two tumor suppressor genes,
p53 and Rb, on the regenerative capacity and fate of adult neural stem cells (aNSCs) in culture.
aNSCs are restricted in number and generate specific types of neurons at a relatively low rate.
Identifying the mechanisms that control their development and expansion will help improve the
regenerative capacity of the brain in case of injury or neurodegenerative diseases.
Introduction: Adult neurogenesis is highly regulated process that is restricted to aNSCs found in
the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) lining the lateral
ventricles in mammals. SVZ-aNSCs have unlimited self-renewal capacity and give rise to
GABAergic interneurons in the olfactory bulb. This process requires a fine control and balance
between cell proliferation and cell death. Loss of p53 was previously shown to enhance the self-
renewal capacity and the rate of differentiation of aNSCs both in vivo and in vitro. Moreover, we
have recently demonstrated that Rb specifically regulates progenitor proliferation and is needed for
the long-term survival of adult-born OB interneurons. Given the above findings, we have examined
here how both genes function together to control the developmental properties of aNSCs and
progenitors in vitro.
Methods: We induced a temporal deletion of Rb (conditional Knock-out, cKO) in
aNSCs/progenitors in p53 null mice (2 months old) carrying the Nestin-CreERT2-YFP cassette and
performed primary cultures of dissected SVZ tissues from p53-null and RbcKO;p53null mice. We
then sorted by FACS the green neurospheres at the second passage and conducted neurosphere and
differentiation assays.
Results: Compared with p53-null cultures, aNSCs/progenitors derived from Rb-cKO; p53null
cultures showed a 3-3.5 fold increase in their self-renewal capacity and amplification rate. Thus,
they generated more primary and secondary neurospheres and seem to have retained a strong
differentiation potential without excessive cell death.
Conclusion: The population of aNSCs/progenitors can be expanded in vitro by manipulation of the
p53/Rb pathways without affecting aNSCs differentiation potential. However, we are presently
testing this in vivo and whether long-term survival of adult-born neurons is affected by the dual loss
of these genes.
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P6. p53 and Rb are indispensable for normal kidney development in mice
JAAFAR Carine1, Zalzali Hassan2, AL LAFI Sawsan1, SAAB Raya2 and GHANEM Noël1*
1 Department of Biology, 2Department of Pediatric and Adolescent Medicine, American
University of Beirut, Beirut, Lebanon
Funding source: Kamal Shair (KS) grant (CRSL, AUB)
Keywords: kidney, development, Rb, p53, renal failure
Descriptive Statement: In this project, we have investigated the combined roles of two tumor
suppressor genes, Rb and p53, in kidney development and found that the dual loss of these genes
leads to several morphological and functional defects during kidney development in mice.
Introduction: The kidneys have a vital homeostatic role in the excretion of nitrogenous waste
products and regulation of blood composition in mammals. Renal development occurs between
embryonic day (E) 8.5 and post-natal day (P) 2. The Rb and p53 pathways are master regulators of
cell division and senescence in many organs. Previous studies have also demonstrated that p53 is
required for early renal development particularly during nephrogenesis, and, metanephroi
differentiation at later stages. p53 knockout mice hence exhibit abnormal metanephric and uterine
development, however, this effect is strain-specific and not embryonic lethal. In contrast, the role
of Rb in kidney development has not been addressed to date. We have recently generated an
inducible deletion of Rb in p53-null mice during development that resulted in perinatal lethality
with almost complete penetrance due to severe kidney failure.
Methods: We have used an inducible Nestin-CreERT2-YFP system to conditionally delete Rb in
p53-null (-/-); Rb flox/flox mice. Nestin is an intermediate filament protein specifically expressed
in neural precursors in the brain, and, in mesodermal cells and theirs derivatives in the developing
kidney. Few animals only survived the dual loss of Rb and p53 at E18.5 but not earlier, and survived
till P40. To characterize the morphological and developmental defects in these animals, we have
performed: 1) histological analysis using Hematoxylin and Eosin staining, and 2) immunostaining
to examine the expression of key developmental genes in the kidney including the podocyte-specific
marker, Nestin and the differentiation marker, NeuN.
Results: We compared the kidney phenotypes in p53-/-; Rbflox/flox mice treated with tamoxifen
or vehicle only (p53-/- as controls). Our results revealed the presence of severe renal developmental
defects manifested by the presence of hypoplastic kidneys with dilated renal tubules and possible
glomerular hypertrophy as well as severe kidney failure as indicated by a 5-6 fold increase in blood
creatinine levels.
Conclusion: Rb and p53 are required for kidney development and control critical developmental
pathways that are indispensable for proper renal morphogenesis and function.
22
P7. HIV Rev mutant R35G-N40V recognizes HIV RNA RRE IIB using
different interactions than wild-type Rev.
Nicole Raad and Colin Smith
Department of Biology, American University of Beirut,
[email protected], [email protected]
The human immunodeficiency virus (HIV) mediates nuclear export of its transcripts, a
crucial step for viral replication, through the binding of the arginine-rich motif (ARM) of its Rev
protein to the Rev-response element (RRE). The Rev ARM double-mutant R35G-N40V binds
RRE’s stem loop region IIB, in spite the absence of critical asparagine 40 hydrogen bonds. Previous
studies also demonstrate the ability of this mutant to bind distinct RRE IIB variants, indicating that
35G-40V must use a distinct recognition strategy, yet no structural insight has been presented. To
elucidate this putative mechanism, important 35G40V residues were sought using scanning
mutagenesis. Through a plasmid-based lambda N-nut antitermination reporter system that reports
on ARM-RNA recognition, libraries of 35G40V residues were screened and distinct positions were
found to be important. Characterization of these important residues paves the way for structural
models governing the 35G-40V interaction. Together, these results support neutral theories of
evolution, and shed light on how macromolecules evolve new recognition strategies.
23
P8. Mechanisms Implicated in the Enhanced Sensitivity of MDA-MB-231
Breast Cancer Cells to Anti-neoplastic Agents Post Sub-lethal HIFU Exposure
Sara Assi1, Wadih Khoury2, Ingrid F. Younes1, Mohamad Mahdi Sleiman1, Hussein Daoud2,
Ghanem Oweis2, Nisreen Alwan3, Diana E. Jaalouk1†
1Department of Biology, Faculty of Arts & Sciences; 2Department of Mechanical Engineering,
Faculty of Engineering & Architecture, American University of Beirut, Beirut, Lebanon; 3School
of Health Sciences, Modern University for Business & Science
Funding & facility support: Dar Al Handassah Endowment Fund, FEA, AUB; AUB’s University
Research Board (URB) Fund; TWAS-COMSTECH, Trieste, Italy, K.A. Shair CRSL Research Lab
Keywords: HIFU, Breast Cancer, Chemotherapy, Caveolin
High Intensity Focused Ultrasound (HIFU) is a non-invasive and non-ionizing therapeutic
method that can be utilized to destroy a variety of solid tumors by destroying tissues that are deep
inside the body. At the focal point where the acoustic waves are intensified, cell death can result
from cavitation and/or thermal ablation effects. However, the effects of sub-lethal HIFU exposure
on cell function are not clearly understood. Previous work from our laboratory showed that sub-
lethal HIFU exposure of MDA-MB-231 breast cancer cells in vitro results in significant alterations
in transcript expression of a number of mechanosensitive genes, including Cav-1 gene which
encodes for caveolin-1 protein. Moreover, there was enhanced cellular sensitivity to suboptimal
cytotoxic doses of Paclitaxel and Doxorubicin. The objective of this study is to identify the
mechanisms implicated in the enhanced in vitro sensitivity of MDA-MB-231 cells to anti-neoplastic
agents post sub-lethal HIFU exposure. We rationalized that sonoporation and/or caveolin-
dependent endocytosis are involved in this enhanced in vitro sensitivity of MDA-MB-231 cells post
sub-lethal HIFU. We utilized a commercial HIFU setup that operates at the fundamental resonance
of 0.5MHz. To examine if sonoporation is implicated in the enhanced drug uptake post sub-lethal
HIFU, FITC-dextran uptake exposure to sub-lethal HIFU was assessed by two methods: cell
fixation followed by flow cytometry or laser confocal microscopic imaging and analysis. To
determine if caveolin-dependent endocytosis was implicated as a mechanism of enhanced drug
uptake, we applied pre-treatment with Genistein, a specific potent inhibitor of this pathway. Cellular
viability was quantified using trypan blue vital stain exclusion assay. As a result, we found no
significant change in FITC-dextran uptake in MDA-MB-231 cells post sub-lethal HIFU exposure
at 30hr prior to the in vitro addition of agents by flow cytometry and laser confocal microscopy.
Likewise, no significant change in FITC-dextran uptake was noted at the 6hr time point by laser
confocal microscopy. Interestingly, pre-treatment with Genistein resulted in a significant increase
in cellular viability in comparison to control group. Our findings indicate that sonoporation does
not seem to play a significant role in enhanced drug uptake post exposure of MDA-MB-231 breast
cancer cells to the sub-lethal HIFU levels that we applied, whereas caveolin-dependent endocytosis
is implicated in this process. Work is underway to validate the latter results using various levels of
exposure and time points.
24
P9. Proteomic Profiling of Nuclei from Lamin A/C-Deficient Mouse Embryo
Fibroblasts by Differential Phage Display Screens
Hind Zahr1, Heinrich zu Dohna1, Jan Lammerding2, and Diana E. Jaalouk1†
1 Department of Biology, Faculty of Arts & Sciences, American University of Beirut, Beirut,
Lebanon; 2 Meinig School of Biomedical Engineering & Weill Institute for Cell and Molecular
Biology, Cornell University, Ithaca, New York † [email protected]
Funding & facility support: AUB’s University Research Board (URB), Lebanese National
Council for Scientific Research (CNRS), K.A. Shair CRSL Research Fund, K.A. Shair CRSL
Research Lab
Laminopathies are a group of genetic disorders including skeletal and cardiac muscular
dystrophy. They are caused by mutations in the LMNA gene which encodes for the nuclear lamina
proteins lamin A/C that anchor other nuclear envelope (NE) proteins to the nuclear membrane. To
date, the molecular mechanisms underlying the phenotypic diversity and the tissue-specific
impaired function in laminopathies have not been deciphered. We rationalized that the phenotypic
and mechanistic differences seen between laminopathies may result from varied expression,
localization, and/or impaired function of a number of nuclear interacting proteins mediated by their
interactions, or lack thereof, with wild-type or mutant lamin A/C resulting in differential
deregulation in critical signaling pathways that are tissue-specific. To address this hypothesis, we
used a phage display - based approach to map nuclear– associated proteomic heterogeneity in the
context of laminopathies. We successfully employed a phage display - based technology termed
Bio-panning and Rapid Analysis of Selective Interactive Ligands (BRASIL) for proteomic profiling
of nuclei isolated from either wild-type (WT) mouse embryo fibroblast (MEF) cells or lamin
A/Cdeficient MEFs. Successive rounds of bio-panning showed significant enrichment in
differential phage binding to nuclei expressing WT A-type lamins as determined by a 2.4-fold
change in relative phage binding units in Round III. No significant enrichment in differential phage
binding to nuclei lacking A-type lamin expression was obtained. Direct phage display bio-panning
performed without pre-clearing the library also showed significant enrichment in phage binding to
nuclei expressing WT A-type lamins and nuclei lacking A-type lamins with a 2.3- and 2-fold
increase in relative phage binding respectively. Nearly 200-300 phage plaques were selected from
various rounds per screen, PCR amplified, and inserts sequenced. Multiple high frequency peptides
were obtained with preferential binding to either type of nuclei. Bioinformatic analysis indicates
differential clustering of enriched peptides. Analysis is underway to identify the natural proteins
that are mimicked by the binding peptides with matching motifs. Hits identified in this project will
offer new insights into the molecular mechanisms responsible for the phenotypic complexity of
laminopathies including a better understanding of the causes for debilitating diseases such as dilated
cardiomyopathy and Emery-Dreifuss muscular dystrophy.
25
P10. Insights into the Deregulation of Rbm20 in Lamin A/C and Emerin
Related Cardiomyopathies
Dana Sedki1, Hind Zahr2, Dima Diab El Harakeh2, Georges Nemer1†, Marwan Refaat1†, and Diana
E. Jaalouk2† 1Department of Biochemistry and Molecular Genetics, Faculty of Medicine; 2Department of
Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon †[email protected]; [email protected]; [email protected]
Cardiomyopathies are among the leading causes of premature sudden death. Their etiology
is genetically heterogeneous with more than 50 genes linked to them. The most substantial
mutations involved in the cardiac phenotypes are those affecting the integrity and structure of the
nuclear lamina; LMNA gene coding for Lamin A/C, and EMD gene coding for the inner nuclear
membrane (INM) protein emerin. Additionally, recent studies identified mutations in the RBM20
gene coding for the intracellular RNA-binding protein as highly implicated in familial
cardiomyopathies. We sought to get a better understanding of how Emery-Dreifuss Muscular
Dystrophy (EDMD) and Dilated Cardiomyopathy (DCM) originate from deficiency and/or
mutations in the LMNA and EMD genes. Accordingly, we aimed to investigate potential
deregulations in Rbm20 transcript and protein expression in addition to intracellular localization in
the context of Lmna and Emd deficiency. For the purpose of this pilot study, we used mouse embryo
fibroblast (MEF) lines that were derived from mice lacking the expression of either Lamin A/C
(Lmna-/-) or emerin (Emd-/Y) which have an EDMD phenotype, or mice expressing the Lmna N195K
homozygote mutation (LmnaN195K/N195K) which have the DCM phenotype versus wild-type (WT)
controls, under baseline conditions. We have also used Lmna null MEFs that were transduced by
retroviral infection to re-express the Lmna WT or different mutant forms that result in EDMD
(E358K, L530P). Real Time PCR quantification, Western Blot analysis, and immunofluorescence
staining were performed on these cell lines to test for alterations in Rbm20 transcript or protein
expression and intracellular localization. Rbm20 showed a significant reduction in the transcript
levels in all the three mutant MEFs which was reversed upon re-expression of Lmna confirming the
direct effect of lamina disruption on the expression of Rbm20. Likewise, the protein expression of
Rbm20 was significantly reduced in the mutant cell lines compared to the wildtype, while there was
no significant alteration in its intracellular localization. Taken together, our findings highlight the
implication of Rbm20 in lamin A/C and emerin related cardiomyopathies. Ongoing work and future
directions will focus on investigating the consequential aberrations in RBm20 – mediated splicing
of a number of targets that mediate key signaling pathways altered in these diseases.
26
P11. Uptake, Delivery and Anticancer Activity of Thymoquinone
Nanoparticles in Breast Cancer Cells
Isabelle Fakhoury1, Walid Saad2, Kamal Bou Hadir3, Peter Nygren4, Regine Schneider-Stock5,
and Hala Gali-Muhtasib1,6
1Department of Biology, 2Department of Chemical and Petroleum Engineering, , 3Department of
Chemistry, American University of Beirut, Beirut, Lebanon, 4Department of Immunology,
Genetics and Pathology, Experimental and Clinical Oncology, Department of Medical Sciences,
Cancer Pharmacology and Computational Medicine, Uppsala University, Sweden, 5Experimental
Tumor Pathology, Institute for Pathology, University Erlangen-Nuremberg, Germany, 6
Department of Anatomy, Cell Biology, Physiology, Faculty of Medicine, American University of
Beirut, Beirut, Lebanon
IF: [email protected], WS: [email protected],
KB: [email protected], PN: [email protected],
RS: [email protected], HM: [email protected]
Background and aims: Thymoquinone (TQ) is a promising anticancer molecule but its
development is hindered by its limited bioavailability. Drug nanoparticle formulation is commonly
used to overcome low drug solubility, limited bioavailability, and nonspecific targeting. This
project aimed at synthesizing different TQ nanoparticles (TQ-NP), characterizing them, and
assessing their uptake and delivery mechanisms, as well as their anticancer potential in a panel of
breast cancer cells.
Methods: TQ-NP were prepared by Flash nanoprecipitation. Dynamic light scattering and scanning
electron microscopy were used for the characterization of the size, morphology and stability of the
NPs. The anticancer activity was assessed by MTT. The uptake and subcellular intake mechanism
of fluorescent TQ-NP were evaluated by both fluorometry and confocal microscopy.
Results: Four different TQ-NPs were formulated. The average diameter size ranged between 45-
130 nm. All TQ-NPs were stable and had high entrapment efficiency (75-80%) and loading content
(36-50%). In vitro, TQ-NP had equal or enhanced anticancer activity effects compared to TQ, in
MCF-7 and aggressive MDA-MB-231 breast cancer cell lines. No significant cytotoxicity of the
blank NP was noted. The uptake of fluorescent TQ-NP occurred in a time and concentration-
dependent manner. Furthermore, treatment with inhibitors of endocytosis revealed the involvement
of caveolin mediated endocytic pathway in TQ-NP uptake. This was also confirmed by subcellular
localization findings, showing the colocalization of TQ-NP with both caveolin and transferrin, as
well as with the early and late markers of endocytosis, EEA-1 and lamp-1 proteins.
Conclusion: Altogether, the results describe an approach for the enhancement of TQ anticancer
activity and uncover the mechanisms behind cell-TQ-NP interaction, uptake and biodistribution.
27
P12. TPEN induces DNA damage in human colon cancer cells: Role of
Chk1/2 and DNA-PK
Omar Rahal1, Maamoun Fatfat1, Carla Hankache1, Bassam Osman1, Hala Khalife2, Khaled
Machaca3, Hala-Gali Muhtasib1*
1Department of Biology, American University of Beirut, Lebanon,2Department of Biology,
Lebanese University, Lebanon, 3Department of Physiology and Biophysics, Weill Cornell Medical
College, Qatar
Key words: Metal chelation; redox cycling; copper; ROS; anticancer
In many cancers, the levels of zinc and copper are elevated. Recently, we showed that the
transition metal chelator TPEN selectively targets colon cancer cells through the redox cycling of
copper. In this study, we investigated if TPEN induces DNA damage and deciphered the role of
Chk1 and DNA-PK in TPEN-induced toxicity in HCT116 human colon cancer cells. In addition,
we determined the role of reactive oxygen species (ROS) and copper redox cycling in TPEN-
induced DNA damage. We found that cell death by TPEN is associated with significant DNA
damage, an effect that was dependent on ROS generation and on the redox cycling of copper, as
evidenced by reversal of DNA damage in the presence of the antioxidants N-acetyl-L-cysteine and
catalase, and the copper chelator Neocuproine. DNA damage by TPEN increased the expression
levels of ɣ-H2AX protein and activated the ATM/ATR signaling molecules p-ATM, p-ATR and p-
Chk1. The pre-incubation with specific inhibitors of ATM and DNA-PK led to the recovery of cells
from TPEN-induced DNA damage. In addition, siRNA silencing of Chk1 and DNA-PK abrogated
the expression levels of ɣ-H2AX and reversed cell death by TPEN, suggesting that Chk1 and DNA-
PK mediate TPEN-induced cytotoxicity in HCT116 cells. This study shows for the first time the
involvement of Chk1 and DNA-PK in TPEN-induced DNA damage and confirms our previous
findings that ROS generation and the redox cycling of copper in response to TPEN are the main
mechanisms by which this compound induces cell death in human colon cancer cells.
We would like to acknowledge Dr. Mouneimne at the KAS CRSL for providing us access to the
instruments to complete our study.
28
P13. Effect of Endotoxin Challenge on Normal, Tumor Initiated, and Invasive
Human Breast Cells
Farah Yassine, Sabreen Fostok, Nataly Naser Al Deen and Rabih Talhouk
Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Lebanon
Breast cancer is the most common female cancer worldwide, recording high incidence rates
caused by risk factors associated with urbanization and economic development. Inflammatory
breast cancer (IBC) is the most lethal type of breast cancer, targeting young women, mainly after
chronic inflammation. Interestingly, endotoxin (ET) is known to simulate inflammation-like
conditions in several in-vitro and in-vivo models including mammary epithelial cells; however, little
is known about the effect of ET-induced inflammation on breast cancer initiation events, In order
to investigate the tumor initiating role of ET-induced inflammation, we monitored inflammatory
mediators’ response and cancer progression events of in-vitro 2D and 3D breast progression models
of both, non-tumorigenic S1 cells, and intermediate stage of tumorigenesis S1-Connexin 43
knockouts (S1-Cx43KO), that can closely mimic the in-vivo mammary epithelial morphology. 2D
cell cultures of normal mouse mammary epithelial cells (SCp2), moderately invasive MCF-7, and
highly invasive MDA-MB-231 human breast cancer cells were exploited in order to determine the
effect of ET treatment on tumor invasion events.
The inflammatory mediators: nitric oxide (NO) and interleukin 1-β (IL1-β), measured using
colorimetric assays and Elisa, as well as matrix metalloproteinases (MMPs) assayed by
Zymography, were upregulated in MDA-MB-231 culture supernatants upon ET-induced
inflammation. An ET treatment for one month prior to performing any assay accelerated the
proliferation rate of MCF-7 and MDA-MB-231 cells as shown by cell count and wound healing
assays. Cell proliferation of normal mammary epithelium, (SCp2 mouse mammary cells) was
enhanced upon long term treatment. ET also increased the levels of MMPs in 2D cultures of normal
non-neoplastic S1 human breast epithelial cells, and slightly initiated S1-Cx43 KO. Immuno-
fluorescent assays investigating ET effect on lumen and polarity disruption events in 3D (matrigel)
cultures are still in progress. Epithelial-to-mesenchymal (EMT) markers will also be monitored
through both, RNA analysis via RT-PCR and protein analysis by western blots.
Consequently, endotoxin-induced inflammation enhanced inflammatory mediators’ response and
tumor progression in normal, tumor-initiated and cancer breast cell lines. Our findings highlight the
role of inflammatory insult on breast cancer initiation events in normal breast cells and whether
such insults can “add-injury” to an already tumor-initiated or already invasive breast cells. Our aim
is to identify inflammatory signals driving early cancer development which can be used as targets
for cancer immune prevention as well as emerging biomarkers for cancer screening and early cancer
detection.
Funding Source: Kamal A. Shair CRSL Research Fund 2015
Acknowledgement of the KAS CRSL Contribution:
The cell culture facility, the fluorescent microscope, Xomat machine needed for western blotting
X-ray film development, the Nanodrop for RNA quantification, the reverse transcription PCR
machine as well as the Real Time PCR machine that are all found in the K.A. Shair CRSL laboratory
and are being used for the performed experiments.
29
P14. The Vasorelaxant Effect of Marjoram in Rat Thoracic Aorta is mediated
Via a PI3-K/Akt/eNOS/cGMP Pathway
Khodor Issa1, Manal Fardoun2, Alaaeldin I. Saleh3, Ali Samaha4, 5, Rabah Iratni6,Elias Baydoun2
Ali H. Eid1
1Department of Pharmacology and Toxicology, American University of Beirut, 2Department of Biology, American University of Beirut
3College of Medicine, Qatar University 4Biomedical Sciences, Lebanese International University, Beirut, Lebanon,
5Faculty of Public Health IV, Lebanese University, Lebanon; 6Department of Biology, United Arab Emirates University, Al Ain, United Arab Emirates.
[email protected]; , [email protected]; [email protected],
[email protected]@liu.edu.lb
Introduction: Cardiovascular disease (CVD) is the leading cause of morbidity and mortality
worldwide. Hypertension remains a major contributor to CVD-associated mortality, despite the
significant therapeutic advances made. This mandated that alternative approaches, such as herbal
medicine, be sought. Although the medicinal value of marjoram, Origanum majorana, is
recognized, its vasculoprotective effects remain poorly investigated. Here, we investigated the
mechanisms underlying the effects of ethanolic extract of marjoram on rat thoracic aortas.
Methods: Thoracic aortic rings were isolated from Sprague-Dawley rats, cleaned of adipose and
adventitial tissue, and set-up in an organ bath system. Noradrenaline (3µM) was used to preconstrict
the vessel segments. Aortic rings were subjected to increasing doses (cumulative) of an ethanolic
extract from leaves of Origanum majorana (OME) in the absence or presence of several different
signaling inhibitors. cGMP levels were also quantified.
Results: In endothelium-intact rings, OME evoked significant (P<0.01) relaxation in a dose-
dependent manner (25µg-1mg/ml). The maximum arterial relaxation (EMax) was significantly
reduced by approximately 45% in endothelium-denuded rings. Pretreatment with L-NAME (a non-
selective inhibitor of nitric oxide synthase, 100µM), or ODQ (an inhibitor of soluble guanylyl
cyclase, sGC, 1µM) of endothelium-intact rings significantly reduced the OME-induced
vasorelaxation by about 40%. OME was also found to increase cGMP content in these vessels.
Importantly, preincubation with wortmannin (0.1µM) , an inhibitor of PI3-K, significantly (p<0.05)
reduced the OME-induced vasorelaxation, where EMax was reduced by approximately 55% .
Preincubation with glibenclamide or tetraethylamonium, blockers of ATP-sensitive or Ca++-
activated potassium channels respectively, did not significantly affect the OME-induced relaxation
(P>0.05). Similarly, treatment with verapamil, a Ca++ channel blocker, or indomethacin, a non-
selective cyclooxygenase inhibitor, did not significantly alter the observed relaxation (P>0.05).
Conclusion: Taken together, our results show that OME induces vasorelaxation via an
endothelium-dependent mechanism. OME appears to elicit its effects by activating the PI3-
K/Akt/eNOS/cGMP pathway. However, the induced relaxation is neither dependent on calcium or
potassium channels nor on the cyclooxygenase pathway. Our findings further support the medicinal
value of marjoram and provide a basis for its beneficial intake. Although consuming marjoram may
have an antihypertensive effect, further studies are needed to better determine the effect of OME
on different vascular beds.
30
P15. Modulation of hippocampal neurogenesis by inflammatory and anti-
inflammatory agents
Lynn Bitar, Farah Chamaa, Nayef Saade and Wassim Abou-Kheir
[email protected]; [email protected]; [email protected]; [email protected]
Neurogenesis is the process of generation of functional neurons from neural stem cells and
progenitor cells. This process continues in postnatal stages yet becomes spatially restricted under
regular conditions to two main neurogenic brain regions; the subgranular zone (SGZ) in the dentate
gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Adult neurogenesis is prone to
alterations by different physiological, pathological and pharmacological stimuli. One flaunting
stimulus is acute or chronic neural inflammation which has been implicated in neurodegenerative
disorders. Subsequently, attempts to reverse inflammation using anti-inflammatory agents may
serve as a possible intervention. Our results reveal an effect of inflammation induced by
Lipopolysaccharide on the behavioral and neurogenic niche of adult rats by decreasing neurogenesis
thus constituting a platform for plausible therapeutic approaches of neural inflammatory disorders.
31
P16. Whole-exome sequencing in Lebanese families identifies novel causes of
autism spectrum disorder
Dikran Richard Guisso 1, Jihane Soueid1, Maria Chahrour2, Rose-Mary Boustany1 1 Departments of Pediatrics and Adolescent Medicine, Biochemistry and Molecular Genetics,
AUBMC Special Kids Clinic, Neurogenetics Program and Division of Pediatric Neurology,
American university of Beirut, Lebanon. 2 Eugene McDermott Center for Human Growth &
Development, Neuroscience, Psychiatry, UT Southwestern Medical Center, Dallas, TX
Funding source: The work is supported by a generous grant from OpenMinds.
Keywords: ASD; Whole-exome sequencing; GABRB2; HUWE1
Descriptive Statement: By using whole exome sequencing, we identified two novel mutations in
genes implicated in neurodevelopmental disorders, but new to autism.
Introduction, background and aims. Autism spectrum disorders (ASDs) are a group of
neurodevelopmental disorders with high heritability. In Lebanon, ASD prevalence in the greater
Beirut and Mount Lebanon areas is 1/66 children (Chaya et al, 2015 JADD). Recent findings by
our group (Soueid et al, Scientific Reports, NPG, Jan 2016) support a highly heterogeneous genetic
etiology, including rare de novo and inherited mutations, chromosomal rearrangements, as well as
double hits.
Methods: We performed whole-exome sequencing (WES) in 4 families having 1 or 2 affected
children in order to identify novel causative genes and autism susceptibility variants. Validation of
WES results was accomplished by Sanger sequencing in the families and in normal Lebanese
controls (n=104).
Results: We identified 2 potentially causative genes in 2 out of 4 families analyzed. These variants
are intronic substitutions in GABRB2 and HUWE1 genes, less than 100 base pairs away from the
exon. They might alter splicing sites, or transcriptional regulatory elements causing a dysregulation
of the pre-mRNA splicing process and dysfunction at the protein level. Mutations were successfully
validated by Sanger sequencing in the family and were absent in 104 controls.
GABRB2 encodes for the β2 subunit of the γ-aminobutyric acid type A (GABAA) receptor, one of
three main classes of receptors activated by GABA, the principal inhibitory neurotransmitter in the
central nervous system. Mutations in genes encoding various subunits of this receptor are implicated
in a number of neurological and developmental disorders, including epilepsy and autism. To date,
genetic studies have implicated mutations in GABRB2 with intellectual disability and epilepsy, but
not autism.
HUWE1 is an X-linked gene and encodes a protein that functions as an E3 ubiquitin ligase.
HUWE1 is required for the ubiquitination and subsequent degradation of the BCL2-related anti-
apoptotic protein MCL1, the p53 tumor suppressor, core histones, and DNA polymerase beta.
Defects in HUWE1 cause mental retardation of the syndromic X-linked Turner type (MRXST).
Recently, a de novo missense variant in HUWE1 was identified in a male ASD proband, but not in
the proband's less severely affected brother.
Conclusion: WES analysis has proven to be an efficient strategy to identify de novo and inherited
mutations that can contribute to ASD risk in the Lebanese. We were able to identify two novel
variants in GABRB2 and HUWE1. Future functional studies in cellular models are underway to
determine the contribution of these mutations to pathogenicity in autism.
32
P17. Flupirtine Analogue Structure-Activity Relationships for Treatment of
Batten Disease
Fadi Saadeh1, Karl Albert Mansour1, Joelle Makoukji1, Paul Trippier2, Rose-Mary Boustany1, 3
1Department of Biochemistry and Molecular Genetics, American University of Beirut Medical
Center, Beirut, Lebanon, 2School of Pharmacy, Texas Tech University Health Sciences Center,
Amarillo, TX, 3Neurogenetics Program, AUBMC Special Kids Clinic and Division of Pediatric
Neurology, Department of Pediatrics and Adolescent Medicine, American University of Beirut
Medical Center, Beirut, Lebanon
Keywords: Batten disease, Neuronal Ceroid Lipofuscinoses, Flupirtine, Neurodegeneration
Descriptive Statement: Flupirtine analogues were tested and compared to flupirtine regarding their
neuroprotective ability in human cells mimicking Batten disease.
Introduction: Batten disease/the Neuronal Ceroid Lipofuscinoses or NCLs are fatal inherited
neurodegenerative diseases with no cure. CLN3 disease is the juvenile and most common. Although
rare the disease often strikes multiple offspring in the same family that carry the defective NCL
gene. Current treatment regimens are symptomatic and supportive but do not target the underlying
disease. The need for disease-modifying drug candidates is urgent. This work aims to address this
requirement by providing lead therapeutic compounds. Previous work from the lab shows that
Flupirtine aborts etoposide-induced apoptosis in NCL and normal lymphoblasts and prevents death
of neurons. The end goal of this application is to generate a full structure-activity relationship map
of flupirtine analogues as applied to NCL and deliver several derivative compounds with enhanced
neuroprotective activity.
Methods: Optimum drug concentrations of flupirtine derivatives were tested by establishing
growth curves under pro-apoptotic conditions of etoposide treatment assessed by trypan blue and
Propidium Iodide. Flupirtine derivatives with desirable activity at the optimum concentration were
evaluated by Trypan blue staining after siRNA knockdowns of CLN3 gene in PC12 cells.
Results: Three of the flupirtine analogues with specific chemical substitutions proved to be
neuroprotective after the application of etoposide to PC12 cells. After knocking down the CLN3
gene in PC12 cells, the same three drugs prevented neuronal cell death. These drugs had a better
neuroprotective effect than flupirtine.
Conclusion: These findings uncover analogous compounds to flupirtine with enhanced activity for
the treatment of Batten disease. These analogues even prove to possess greater neuroprotective
activity than flupirtine.
33
P18. MicroRNA Expression in Lebanese Breast Cancer Tissues: A
Comparative and Integration Analysis
Farah Nassar1, Rabih Talhouk1, Nathalie K. Zgheib2, Arafat Tfayli3, Maya El Sabban4, , Fouad
Boulos5, Mark Jabbour5, Claude Chelala6, Rose-Mary Boustany7, Nagi El Saghir4, Brian Joyce8,
Zhou Zhang8, Yinan Zheng8, Lifang Hou8 , Ali Bazarbachi4, George Calin9, Rihab Nasr3 1Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut,
Lebanon; [email protected] 2Department of Pharmacology, Faculty of Medicine, American University of Beirut, Beirut,
Lebanon
3Department of Internal Medicine, Faculty of Medicine, American University of Beirut, Beirut,
Lebanon
4Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American
University of Beirut, Beirut, Lebanon 5Department of Pathology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon 6Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London,
London, UK 7Departments of Pediatrics, Adolescent Medicine, Biochemistry and Molecular Medicine,
American University of Beirut Medical Center, Beirut, Lebanon 8Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University 9Department of Experimental Therapeutics, Division of Cancer Medicine, University of Texas MD
Anderson Cancer Center, Houston, Texas, USA
In Lebanon, breast cancer (BC) is the most common type of cancer among women, and it has a
higher reported incidence in younger patients compared to those in the West. microRNA (miRNA)
are small noncoding RNA that act as master players at all stages of BC development. We have
recently shown that expression of certain miRNA in Lebanese BC tissues was different than that
reported in the West. This could reflect an ethnic difference and thus suggested the necessity of a
global miRNA profile of BC tissues from Lebanese patients. Hence, the aim of this study is to
examine the miRNA expression in formalin fixed paraffin embedded Lebanese BC tissues using
microarray, perform a comparative miRNA profile analysis with American samples and predict the
role of dysregulated miRNA through miRNA-mRNA integration analysis after mRNA profiling. A
total of 74 miRNAs were significantly dysregulated between tumor and normal adjacent breast
tissues. The reliability of microarray data was confirmed using quantitative reverse transcription
real time PCR. mRNA profiling showed that the BC samples were mainly of luminal B subtype.
Using ingenuity pathway analysis, integration results identified 719 potential mRNA that could be
targeted by 51 miRNA with miR-183 and miR-182 having the highest number of targets in an
inversion correlation analysis of mRNA and miRNA. Although most of the dysregulated miRNA
in Lebanese BC patients were similar to American patients, some differences were noted that could
reflect either the patient’s age at diagnosis or potential epigenetic regulation of miRNA expression.
mRNA-miRNA integration analysis revealed a potential miRNA role at early stage of BC in
regulating important dysregulated tumor suppressive or oncogenic mRNA mainly involved in
increasing cellular proliferation and decreasing migration and invasion. Further investigation of the
dysregulated miRNA is needed to comprehend BC onset especially in young patients.
Acknowledgement: Kamal Shair Central Laboratory (for real time PCR and funding) and
Neutrogenetics Core Facility (for microarray). Farouk Jabre, Medical Practice Plan, Lebanese
National Council for Scientific Research, and Richard and Susan Kiphart Northwestern Global
Health Research Fund.
34
P19. Ingestion of phosphorus containing high carbohydrate meal increases
postprandial energy expenditure.
Mariam Assaad, Carla El Mallah, Ammar Olabi, Omar Obeid
Department of Nutrition and Food Science, American University of Beirut, Beirut, Lebanon
[email protected]; [email protected]; [email protected]; [email protected]
Funding source: University Research Board (URB)
Keywords: Diet induced thermogenesis (DIT), postprandial energy expenditure, adenosine
triphosphate (ATP), phosphorus.
A. Descriptive Statement: Phosphorus is required for ATP production and is known to be involved
in energy metabolism. However, it’s not clear whether phosphorus ingestion with meal can affect
energy expenditure.
B. Introduction: background and aims :
Both overweight and obesity are increasing globally and recognized to cause main health
problems nowadays. Weight gain results from an imbalance between energy intake and energy
expenditure. Diet induced thermogenesis (DIT) accounts for 5-15% of total energy expenditure and
is mainly related to ATP production that depends on phosphorus (P) availability.
Our objective was to determine the effect of P ingestion with high carbohydrate meal on
postprandial energy expenditure. We hypothesized that P ingestion increases diet induced
thermogenesis of the subjects.
Methods: A cross over study was conducted on six lean male subjects. Subjects received a 500 Kcal high
carbohydrate meal with (500 mg of P) or without P. Energy expenditure was measured at baseline
and at 30 minute intervals for 4 hours following meal ingestion using a ventilated hood and canopy
system COSMED QUARK CPET unit.
Results: Postprandial energy expenditure of meal containing P was significantly higher than that
of placebo (p=0.007). This increase was associated with a significant rise in fat oxidation (%) (p=
0.022), while carbohydrate oxidation (%) was decreased (p= 0.023).
Conclusion: P was able to increase postprandial energy expenditure mainly due to increased fat
oxidation. This data may have promising effect for the management of obesity.
35
P20. Classification and Analysis of Notices under the 1999 FIDIC’s General
Conditions of the Construction Contract
Salam Khalife, Industrial Engineering and Management Department;
Dr. Mohamed-Asem Abdul-Malak, Civil and Environmental Engineering Department;
American University of Beirut ; Beirut- Lebanon
[email protected] [email protected]
The rights, obligations, and responsibilities of the parties to a construction contract are
described in the conditions of contract through both the general and particular conditions. These
provisions need to be well comprehended and administered by contract administrators, working
with each of the parties. Notice provisions are viewed as main contractual provisions, as these, in
general, preserve the right of knowledge by either party about events arising during the construction
phase and serve to highlight the following or consequent actions in response to such events. Under
certain clauses, notices are sometimes regulated by time bars for their timely issuance; one well-
known example is the notice of intention to submit a claim for an additional compensation or an
extension of time upon encountering certain events during the course of construction. Yet, if the
notice is not served within the regulated period set by the contract conditions, the contractor, under
many jurisdictions, loses his right for such a claim. Contract administrators encounter various
events that necessitate the issuance of notices, all in accordance with contract stipulations. The
problem is that contract administrators often deal with notices in a negligent way, thereby exposing
the contract to major risks. This is because of the lack of material that explains the notice provisions
and their importance. The objective of this paper is to look into the wide range of notices and study
their relevant provisions. For that purpose, the globally used 1999 standard contract conditions,
issued by the International Federation of Consulting Engineers (FIDIC), are examined. The 1999
FIDIC conditions are said to represent a set of balanced conditions that fairly distribute the possible
project risks between the parties to a construction contract. These conditions call for a wide range
of notices to be issued, related to work progress and other vital aspects of the construction process.
The twenty clauses and their corresponding sub-clauses are examined and those prescribing the
need for issuing notices are filtered out. A classification matrix, which includes general information
deduced about each filtered sub-clause, is then constructed. The matrix construction is based on a
number of selected descriptors that help distinguish among the various notices encountered.
Detailed analysis of the information offered by the matrix is then carried out to reveal the underlying
needs for such notice provisions. The research aims at providing guidelines for practitioners,
particularly contract administrators, regarding notices and their regulating time bars, which shall
help improve the way contract administration responsibilities are fulfilled during the construction
phase of projects.