Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1

Background: Breakdown of blood-retinal barrier (BRB) and subsequent hyperpermeability is a cardinal feature of early diabetic retinopathy (DR) and leading cause of blindness. Fatty acid metabolism is implicated in several biological functions via multiple signaling pathways including the synthesis of bioactive metabolites. Our previous studies established 12/15-lipoxygenase (12/15-LOX)-derived 12- and 15- hydroxyeicosatetraenoic acids or HETEs as proangiogenic mediators during DR via disrupting the retinal levels of vascular endothelial growth factor and pigment epithelium derived factor (PEDF). Purpose: 1) To screen the impact of high glucose (HG) treatment on the levels of bioactive lipids including 12/15-LOX metabolites in cultured human retinal endothelial cells (HREC), 2) To test the impact of 12/15-LOX deletion on BRB function and explore the underlying mechanism in particular the role of NADPH oxidase as a major source of oxidative stress during DR. Material and Methods: Liquid chromatography-mass spectrometry (LC/MS) was used in a comprehensive lipidomic screen of HRECs treated with or without HG (30mM D-glucose). Effect of 12- or 15-HETE (0.1 M) on leukostasis and angiogenesis (tube formation) was examined in HRECs transfected with siRNA against the catalytic subunit of NADPH oxidase (NOX2) or the scrambled siRNA or treated with or without the NADPH oxidase inhibitor (apocynin, 30°M). Human leukocytes (PMNs) labeled with lipophilic fluorescent probe were used for leukostasis assay. Migration assay was performed using the Electrical Cell Impedance Sensor (ECIS). Retinal vascular changes were examined by fluorescein angiogram (FA) and Optical Coherence tomogram (OCT) in streptozotocin-induced diabetic wild type (WT) or 12/15-LOX-knockout mice and in mice received intraocular injection with 12-HETE or 12-HETE with PEDF. Western blotting, immunofluorescence and multiplex system were used to test the changes in the levels of inflammatory markers. Results: Out of 126 bioactive lipids screened, 70 were undetected, 47 un-significantly changed, and 9 metabolites were significantly up-regulated by HG compared to osmotic control (5mM D-glucose+25 mM L-glucose). Intriguingly, six metabolites among the 9-increased are the products of 12/15-LOX pathway (15-HETE, 11-HETE, 8, 15-DiHETE, 12-HETE, 17-HDoHE, and 13-HODE). Of these 15-HETE has the highest fold increase (p value= 0.004). Thereafter, we pursued to determine whether the altered metabolites of 12/15 LOX play a role in BRB dysfunction during diabetes. Retinal vascular leakage was significantly reduced in diabetic mice deficient in 12/15-LOX as evidence by FA and less albumin leakage compared with diabetic wild-type littermates. Furthermore, intravitreal injection of 12-HETE per se in normal WT mice resulted in a marked increases in the vascular leakage and expression of inflammatory markers such as ICAM-1, VCAM-1, CD45 as well as the NOX2 compared to vehicle-injected control or to mice received both 12-HETE and PEDF. In vitro, 12/15-HETEs induced HREC leukostasis, migration, and tube formation. These effects were inhibited by the concomitant use of apocynin and by silencing NOX2. Conclusion: Retinal endothelial 12/15-LOX is activated by hyperglycemia causing overproduction of 12- and 15-HETEs. These metabolites are implicated in the pro-angiogenic, -oxidative and -inflammatory effects of hyperglycemia in HREC. Thus, targeting this signaling system represents an attractive therapeutic strategy to prevent and treat DR.

Neuroblastoma is a solid malignant embryonic tumor. It is usually diagnosed in the infancy and rarely found after the age of 10 years. In the current treatment options, CDDP and TOPO are included. Methods: Neuroblastoma (SH SY-5Y) cell death was investigated after treatment with CDDP and TOPO (0.1nM-1μM) using Trypan Blue Cytotoxicity Test and Total Cytotoxicity and Apoptosis Detection Kit (FACS). These data are compared to the drug triggered increase of the intracellular calcium ([Ca2+]i) concentration using live calcium imaging with the calcium sensitive dye Fluo-4 AM. Furthermore, we analyzed changes of gene expression of selected [Ca2+]i signaling related genes such as the calcium binding protein S100A6, IP3 receptors, Ryanodine Receptors, Calmodulin (CALM) and the Calmoduling Binding Transcriptor Activator (CAMTA1). Results: With increasing concentration of either of the two substances the amount of viable cells was reduced after 24h of application. The tests revealed a higher cytotoxicity of TOPO (about 25% cell survival at 0.1μM) compared to CDDP (more than 80% survival). With increasing concentrations the percentage of cells which were in the “late apoptosis” stage increased for both drugs but were more pronounced for TOPO. A similar observation was made when the incubation time for the two drugs was prolonged to 48h or 72h when the amount of viable cells was further reduced. Individual (but not all) neuroblastoma cells increased their [Ca2+]i after the application of either CDDP or TOPO (both 1 μM). Over the period of three to four hours the fluorescence increased about 40%. The increase was time and concentration dependent for CDDP and TOPO, while a concentration of 0.01μM was most efficient to increase [Ca2+]i. CDDP and TOPO influence the gene expression of SHSY-5Y cells, targeting specific calcium signaling related genes. In this context, a concentration and time dependent increase in the expression of S100A6 was observed in the cells treated with CDDP. TOPO showed a more pronounced effect than CDDP in increasing the mRNA expression of S100A6. The expression of ITPR1 and ITPR3 genes encoding the type 1 and 3 of the IP3R were found deregulated in the neuroblastoma cells treated to CDDP and TOPO. The 1μM CDDP exposure for 72h was responsible of ITPR1 and ITPR3 down regulation however; the same concentration applied for 24h transiently increased the mRNA expression of ITPR3. The application of TOPO did diminished concentration and time dependently the mRNA expression of ITPR1 after 24h and 72h exposure and of ITPR3 after 72h exposure, however 24h exposure to TOPO did transiently increased the mRNA expression of ITPR3 for the lower concentrations tested. The expression of Ryanodine Receptor genes RYR1 and RYR3 was found deregulated in the neuroblastoma cells treated to CDDP and TOPO. Conclusion: Time and concentration dependently, the treatment of SHSY-5Y cells with TOPO and CDDP increased the cell death, at clinical relevant concentration. That correlates with increased [Ca2+]i levels and with the deregulation in gene expression of calcium signaling related genes suggesting that [Ca2+]i regulation is involved in the anticancer effects of TOPO and CDDP.

Biocompatible Graphene Oxide with Anchored Zwitterionic Moiety - Synthesis, Characterization and Application Markéta Ilčíková1, Zuzana Kroneková2, Anna Záhoranová2 and Peter Kasák1* 1 Center for Advanced Materials, Qatar University, P.O.Box 2713 Doha, Qatar 2 Polymer Institute, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia *Corresponding author: [email protected] The novel biocompatible graphene oxide (GO) bearing zwitterionic moiety was synthesized and characterized. The zwitterionic structures have recently gained attention in biomedical applications as materials for development of ultra-low fouling surfaces. Generally, zwitterions contain both negative and positive charge in their structures; however the overall charge is neutral. That impart them highly hydrophilic performance; the adsorbed water then prevent the cells or bacteria to interact with surface. Graphene is two dimensional filler interesting predominantly due to high electrical conductivity. In oxidized form the electrical conductivity is compromised due to presence of hydroxyl, epoxy and carboxyl functional groups. In this work, the hydroxyl groups were utilized for anchoring the zwitterionic moiety onto the GO surface. The reaction was performed in two steps. First the silanization with (3-mercaptopropyl)trimethoxysilane was preformed to introduce the thiol functionality onto the GO surface that was reacted with sulfobetaine monomer (3-((2-methacrylamidoethyl)dimethylammonio)propane-1-sulfonate) through thiolene click reaction in the following step. The successful modification was confirmed by FTIR and TGA. Compared to neat graphene, the presence of sulfobetaine improved the graphene biotolerability for fibroblasts and pancreatic beta cells. Modification of GO surface with zwitterionic moiety prevents its negative effect on cell viability. Thus the adsorption of cells on the surface and the cell - GO surface interaction is effected. The modified GO will be used for modification of electrode at biochips construction, and the electrorheological response will be discussed as well. Acknowledgement: This publication was made possible by NPRP grant # NPRP-6-381-1- 078 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors.

One goal in the field of soft tissue biomechanics is to understand and to model the properties of collagen-fibers in biological tissues such as blood vessels, brain tissues, and skin which undergo growth and remodeling processes. Experimental studies of Bhole et al.\ [1] suggest that the mechanical properties of fibers in biological tissues depend not only on the current state of the deformation but also on the deformation history. Demirkoparan et al.\ [2] developed a hyperelasticity based framework taking this effect into account. The framework developed in [2] uses an additive hyperelastic material model for the strain energy density, $W = W_m + W_f$. The matrix contribution $W_m$ does not take time-dependent changes of the material properties into account yet the fiber contribution $W_f$ changes over time as it is modeled in terms of a constant creation rate and a deformation dependent dissolution rate of the fibers. Then Topol et al.\ [3] have investigated the predictions of such a model by studying the effect of fiber deformation state at the time of their creation on the overall mechanical properties of the material. This research investigates the relation between the change in the fiber density, the Cauchy stress development, and the deformation of the material.

Diabetic retinopathy (DR) is a leading cause of blindness among working diabetic patients worldwide. The current therapeutic strategies are limited by several side effects. Thus, it is important to investigate novel approaches for therapeutic intervention. Early inflammatory response and late neovascularization (NV) are cardinal clinical signs of the DR. We have previously demonstrated the involvement of bioactive lipids especially the products of omega-6 polyunsaturated fatty acids (PUFAs) such as arachidonic acids in the development of retinal hyperpermeability and NV during DR. Hyperglycemia is a major risk factor for endothelial cell activation and development of microvascular dysfunction during DR. Overwhelming evidences suggest that omega-3 PUFAs like DHA (C22:6n-3) are protective in proliferative DR. In the present study, we investigate the effect of exogenous supplementation of DHA on human retinal microvascular endothelial cells (HREC) under hyperglycemic conditions. Cells were treated with or without high glucose (HG, 25 mM) for 36 hrs in the presence or absence of the DHA (30 µM). To study the early inflammatory response, cells were analyzed for cell barrier function by measuring the changes in transcellular electrical resistance (TER) using the Electrical Cell-substrate Impedance Sensing (ECIS). Hyperglycemia resulted in a significant decline in TER while it was significantly improved by DHA. Furthermore, to investigate the effect of DHA on the proangiogenc properties of hyperglycemia in HREC a scratch-wound repair assay was carried out in cultured HREC cells treated with or without HG in the presence or absence of DHA. The migration and proliferation of cells induced by HG was reduced on supplementation of cells with DHA. Levels of apoptosis and oxidative stress were also monitored under the effect of hyperglycemia. While HG treatment did not result in any change in level of apoptosis, it resulted in sharp induction of oxidative stress in cells, which however was ameliorated under the effect of DHA treatment to hyperglycemic cells. Real-time PCR revealed significantly enhanced transcripts for the vascular endothelial growth factor (vegf) in hyperglycemic cells while reduced expression of the pigment epithelium derived factor (pedf) and the peroxisome proliferator activated receptor gamma (PPAR) was observed. These changes were prevented by DHA. Anti-inflammatory properties of DHA were further evinced by monitoring the levels of several inflammatory cytokines and angiogenic factors by multiplex enzyme-linked immunosorbent assay. Glucose treatment resulted in upregulation of various pro-angiogenic factors like angiogenin, angiopoietin, pdgf, pigf and this effect of HG was prevented by DHA treatment. Similarly, inflammatory cytokines like TNF-alpha and MCP1, which are involved in promotion of angiogenesis and inflammation were induced by HG and was attenuated in the presence of DHA. Interestingly, anti-inflammatory cytokines like IL-1 beta and IL-6 were markedly increased by DHA as compared to HG alone. These results suggest that DHA protects HREC against the pro-angiogenic, -inflammatory and -oxidative effects of HG and thus it can be proposed as alternative or supplementary treatment to the current therapeutic strategies of DR.

General consensus exists that biomedical research is a key determinant of innovation, health, and economic wellbeing. However, the answer to the question how do we determine and quantify the value and the impact of research is apparently simple, while a thoughtful and analytical answer quickly becomes very probing and difficult to address. Qatar has dedicated itself to transitioning from a resource-based to a knowledge-based economy and Sidra Medical and Research Center is envisioned to become a beacon of learning and a world-class institution of its kind in the MENA region. A unique opportunity exists at Sidra for the development of a comprehensive system to capture the return on investment (ROI) for research on knowledge, the economy, and on health outcomes. This would enable Sidra to establish itself as a leader in the field of impact assessment, ahead of many other countries also grappling with this thorny problem. In fact, Sidra's status as a startup creates a rare situation where measurement and analysis of ROI can be initiated from a baseline. Work can then be done prospectively rather than trying to gather information retroactively, a difficult obstacle that institutions that have attempted to implement similar systems have had to try to overcome. In order to take advantage of this opportunity at Sidra, we propose creating a core system consisting of a smart, web-based, auto-populated "Sidra Biosketch". This system will interact with internal enterprise resource planning, grants and proposal management systems, as well as external publication, patent, startup and commercial license databases (e.g., PubMed). This system takes into consideration two critical factors. First, it minimizes the administrative burden on investigators and administrators. Second, it does not solely measure stochastically by counting numbers of publications, citations, etc. Rather, it focuses and relies heavily on content and contextual assessment to enable Sidra to fully understand exactly what research is being done, where it is being done, by whom, and with what collaborators. The impact on knowledge, economic and health outcomes can then be mapped through dynamic network analyses, natural language processing, and topic modeling approaches. This approach to measuring the impact of Sidra research will maximize efficiency of input and analysis as well as the quality of outputs and results. Meanwhile, this system will also enable Sidra to make decisions that will enhance resource allocation and allow for better assessment of its contribution to progress in science, the economy, and health outcomes in Qatar and worldwide.

Nonlinear continuum mechanics is commonly used to study the response of highly de- formable materials. Nowadays, the proposed material models often include internal ma- terial variables in order to capture realistic engineering behavior. The theoretical back- ground is mature and its implementation in the frame of nite element method is well established. Solely, these material constitutive models are expressed in terms of defor- mation gradient F, its derived quantities, and various multiplicative decompositions such as F = ^F F (1) This multiplicative decomposition serves to pertain particular portions of F to specic parts of the material response. The usual scheme is then that ^F models elastic response and it is associated to the rules of variational calculus. The F portion then models inelastic response, usually by means of a time dependent evolution law. Recently, the arguments of variational calculus have been applied to both portions of the deformation gradient decomposition for incompressible hyperelastic material [1]. The decomposition itself is then determined by an additional internal balance equation that is generated by such a variational treatment. The internally balanced compressible hyperelastic material response is presented in [2]. Fundamental studies of this type are key to better physics-based modeling of complex biomechanical processes in soft tissue, including long time scale processes of growth, and short time scale processes of wound healing. The FE formulation of this internal balance multiplicative decomposition is naturally achieved by linearizing the weak form that is obtained by the variation with respect to both portions of the multiplicative decomposition. In this work, we would like to present the Updated Lagrange Formulation. This formulation will be implemented into com- mercial nite element package FEAP that will facilitate in future studying engineering application in the eld of biomechanics. The correctness of implementation will be ex- amined by simulating homogeneous deformations such as uniaxial loading, dilatation and simple shear. References [1] H. Demirkoparan, T. J. Pence, and H. Tsai. Hyperelastic internal balance by multi- plicative decomposition of the deformation gradient. Archive for Rational Mechanics and Analysis, 2014. DOI: 10.1007/s00205-014-0770-9. [2] A. Hadoush, H. Demirkoparan, and T.J. Pence. Modeling of soft materials via mul- tiplicative decomposition of deformation gradient. In USNCTAM, 2014.

Background Islet cell death is a common feature of type 1 diabetes (T1D), including after islet cell transplantation, as well as of type 2 diabetes (T2D). As of today, we lack tools to quantitatively detect islet cell loss prior to the clinical onset of diabetes, or to predict the progression of established diabetes. Our preliminary data demonstrates that a signature of 20 different non-coding (nc) RNAs (including microRNAs) reflects beta cell death (RAPID: RNA-based Analysis for Prediction of Islet Death signature). Objectives 1)To identify a high-throughput platform for detection of ncRNAs/microRNAs 2)To validate the RAPID signature of 20 ncRNAs using this high-throughput platform 3)To test the suitability of these ncRNA biomarkers in predicting the progression to T1D Method Next Generation Sequencing (NGS) studies on developing human pancreas have led to the identification of specific ncRNAs that are found to be expressed specifically in the human pancreatic islets. We use combined FISH and immunostaining to confirm the localization of each miRNA in human islets. TaqMan-based real-time PCR on plasma from at-risk diabetic individuals and age/gender matched controls in a longitudinal study, is used to measure the abundance of specific ncRNAs in plasma-EDTA samples of individuals at risk of T1D. I will discuss the comparison of 2 different ultra-high-throughput TaqMan real-time PCR platforms that we use to analyze these samples and present the data demonstrating the suitability of these RNA-based biomarkers in predicting the progression to T1D. Results Present study has allowed us to validate a microRNA-based signature of islet cell death in diabetes. Using a cohort of kids at risk of T1D, we demonstrate the suitability and advantages of these ncRNA biomarkers over traditional / conventional antibody- and glucose-based detection. The development of an assay for early detection of islet injury and death has direct applications in clinical medicine. Hopefully, this study results will inform medical researchers as to how to predict the development of Type 1 diabetes, monitor response to interventions such as islet transplantation, vaccines and drugs that aim to retard beta cell loss or promote beta cell regeneration.

Over the last two decades the prevalence of obesity has reached epidemic levels worldwide. Secondary major health risks, such as hypertension, insulin resistance, type 2 diabetes and cardiovascular diseases, can be summarized as the metabolic syndrome and are highly associated with obesity. In cases in which energy intake e.g. through high caloric food intake exceeds energy expenditure the overall energy homeostasis is imbalanced. White adipose tissue depots mainly act as the body's main energy storage and additionally act as an endocrine organ by releasing adipokines and cytokines into the body. Additionally, functional brown adipose tissue (BAT) has been discovered through radiological detection in the last years in substantial amount in adults. Formerly overlooked BAT, which was thought to be absent in the human adult, has therefore recently become an interesting anti‐obesity target due to its ability to dissipate energy in form of heat. We extensively characterized human brown PAZ6 adipocytes in comparison with a white adipose cell line SW872. In addition, human SGBS adipocytes were included in the analysis. Brown and white adipocyte markers were tested by quantitative Real-time PCR. Fluorescent and Oil‐Red staining assessed the quantity and quality of the differentiation process. Next generation RNA sequencing of undifferentiated and mature SGBS and PAZ6 cells was performed in order to elucidate pathways distinctly activated in white vs. brown human adipocytes. Functional assessment of oxidative rates of each cell line was conducted using the Seahorse technology. Whereas PAZ6 and SW872 cells showed classical molecular and phenotypic markers of brown and white adipocytes, respectively, SGBS cells presented a versatile phenotype of adipocyte. 14 days after initiating the differentiation process the expression of classical brown marker such as UCP‐1 and PPARγ peaked and declined until day 28. The white adipocyte marker Tcf21 however, showed reciprocal behavior. Interestingly, Leptin levels peaked at day 28 whereas the highest adiponectin mRNA levels were monitored at day 14. Phenotypic analysis of the abundance and shape of lipid droplets were consistent with the molecular patterns. On day 14, SGBS cells showed multiple small droplets, however the number of droplets decreased and the size increased until day 28 as expected for a white adipocyte phenotype. Lastly, functional metabolic analysis showed the highest oxidative rate of mature SGBS cells on Day 14, which remained consistent or slightly decreased until day 28. SGBS cells are widely used as a model for white adipocytes. Our data suggest that the cell line harbors a versatile phenotype, which changes throughout their mature stage. Many reports suggest recently that the traditional classification of adipocytes is not exclusive and the existence of beige/brite adipocytes has been shown. We are presenting data derived from a human cell line model, which harbors characteristics of both distinct phenotypes. This knowledge will be of importance for studies aimed to increase brown fat depots in order to increase energy expenditure in obese subjects with the ultimate goal of weight reduction.

Background: Polycythemia Vera (PV), Essential Thrombocytosis (ET) & Primary Myelofibrosis (PMF) are Philadelphia negative Myeloproliferative Neoplasms (MPNs) characterized by overproduction of one or more myeloid cell lineages. MPNs are associated with the presence JAK2 V617F mutation in 95% of PV & 50% of ET & PMF patients. Several molecular methods such as RQ-PCR, HRM & Sequencing are currently used to detect common mutations. However, there are still significant numbers of MPNs are negative to the most common genetic anomalies. The advent of Next Generation Sequencing (NGS) gives the opportunity to study relevant mutations in several genes. Aim: Utilizing NGS to identify potential genetic anomalies causing familial MPNs patients in Qatar. Methods: 6 MPNs patients from consanguineous families & 5 healthy individuals were consented into the study gDNA was extracted & used for multiplex amplification of amplicons targeting cancer associated mutations in 28 key genes using the Ion AmpliSeq Kit. NGS was performed via the Ion Torrent using the 318 chip & data was analyzed with the Torrent Suite. The confirmation of NGS data was done by RQ-PCR or Sequencing. Results: NGS identified novel deleterious mutations in MPNs patients. Out of 6 familial cases, 5 patients (P1- P5) were ET & 1 patient (P6) was PV. P1 had JAK2 V617F, ASXL1 T600P, CBFB G180S, THPO S184R & ITGA2 R76Q, P2 had JAK2 V617F, MPL A554G & ATM F582L, the other three Patients (P3, P4 & P5) had CLAR K385fs*47 & one PV patient (P6) had TYK2 E1163G, ASXL1 P808H, PDGFRB P4L TERT G300fs. In patients & healthy individuals, mutations/SNVs such as MPL P106L, K553N, SH2B3 L476F, ATM F1036F KIT N564S & TET2 T730R were also found Discussion & conclusion: In this study, several deleterious somatic/germ-line mutations/SNVs were identified using Targeted Exome Sequencing approach. A complex combination of mutations occurred in ET patients & coexistence of several oncogenic events occurred in PV patient. This finding may also suggest that the MPNs phenotype may depend on presence of other mutations. It is worth to mention that the presence of ATM variant in P2 is associated with increased risk of CLL. Somatic CALR type-2 mutation was identified in 3 ET (nonmutated JAK2 or MPL) patients. This mutation is 5-bp TTGTC insertion in exon 9 that generates a mutant protein with a novel C-terminal (p.K385fs*47). In patients & healthy individuals, a heterozygous germ-line mutation in exon 3 of the MPL gene (MPL P106L) has been observed and previously described as a rare autosomal-dominant disorder. However, this mutation is considered to be frequent in Arabic populations also several unreported variants of uncertain significance were identified. Our finding suggested that familial MPNs patients in Qatari tribes have several potential disease- associated variants/mutations which represent a unique interest for the scientific community worldwide and present an unprecedented opportunity for local and international collaborations to carry out WES in society with its unique coherence and genetic pool (founder effect), this would offer invaluable resources to unravel the genetic etiology and pathogenesis, delineate the mechanisms behind MPNs phenotypic diversity.