Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1

Background and Objectives Impaired cholesterol and fat metabolism contributes to obesity, type 2 diabetes and atherogenic cardiovascular disease; major chronic conditions that are increasingly prevalent in Qatar. There is thus an urgent need for new treatment modalities to combat the rise in these diseases. Understanding how cholesterol/lipid homeostasis is achieved and maintained is among the very first critical steps towards developing new strategies for better treatments. Despite the intricate underlying mechanisms, many regulatory factors have been found to be involved in the metabolic regulation of lipids. Recent studies found that microRNAs (miR), short 22-nucleotide RNA molecules that control gene expression by silencing target mRNAs at the translational level, hold promise as therapeutic targets since they contribute to the etiology of many pathological conditions. We found that microRNA 33a (miR-33a) embedded within the intronic sequence of SREBP-2 gene, the master regulator of cholesterol metabolism, acts as a key modulator of intracellular cholesterol trafficking pathways. Here, we report our new findings on the 5p and 3p strands of miR-33a (miR-33a-5p and miR-33a-3p) in regulating cholesterol homeostasis. Methods Using computational algorithms we identified potential target genes of both strands of miR-33a that are involved in cholesterol metabolism. We then used luciferase reporter assays, wherein the 3'-UTR of the target gene was fused to a luciferase reporter gene, to validate the predicted targets of miR-33a. Positive associations between the microRNAs and their targets were further confirmed by mutating the binding sites on the target gene 3'-UTR and performing luciferase assays with the mutant constructs. Overexpression and knockdown of the microRNAs in human liver cell lines were carried out with mimics and inhibitors, respectively, and their effect on target gene expression was studied by RT-qPCR and western blotting. Cholesterol uptake and efflux assays in hepatocytes were exploited to better assess the functional roles of miR-33a. Results Intriguingly, we found that in addition to our pervious discovery of miR-33a-5p reducing cholesterol efflux from hepatocytes and macrophages, miR-33a-3p promotes intracellular cholesterol uptake. While miR-33a-5p inhibits ABCA1, a cholesterol efflux pump instrumental in raising plasma HDL-cholesterol levels and reverse cholesterol transport, miR-33a-3p activates LDL-receptor (LDLR) by repressing both Idol and PCSK9, two major negative regulators of LDLR. Accordingly, in an SREBP-2 active condition antisense-mediated inhibition of miR-33a-3p, but not miR-33a-5p, led to decreased LDLR expression and LDL uptake in human hepatocytes. Conclusions Mutations in the LDLR gene are well known to be highly associated with atherosclerosis and familial hypercholesterolemia. Our data unravels a novel regulatory circuit by which LDLR, which is transcriptionally activated by SREBP-2, is further protected from degradation at the post-translational level by miR-33a-3p. This indicates that both the 5p and 3p strands of miR-33a are mutually exclusive in elevating intracellular cholesterol levels along with their SREBP-2 host gene. These findings provide impetus for further characterization and development of new therapeutic interventions in cardiovascular disease.

Thymidylate kinases (TMKs) play a central role in the production of nucleotide precursors that are required for the replication of DNA. Consequently, this enzyme is a potential drug target for the discovery of anti-bacterial, anti-fungal, and anti-parasitic drugs. In addition, TMKs are also involved in the activation of prodrugs. In particular, the anti-HIV drug AZT is activated by human TMK (huTMK) and the low efficiency of huTMK towards AZT is a significant problem in the use of AZT in the treatment of HIV. Finally, nucleotide precursors are required in large amounts by cancerous cells, thus the inhibition of huTMK by chemotherapeutic agents may enhanced the arsenal of drugs that are used to treat cancer. Although there has been some effort to develop inhibitors of TMKs, these efforts have been hampered by the difficulty in performing high throughput screening using compound libraries. In addition, the characterization of TMK-drug complexes has been limited to X-ray diffraction studies which provide static information about the enzyme-drug complex. There have been no attempts to apply high-resolution multi-nuclear NMR techniques to determine the fundamental dynamic properties of these enzymes and how the structure and dynamics of the enzyme are altered by the binding of substrates or inhibitors. As a preliminary step in characterizing these enzymes by NMR we have over-expressed TMKs from yeast, human, and two pathogens - Plasmodium falciparum and Candida albicans. Expression of these TMKs was optimized by the design of synthetic genes for expression in bacteria. In the case of the human enzyme, we are able to routinely produce 250 mg of the enzyme/L of culture. Preliminary NMR spectra of the yeast, human, and plasmodium enzyme show that the protein is a homo-dimer in solution, as anticipated from X-ray studies. The amide and methyl spectra are well resolved, indicating that resonance assignment by traditional TROSY based methods will be feasible for both the amides and the methyl resonances. In particular the high sensitivity and dispersion of the methyl spectra will facilitate characterization of the dynamic properties of these enzymes by carbon and deuterium relaxation. Ligand induced changes in the dynamics and structure of huTMK in solution will be characterized using NMR methods. These studies will provide additional insights into the inability to huTMK to effectively activate AZT. The entropic component of the thermodynamics of substrate binding to TMK from the parasite that causes malaria will also be characterized by determining dynamic changes by NMR methods. The development of NMR methods to study these enzymes also provides a method for high throughput screening of compound libraries by detecting chemical shift changes in the NMR spectral of the enzyme due to binding of a potential lead compound.

Identification of post-translationally modified α-Synuclein protein in biofluids of Parkinson's disease patients using a targeted and quantitative mass spectrometry approach. Céline Vocat1, Bruno Fauvet1, Michel Prudent4, Adrien W. Schmid3, Hilal A. Lashuel1&2. 1 Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2. Qatar Biomedical Research Institute, 5825 Doha, Qatar. 3. Proteomics Core Facility, Ecole Polytechnique Fédérale Lausanne (EPFL), Switzerland. 4. Service Régional Vaudois de Transfusion Sanguine, Unité de Recherche et Développement, Switzerland. Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopaminergic neurons and the presence of intracellular protein inclusions (Lewy Bodies) found in the brain of affected patients. Protein aggregation and post-translational modifications (PTMs), such as the site specific phosphorylation of alpha-Synuclein (α-Syn) protein have been reported to be strongly linked to PD pathogenesis. Therefore, pathologically modified α-Syn species represent a primary target for the diagnosis and treatment of PD. In this work, we aimed at conducting a comprehensive study, using multiple mass spectrometry and proteomics based approaches, to assess the chemical heterogeneity of α-Syn and to identify and map the pattern of α-Syn PTMs in plasma and red blood cells from PD and dementia with Lewy bodies (DLB) patients compared to healthy, age-matched control subjects. More specifically, we focused on the pattern of PTMs in the blood in order to identify if these modifications correlate with α-Syn PTM's observed in the brain and cerebrospinal fluid (CSF) during disease progression. The use of full-length, heavy isotope-labelled (15N) α-Syn protein and peptide standards with site-specific modifications, which mirror the key pathological PTMs of α-Syn found in PD, with targeted proteomics and selected reaction monitoring (SRM) mass spectrometry have enabled us to specifically identify and monitor single or multiple site-specific phosphorylations, N-terminal acetylation, truncations and splice variants of α-Syn. We have developed a multiplexed SRM assay which allows us to monitor several PTMs during a single analytical run. The identification of a specific isoform or PTMs pattern that correlate with PD or DLB could provide novel insights into the mechanism of the disease development, contribute to the identification of novel therapeutic targets and most importantly, could provide a diagnostic marker to detect and monitor the progression of PD and related synucleinopathies.

It is well established that intermittent exercises are very specific for performance in field and combat sports. However, few studies have examined the effect of sinusoidal oscillation in exercise intensity could maintain or ameliorates energetic coast. The aim of this work was to investigate if the variation of exercise allures (constant speed (CT-sp) vs. sinusoidal speed (SIN-sp) on physiological responses during submaximal exercise. Ten male footballers (182.6 ± 6.2 cm and 79.6 ± 6.4 kg) were volunteered to participate to this study. After measuring maximal aerobic velocity (MAV) and corresponding maximal oxygen uptake (VO2max) during the University of Montreal incremental test', subjects performed, in a randomized order, six test sessions of 10 min at different intensities (65, 75 and 85% VMA) in either CT-sp (a constant distance of 12.5 m between cones) or SIN-sp with an amplitude of 3 km.h-1 (alternating distances of 9.85 m and 15.15 m in each speed). Heart rate (HR), blood lactate concentration [La] and oxygen uptake (VO2) were determined during each test session. Results showed that HR, [La] and VO2 were higher during SIN-sp than CT-sp in the different exercise intensities. In addition, multiple linear regression was performed as below: Y = a *X1+ b * X2+ C with X2 corresponding to exercise allure (EA) (Vcte vs Vsin) as independent variable (taking the value of 0 or 1 ) to study the relationships: VO2 / VO2max = a * HR / HR max + b * EA + C, [La] / [Lamax] = a * HR / HR max + b * TE + C and [La] / [Lamax ] VO2 = a * / b * VO2max + TE + C. The statistical analysis shows that only the VO2/ VO2max and HR / HRmax were significant (P <0.001). The results of this study raise the question of the effectiveness of sinusoidal training allure on cardiorespiratory and metabolic parameters in football players.

Background & Objective: Cerebral aneurysms are one of the prevalent and devastating cerebrovascular diseases of adult population worldwide. The resulting effect is subarachnoid hemorrhage, intra- cerebral hematoma and other complications leading to a high mortality rate. When the aneurysm is fusiform, having wide neck or is large in shape, deploying stent in the parent artery to bypass aneurysm is considered as the most suitable treatment. The stent graft is designed to seal tightly with your artery above and below the aneurysm. The graft is stronger than the weakened artery and it allows your blood to pass through it without pushing on the bulge. So that blood cannot flow through the aneurysm to cause any future complication including rupture. Therefore, separation of parent artery from the aneurysm is immensely desired. This paper presents a method to separate parent artery from the aneurysm. Method: It has been challenging to distinguish the parent artery from the aneurysm geometry using a computer algorithm [1]. To date, only a few approaches to accomplish this task have been proposed. In our method, an initial surface mesh of the parent artery with aneurysm is first generated. Then the following steps are subsequently performed to separate the artery from the aneurysm. Step 1. The user specifies foreground and background on the mesh by placing centerline (Figure (a)) on the parent artery and aneurysm; it is also useful for generating Voronoi diagram (Figure (b) and (c)). Step 2. A feature preserving harmonic field based on the user specification is generated (Figure (d)). The resultant harmonic (i.e., "intensity") field over the artery geometry contains large variations not only at these concave and high curvature regions but also at the borders between the normal parent artery and the aneurysm. Since the parent artery centerline is nominally influenced by the presence of an aneurysm, the parent centerline is reconstructed; deviation is recorded and used while finalizing the average isoline or cutting boundary. Step 4. The isolines are generated with the help of Voronoi diagram (from which the average isoline is extracted); see Figure (e). We consider the isolines of the resultant harmonic field as the potential cutting boundary of the parent artery from the aneurysm. Along an isoline, the field variation is minimum. Step 5. A graph-based technique [2] is applied on the harmonic field to segment the parent artery from the aneurysm utilizing the average isoline and distance metric, where we define the energy function according to the harmonic field on the mesh. Results & Conclusion: For testing the method, we collected CTA slices with average thickness of 0.29mm, pixel spacing of 0.29mm x 0.29mm, and matrix size 512x512 on five subjects at the Hamad Medical Corporation using the Siemens Axiom Artis Interventional suite. The average time required by MATLAB R14 to perform segmentation is 2 m for one subject by a 2 GB RAM and core2duo processor (without optimization). Experimental results have shown satisfactory results for meshes with either simple or complicated model.

Background: Diabetic nephropathy (DN) is a chronic and serious complication associated with diabetes. The standardization of an in vitro model to best represent DN is very challenging due to the chronic nature of the condition. Therefore, two different renal tubule cell lines - Madin-Darby canine kidney cells (MDCK) and Normal rat kidney cells (NRK-52E) - were used to investigate the effects of high glucose on kidney cells. Objective: To determine the effects of high glucose concentrations on cell viability (using MTT assay), oxidative stress (using dichlorofluorescein (DCF) staining), and expression of proteins activated in DN such as aldose reductase and glucose-regulated protein-78 (GRP78), an endoplasmic reticulum chaperone (using western blotting). Results: MDCK cells showed a subtle decrease in viability when exposed to high glucose concentrations (30 mM and 1% FBS) for 48 h. Furthermore, there was a slight increase in aldose reductase expression after 48 h of high glucose exposure, however; the GRP78 levels remained unchanged. NRK-52E cells showed more consistent decrease in viability after 48 and 72 h of high glucose exposure (30 mM and 1% FBS). In addition, the DCF staining also demonstrated an increase in oxidative stress after 24 h of high glucose exposure. Furthermore, a 30% increase in aldose reductase expression has been observed after 48 h of high glucose exposure. Conclusion: Although the 48 h high glucose exposure in MDCK cells can be used as a model for in vitro DN, the results are less reproducible, whereas NRK-52E cells seem to be a better and more reliable cell line to mimic the features of DN in vitro. Key words: Diabetic nephropathy; In vitro; Kidney; Oxidative Stress; ER Stress.

Background. Insulin resistance and the prevalence of diabetes are high in the Middle Eastern population and in people of South Asian origin. Because of epidemic proportions that diseases have reached in these populations. It is important to find early markers along with preventative interventions. Recent data indicates that the metabolic defect in the pre-diabetic condition relates more strongly to post-prandial deficiency than to the fasted state. Women have lower levels of risk factors for metabolic disease when assessed in the post-absorptive state. However, very few reports have investigated these in the post-prandial state, especially amongst an Arab population. Objectives. This study investigated systemic cardiovascular risk factors both in the fasted and post-prandial state in a healthy, non-diabetic female local population. Methods. A cohort of young female, non-diabetic subjects representative of a general Qatari population was recruited. The study was approved by the national ethical committee and all subjects gave written informed consent. Subjects attended after an overnight fast and blood samples were taken prior to and 30 and 120 minutes after ingesting a liquid mixed meal. Anthropometric measures included age, height, weight, blood pressure and pulse. Bioimpedance was used to measure body fat (%, mass and distribution) andBasel Metabolic Rate {BMR},(Tanita MC-980). Glucose (hexokinase, Roche), lipids (Roche), insulin and proinsulin (Mercodia),Glucose like peptide1 {GLP-1},(Millipore) and adipokines (R & D Systems) were all determined. Data were analysed by SPSS version 22.0 for windows. Parametric tests were used for normally distributed data and non-parametric analysis for skewed dataare shown in the text as Mean {SD} or Median {Interquertal range }. Results. The subjects were young (Age 29.8 {4.9} years), non-obese (BMI 25.6 {4.7} kg/m2}, normotensive (systolic BP 109 {10} and diastolic BP 72 {6} mmHg) and normolipidaemic (Total-cholesterol 3.5 {0.7}, LDL-cholesterol 2.0 {0.4}, HDL-cholesterol 1.3 {0.3}, triglycerides 0.6 {0.2} mmol/L). Their total body fat was relatively high (34 {6.5} %), which was reflected by elevated levels of systemic leptin (30.8 {17.5-53.0} ng/ml) and lower adiponectin (8.1 {6.0-11.3} ?g/ml). Despite no apparent fasting or post-prandial hyperglycaemic and HOMA-IR levels being normal, post-prandial hyperinsulinaemia and hyperproinsulinaemia were significant (see Table). Proinsulin also constituted 19% of total insulin-like molecules. Discussion. Young normal-weight Qatari women with no apparent metabolic disease are hyperinsulinaemic and hyperproinsulinaemic in the fed state. Thus, elevated post-prandial levels of insulin-like molecules may be an early, sensitive marker for the metabolic defect that precedes cardiometabolic disease in this population. Table. Fasting and post-prandial concentrations of insulin-like molecules VariablesFasting30 minutes120 minutes Glucose (mmol/L)4.6 (0.3)5.0 (0.7)4.3 (0.5) Insulin (mIU/L)5.1 (4.0-6.3)50.5 (31.5-57.9)28.8 (23.2-37.4) Proinsulin (pmol/L)8.1 (5.7-11.4)22.3 (14.0-38.7)34.3 (22.0-49.6) GLP-1 (pmol/L)2.0 (1.8-2.4)8.4 (6.0-11.3)7.0 (3.3-9.6) Data ar shown as mean (SD) or median (interquartile range).

ABSTRACT Introduction Over the last 30 years the incidences of cancer and diabetes have been increasing progressively and there is both epidemiologic and experimental data linking diabetes and various cancer outcomes. Previous studies has shown that physical activity, height, and obesity; measured by weight, waist circumference, waist-to-hip ratio and body mass index were associated with the risk of diabetes and common cancer outcomes such as breast, colorectal and prostate cancer. However, there is little data on whether these modifiable risk factors are predictive of these malignancies among diabetics. The identification of factors that modify the risk of cancer among diabetics could lead to better surveillance or intervention to reduce cancer incidence among those at highest risk. Aim The aim of this study was to observe the relationship between anthropometric measures, including weight, height, and adiposity and physical activity with the risk of breast, colorectal and prostate cancer among diabetic individuals within the European Prospective Investigation into Cancer and Nutrition cohort. Methods Data from 27,365 diabetics from nine European countries between the ages of 20 to 85 years and a mean follow-up of 11.1 years from the European Prospective Investigation into Cancer and Nutrition (EPIC) was used. Of the 27,365 diabetics, 546 developed breast cancer, 363 developed prostate cancer and 308 developed colorectal cancer. Hazard ratios (HR) and 95% confidence interval (CI) were estimated using a Cox proportional hazard model, stratified by age in one year increments, gender and centre and adjusted for smoking status, alcohol consumption, education, BMI and physical activity. The HR was used to examine the association between anthropometric measures at recruitment and PA estimated from questionnaires with breast, prostate and colorectal cancer. Results There were no significant associations between height, weight, waist circumference waist-to-hip ratio, BMI and physical activity with breast, prostate and colorectal cancer when comparing the highest and lowest quartiles. However, there was a significant association between height and breast cancer when comparing the third quartile to the first (Q3 vs Q1, HR: 3.34, 95%CI: 1.09-10.22). Moreover, there was a suggestive inverse association between physical activity with breast and prostate cancer (P-trend 0.085 and P-trend 0.07, respectively). There was also a suggestive positive association between abdominal obesity and colorectal cancer (P-trend 0.04 for waist circumference and P-trend 0.08 for waist-to-hip ratio). Conclusion In this study of diabetic patients nested within the EPIC cohort, there was little evidence for an association between anthropometric measures and physical activity with breast, prostate and colorectal cancer. However, there was a suggestive association between physical activity with breast and prostate cancer and a suggestive association between abdominal obesity and colorectal cancer. Due to the limited number of cases in this study, further investigations between the associations of these modifiable factors with these cancers among diabetics are required.

This project focuses on developing a complete telemetry system in response to the growing demand for efficient, mobile and inexpensive system for detecting cardiovascular abnormalities. Our team has already developed two algorithms. The first algorithm detects various critical points on Electrocardiograph (ECG) waveforms such as: P-wave, QRS complex, T-wave and ST elevation. Based on these points, the second algorithm detects Myocardial Infractions (MI) in a patient. Currently, we are developing a telemetry system that comprises of an Arduino microcontroller and a smartphone. The 12-lead ECG signals are collected from a patient or an ECG simulator. The signals are then sent to an electronic circuit for amplification and filtration. The amplified and filtered signals are collected in the Arduino microcontroller. The microcontroller sends the signals, via Bluetooth, to an Android smartphone application developed by our team. The ECG data is sent from the smartphone to a webserver using 3G or Wi-Fi connection. The data is securely processed and analyzed in the webserver using the algorithms developed by our team. The processed ECG waveforms and the results of its analysis are sent back and displayed on the smartphone application. The analyzed results display whether the patient has a likelihood of having an MI. Furthermore, the analyzed results are stored in a secure database for future reference. Using the real-time analyzed ECG waveforms, the integrated system is designed to provide early warning of cardiac risk, thus saving valuable time and effort in patients' treatment. Along with that, this system will be efficient in terms of cyber security, cost management and reliability.

Background: Breast Cancer is one of the leading causes of cancer related mortality in women, both in Qatar and the world. Despite the available treatments the incidence of breast cancer is increasing. This highlights the need for new approaches for cancer. One of the fields that is gaining attention nowadays is herbal medicine. Herbs are known to have bioactive compounds that affect many diseases one of which is cancer. Origanum syriacum is an herb that is frequently used in Mediterranean region. Recently, it has been established that O. syriacum possess anti-proliferative activity in non-invasive breast cancer. Although it has some medicinal values, it remains poorly investigated. Here we tested the anti-tumor activity of O. syriacum extract (OSE) on the aggressive human breast cancer cell line, MDA-MB-231. Methods: The extract was prepared by dissolving the leaves of Origanum syriacum in water and drying it using rotarvapor. MDA-MB-231 cell viability was tested by MTT assay as well as trypan blue exclusion in the presence or absence of increasing concentrations of OSE. Scratch assay as well as Boyden-chamber were used to determine effect of OSE on migratory capacity. Furthermore, the ability of MDA-MB-231 to adhere to fibronectin was investigated using adhesion assay. Phosphorylated ERK1/2 was measured using Western blotting. Results: OSE reduced proliferation of MDA-MB-231 cells in a concentration and time dependent manner. The optimum concentration was determined according to the significance of decrease in viability. Also, in the presence of OSE, there was a decrease in migration of cells. Furthermore, a dose-dependent inhibition of adhesion was seen in MDA when treated with OSE. Moreover, preliminary results indicate that OSE decreased ERK1/2 phosphorylation in MDA-MB-231 cells. Conclusion: O. syriacum may be considered a supplementary drug for patients with malignant breast cancer. Further studies should be conducted to elucidate the molecular mechanism of the anti-cancer property exerted by OSE.