Qatar Foundation Annual Research Forum Volume 2012 Issue 1

Background and Objectives: The epidemiology of road deaths in Qatar has not been fully described. This study will analyze and compare the age-specific death rates from motor vehicle crashes (MVC's) and make recommendations for targeted injury prevention programs for road safety in Qatar. Method: Data from the Qatar Statistic Authority (QSA), for the year 2010 was collected and analyzed. All deaths classified as "motor- or nonmotor-vehicle accident, type of vehicle unspecified" were included. Age group populations were computed from age-specific crude death rates. Results: There were 247 MVC deaths in Qatar in 2010. An overall death rate was computed at 14.2 deaths per 100,000 population. The RRRM varied over ten times amongst different populations with Qatari males (QM) having an increased RRRM from 10 years of age. The QM's aged 20-29 had the highest RRRM of 10.2. The lowest RRRM was for Qatari females who did not have a single road fatality in 2010. Other populations with elevated RRRM (i.e. RRRM >1.0) were non-Qatari males ages 10 to 19 and older than 50 years. Conclusions: Qatari males have an elevated RRRM from the age of 10 onward, definite programs must be implemented to reduce these unnecessary deaths amongst the populations at the highest risk. Multidisciplinary approaches must be implemented and their efficacy evaluated.

Background and Objectives: Recent technological advancements in liquid chromatography coupled with high resolution mass spectrometry have facilitated clinical biomarker discovery, verification and validation. Targeted metabolic profiling refers to the precise quantitative measurements of metabolites for validation of markers identified via untargeted metabolomics. Specifically, multiple reaction monitoring mass spectrometry has enabled the reliable quantification of biomarkers in hundreds of samples, in a multiplexed manner. Methods: In this clinical study we have employed SID-MRM-MS (stable isotope dilution - multiple reaction monitoring - mass spectrometry) to quantify different classes of endogenous metabolites including intermediates of TCA cycle and lipids, on a triple quadrupole mass spectrometer. NPRP funded project enabled us to recruit a total of 169 controls and T2DM (type 2 diabetes mellitus) patients at the Hamad Medical Corporation in Doha, Qatar. Based on the discovery mode metabolomics profiling experiments, ten target molecules were chosen for biomarker validation using a cohort of 72 (control and diabetic) urine and plasma samples spiked with a known concentration of stable isotope labeled standard for each metabolite. The normalized peak area ratios were used to calculate the levels of deregulated metabolites in the T2DM cohort. We also performed extensive quality control experiments to check for retention time variation, matrix effects and metabolite degradation during sample processing. Statistical analysis was performed using GraphPad Prism (v5.0). ROC curves and interactive plots with a stringency cut-off of P ≤0.05 were plotted to test biomarker specificity and sensitivity. The cut-off values were selected to maximize the Youden index. Results: Statistically significant downregulation of succinate, xanthurenic acid, α-ketoglutaric acid and kynurenic acid were observed in the T2DM group while the concentrations of serotonin, PG (18:0/18:1), PC, sphingosine-1-phosphate and pyruvate were determined to be significantly higher in the diabetic group. The OPLS-DA model for the target metabolites had two orthogonal components and the R2 and Q2 were 0.88 and 0.87 respectively. Conclusions Our results underscore the clinical and translational utility of the MRM-MS approach. Further validation and characterization with larger cohorts, is likely to provide valuable insights into clinical potential of these markers and their correlation with T2DM associated complications.

Background and Objectives: The World Health Organization states that cardiovascular disease is the leading cause of death worldwide. The increasing prevalence of obesity and diabetes indicate that cardiovascular disease will remain a major health concerns for decades to come. The Gulf States including Qatar, the Middle East and the North African (MENA) region as whole, but also elsewhere in the world, face a pandemic of obesity and diabetes. Hyperglycaemia is the common denominator of both type-1 and type-2 diabetes and results in "glucose toxicity" that is closely linked to the high cardiovascular morbidity and mortality associated with diabetes. A reduction in the generation of NO -an important contributor to the endothelium regulation of blood flow and coagulation- in response to hyperglycaemia, is an early indicator of the onset of vascular disease. NO bioavailability is determined through the balance of its generation by eNOS and its quenching by reactive oxygen species, ROS. In response to hyperglycaemia and a variety of metabolic perturbations, eNOS produces predominately superoxide anions rather than NO and there is therefore an increase in oxidative stress and reduced bioactivity of NO. MicroRNAs (miRs) are small non-coding RNAs that inhibit gene expression and have also been implicated in the regulation of endothelial cell biology. A number of miRs including 221 & 222 have been implicated in endothelial cell function. They are involved in post-transcriptional control of major regulatory pathways. Methods: Mouse microvascular endothelial cells (MMECs) were cultured in normal (11mM) or high glucose (HG, 40mM) media and the ratio of coupled (dimeric) to uncoupled (monomeric) eNOS determined by immunoblot. ROS, NO and the expression of miR-221/miR-222 were also measured. Results: HG enhanced ROS, reduced the eNOS dimer/monomer ratio and reduced the generation of NO. Our data indicate that HG-induced ROS generation can be reversed in the presence of inhibitors of miR-221/miR-222. Conclusion: Hyperglycemia is associated with increased oxidative stress and decreased NO bioavailability. miR-221 and miR-222 play an important role in high glucose-induced increased oxidative stress and endothelial dysfunction.

Background & Objectives Point Prevalence Surveys (PPS) are used internationally for identifying antibiotic prescribing practices and evaluating antibiotic stewardship programs. The objectives of this study are to develop a PPS tool to be used in Qatar, to determine prevalence of antibiotic consumption in the National Center for Cancer Care and Research (NCCCR) inpatient population, and to characterize antibiotic prescribing practices. The secondary goal is to identify targets for antibiotic stewardship programs to improve prescribing practices. Methods A chart audit tool was designed based on the available literature and piloted for face validity. All adult inpatients receiving active systemic antibiotic prescriptions at 8:00AM on each audit day were surveyed. Data were collected on 3 separate days over a two week period from 26 April through 3 May 2012 at NCCCR. Collected data obtained from electronic and paper-based charts included: diagnosis, type, dose and frequency of antibiotics, route of administration, and duration of therapy. Further information such as compliance to the available local guidelines and microbiology results were also assessed. Results The overall prevalence of antibiotic use during the audit was 43% (25/58). A total of 33 antibiotics were prescribed to the 25 patients receiving systemic antibiotic therapy. An indication for antibiotic prescribing was documented in 80% (20/25) of patient charts, however, only 20% (5/25) reported duration of antibiotic therapy. The most frequently used antibiotics were Penicillin/β-lactamase inhibitor combinations 40% (13/33), followed by carbapenems 15% (5/33). In 2 out of 6 febrile neutropenic patients, local febrile neutropenia guideline was accurately implemented. Sixty one percent (20/33) of prescriptions complied with local antibiotic restriction guideline. Pre-therapy cultures were performed for 96% (24/25) of patients and all antibiotic choices matched results of available sensitivity tests performed for these patients. Conclusions The findings of this study demonstrate frequent antibiotic consumption in the immunocompromised population highlighting the importance of development, implementation, and expansion of antibiotic stewardship programs at NCCCR.

Background and Objectives: Obesity is a major risk factor for the development of liver disease and diabetes and there is currently a pandemic of diabetes that is associated with a very high incidence of non-alcoholic fatty liver disease and consequent deregulation of liver function. The overall objective of this project is to determine whether non-alcoholic fatty liver disease and hyperglycemia affect calcium homeostasis via altering the expression of the plasma membrane calcium release-activated calcium channel protein, Orai1, the endoplasmic reticulum calcium sensor protein, STIM1, and store-operated Ca2+ entry, SOCE, in rat hepatocytes. Methods: Rat H4IIE liver cells exposed to amiodarone were used as a cell culture model of steatosis and Western Blot, RT-PCR and fura-2 techniques were used to assess protein, mRNA and changes in Ca2+ homeostasis, respectively. Rat H4IIE liver cells were grown either in normal glucose (5.5 mM) or high glucose (25 mM) for 24 hours, then each group was treated with either vehicle (methanol), amiodarone for 24 hours, or amiodarone for 24 hours followed by normal glucose for another 24 hours. Intracellular calcium [Ca2+]i was measured using fura-2am. Results: Expression of both STIM1 and Oai1 increases after treating with amiodarone for 24 hours in both normal and high glucose, but decreases after treating with amiodarone for 24 hours when followed by normal glucose treatment. In addition high glucose enhances SOCE whereas amiodarone treatment suppresses SOCE. Conclusion: Changes in the concentration of intracellular Ca2+ in hepatocytes play a central role in mediating the actions of insulin, glucagon, catecholamines and other hormones and growth factors on carbohydrate, lipid and protein metabolism in the liver. Both hyperglycemia and steatosis result in alterations in Ca2+ homeostasis suggesting that such changes may be contributing factors by which hyperglycemia, obesity and fatty liver result in insulin resistance in the liver. The results from this UREP project may lead to a better understanding of the mechanisms whereby diabetes, obesity and fatty liver disease result in liver malfunction.

Hepatocellular carcinoma (HCC) is the most common liver cancer and one of the commonest solid malignancies. High mortality rates and poor prognosis of the disease are mainly due to late diagnosis, underlying cirrhosis and resistance to chemotherapy. The risk factors of HCC include infections with hepatitis B and C viruses, along with other conditions that cause cirrhosis like: alcoholism and non-alcoholic steatohepatisis. Wnt/ß catenin signaling pathway plays a vital role in regulating the cell fate during embryogenesis and cell proliferation in adult tissues. In the absence of the Wnt ligand, beta-catenin is phosphorylated through interaction with GSK-3b, APC, axin and subsequently degraded by the ubiquitin-proteasome system. However, when the Wnt ligand binds to the receptor complex of the pathway, the destruction complex is inactivated leading to the accumulation of beta-catenin in the cytoplasm and its translocation to the nucleus where it forms complexes with TCF/LEF family that causes transcriptional activation of target genes. Recent studies suggest that cancer stem cells play a key role in liver carcinogenesis. Mutations in beta-catenin and activation of Wnt/ß-catenin signaling pathway is likely to cause abnormal proliferation and enhanced self-renewal of hepatic progenitor cells resulting in their transformation into cancer stem cells. Thus understanding the characteristics and function of liver cancer stem cells and their response to beta-catenin inhibitors is crucial to the development of novel, more effective drugs and improving patient survival. In this study we have used various drugs to target Wnt/beta-catenin signaling pathway in hepatic carcinoma cell lines. We assessed the effect of the inhibitors of beta-catenin on the expression of stem cell markers in these cell lines and will present the results of the study.

Background and Objectives: Congestion in outpatient departments (OPD) in Qatar's public hospital system is a major problem due to the demographics of the country. Despite the congestion in the general registration and assessment areas, the utilization of doctors' time is considerably low. This paradigm is propagated by (i) the inefficiencies in the appointment system, (ii) the allocation of the resources in the service design, and (iii) behavior patterns of the patients. Methods: We investigated the problem through a case study in a public hospital. First, through a careful walk-through of the patients' flow process, we identified the disruptions in the patient flows using lean principles. Simultaneously, through extensive data collection, we measured the patient flow times and resource utilization, and classified the value added- and non-value added times for the patients. Based on the observations and data collected, we propose changes for the appointment systems, patients' flow process, and allocation of resources. We benchmark the impact of our suggestions through simulation. Results: The improved solutions reduce the patient flow times by 30%. Conclusions: The patients' flow experience in public hospitals' OPDs can be significantly improved through better design of the appointment system and by eliminating wasted time in the patient flow process without investing in more resources.

Background: Previous studies indicated changes of adiponectin levels during gestation. The adiponectin gene ADIPOQ is located on chromosome 3q27. The association of two single nucleotide polymorphisms (SNPs) (rs1501299 and rs2241766) in ADIPOQ gene with risk of gestational diabetes mellitus (GDM) was investigated among Arab pregnant women residing in Qatar. Methods: A case-control association study was performed on 115 pregnant women with GDM and 130 controls from Qatar. Genotypes were determined using TaqMan real time PCR assay. Fasting serum c-peptide, leptin and adiponectin levels were determined using Multiplex ELISA technique. Results: All SNPs were within the Hardy-Weinberg Equilibrium (HWE). The frequency distribution of the genotype 276 G-T (rs1501299) revealed that (40.9%), (45.6%), had GG and (53.0%), (44.0%) had GT, and (6.1%), (10.4%) had TT between GDM and control, respectively with P value= 0.261. The frequency distribution of the genotype 45T-G (rs2241766) revealed that (20.8%), (16.1%), had TT and (69.6%), (65.4%) had TG, and (9.6%), (18.5%) had GG between GDM and control, respectively with P value= 0.117. T and G alleles were the minor alleles for 276 G-T and 45T-G with a frequency of (0.11) and (0.22), respectively. Using recessive genetic model, the logistic regression analysis reveled that the Odds ratio and 95% CI was 2.14 (1.01-4.95), p=0.04 for 45T-G and was 1.79 (0.69-4.65), p=0.22 for 276 G-T. Serum c-peptide (ng/ml), and leptin (ng/ml) was significantly lower in subjects having GG versus TT+TG alleles of 45T-G with mean and SEM (7.6±1.8 Vs. 17.3 ±1.2, p= 0.002), and (21.5± 2.5 Vs. 42.1± 2.8, p=0.035). Similar significant findings were observed for TT versus TG+GG genotypes for SNP 276G-T. Adiponectin levels (µg/ml) were not significantly different in subjects having GG versus TG+TT genotypes for 45T-G and for TT versus TG+GG genotypes for 276G-T with mean and SEM (17.1± 1.8 vs. 17.2±2.3, p=0.892 and 16.5±3.24 Vs. 19.8 ±1.8, p= 0.478), respectively. Conclusion: GG carriers of 45T-G may increase the odds of getting GDM among Arab residents in Qatar. Decreased pancreatic secretory function (C-peptide) with increased adiposity (leptin) among GG carriers of 45T-G and TT carriers of 276GT may explain its roles in the development of GDM.

An efficient method to generate cardiac tissue from other tissues has great therapeutic potential for patients suffering from cardiovascular disease. Pluripotent stem cells, such as embryonic stem (ES) cells, and so-called induced pluripotent stem (iPS) cells can be differentiated into multiple cell types, including cardiac myocytes. However, therapeutic use of these cells has several important risks, including cancer as well as loss of differentiated cell identity and function or "drift." Transdifferentiation, the generation of cell types from other differentiated cell types, is an attractive alternative because it poses little risk of cancer and may give rise to cells whose identity is locked in. However, the direct reprogramming of adult fibroblasts into cardiac myocytes is inefficient and the transcription factors that drive this process are unknown. To circumvent these hurdles we hypothesized that fetal derived cells may be more amenable to reprogramming into cardiac myocytes with defined transcription factors. To this end, fetal derived fibroblasts and mesenchymal cells were transduced with transcription factors that have been shown to play a role in myocyte specification and maintenance, including GATA4, Mef2c, Tbx5 and Nkx2.5. To test this hypothesis, the transcription factors were cloned into lentiviral vectors, which were used to infect fibroblasts and mesenchymal cells. Infected cells were then cultured in different media. To assess the efficiency of the reprogramming, RNA was extracted from infected and uninfected cells, and quantitative RT-PCR was used to evaluate the expression levels of the transcription factors and known cardiac genes. We expected to observe higher levels of the transcription factors and cardiac markers in the infected cells relative to the uninfected cells. The results showed that the infected cells expressed higher levels of the transcription factors and a few cardiac markers relative to the uninfected cells. However, due to the small increase in the levels of only a few cardiac markers, the reprogramming was concluded to be inefficient. To make the reprogramming more efficient, other as yet unrecognized transcription factors possibly in the GATA family of transcription factors and culture conditions are currently being considered.

Background: Calnexin is a lectin-like chaperone in the endoplasmic reticulum (ER) lumen, which along with calreticulin are involved in folding, maturation and trafficking of many glycoproteins. Insulin receptor and glucose transporters are among some of the proteins which are synthesized and folded in the ER. Previously, calreticulin was reported to play a role in the folding of GLUT-4 transporter as well as its stability. Furthermore, our lab reported that loss of calreticulin function results in increased insulin receptor synthesis, increased phosphorylation of Akt upon stimulation by insulin and increased glucose uptake. To date no data are available on the changes in insulin receptor pathway upon loss of calnexin chaperone. Objectives: The aim of this study is to examine changes in insulin receptor expression and insulin-mediated phosphorylation of Akt upon loss of calnexin function. Methods: Mouse embryonic fibroblast cells were serum starved overnight, then stimulated with insulin for 10 mins at 37°C. Cell lysate were prepared and Western blots with antibodies of different proteins in insulin signaling pathways were performed. Results: Our results showed a significant increase in insulin receptor expression in calnexin deficient cells. Furthermore, we observed a significant increase in the phosphorylation of Akt illustrating activation of insulin receptor pathway upon loss of calnexin function. Conclusions: In conclusion our data illustrates that loss of either of lectin like chaperones (calreticulin and calnexin) results in the activation of insulin receptor pathways via release of a negative suppressor of insulin receptor gene expression. Further research is needed to decipher the mechanism of this regulation.