Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1

In Egypt, β-thalassemia is the most common hereditary hemolytic anemia. Cardiac dysfunction, secondary to iron overload with formation of oxygen free radicals, is the most common cause of death in β-thalassemia patients. This study was designed to determine whether the allelic genotype of apolipoprotein E (Apo E), which exhibits antioxidant properties, could represent a genetic risk factor for the development of left ventricular (LV) dysfunction in β-thalassemia major. Fifty Egyptian β-thalassemia major patients were subjected to echocardiography to assess LV function. Apo E genotyping by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was done for all patients in addition to 50 age and sex matched healthy control subjects. Patients were classified into three groups. Group I and II were clinically asymptomatic. Group II subjects had evidence of LV dilatation, while Group III patients had clinical and echocardiographic findings of LV failure. Apo E4 allele was significantly higher among Group II and III than in controls. In conclusion, Apo E4 allele can be considered as a genetic risk factor for LV dysfunctions in β-thalassemic patients. It could be used as predictive indicator for additional risk of LV failure, particularly in asymptomatic patients with LV dilatation, requiring a closer follow-up, to prevent further disease progression

Environment Gene Interaction in Parkinson's disease (EGI-PD) Mohamed M. Salama1, Thomas W. Rösler2, Ali Shalash3, Abdelhaleem Tantawy4, , Günter U. Höglinger2,5 1Toxicology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt 2Department for Translational Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 3. Department of Neurology, Faculty of Medicine, Ain Shams University, Cairo, Egypt 4. Department of Neurology, Faculty of Medicine, Mansoura University, Mansoura, Egypt 3 Department of Neurology, Technical University, Munich, Germany Introduction: The prevalence of PD is increased in Egypt compared to reports in other countries. Both genetic and environmental causes might be responsible. E.g. a high rate of LRRK2 mutations has been reported in other North African countries, but has not been systematically studied in Egypt. Also, several epidemiologic studies have supported the hypothesis that broadly defined pesticide exposure may increase the risk of PD. A high degree of pesticide use occurs in Egypt due to the limited availability of agricultural areas in the country. Moreover, lack of precautionary measures on applying pesticides leads to increased pesticide exposures. We aimed to study the link between genes encoding detoxifying enzymes and PD-risk genes on one hand and exposure to pesticides on the other hand in the etiology of PD in Egypt. Methods: This work is undertaken in collaboration between the Technische Universtität München, Germany and a network of 16 Egyptian Universities leaded by Mansoura University (Egyptian Network of Neurodegenerative Diseases [ENND]). PD cases from all over Egypt are recruited. Half of the cases were chosen in areas of low pesticide exposure, the other half with high pesticides exposure (farmers 50 years of age or older). Similar numbers of non-PD controls are chosen from two sources; first, patients arriving to outpatient clinic who are not suffering from any other neurodegenerative disorder; secondly, persons accompanying participating PD patients. Controls are further stratified into low pesticides and high pesticides controls, as described for the PD cases. Patients are assessed with a standardized questionnaire to evaluate past exposure to pesticides or other Parkinson-related environmental factors, a standardized clinical neurological examination to verify presence of PD (UK brain bank criteria) and to quantify disease severity (Hoehn & Yahr stage, UPDRS motor part) and genotyping for presence or absence of risk-carrying alleles in detoxification and Parkinson's associated genes. Results: We were able to establish ENND which is the first collaborative network of this type in Egypt. Moreover, we established a functional Standard Operation Procedure to collect and ship clinical data and blood for analysis to the German cooperation partner at TUM. So far, we collected blood and epidemiological data from 70 cases and 80 controls. Preliminary data on the analysis of gene-environment interaction analysis will be presented. Conclusion: This work will provide further insights into the mechanisms of gene environment interplay in the etiology of PD and hopefully insights which allow the establishment of primary preventive measures. Funding: DAAD-funded Al-Tawasul project EGI-PD

Objective: We hypothesized that nebulized magnesium sulfate added to combined bronchodilator and systemic steroid therapy would shorten time to discharge without undue risk. Study design: Patients aged 2 to 14 y with moderate and severe asthma (PRAM severity score >4) admitted to infirmary care were randomized double-blind to 800 mg nebulized isotonic magnesium sulfate or normal saline placebo via Aeroneb Pro and Idehaler, after intensive therapy with combined albuterol-ipratropium and intravenous methylprednisolone. Time to medical readiness for discharge was the primary outcome. Improvement over time in PRAM severity score and other secondary outcomes were compared for the overall group and severe asthma subset. Results: 191 magnesium sulfate and 174 placebo patients met criteria for analysis. The groups were similar with mean baseline PRAM scores>7. Blinded active therapy significantly increased blood magnesium level 2 h post-treatment 0.85 (SD 0.07) vs 0.82 (SD 0.06) mmol/L, p=0.001). There were no important adverse effects. Accelerated failure time analysis showed a non-significantly relatively shortened time to medical readiness for discharge of 14% favoring the magnesium sulfate group, OR=1.14, 95% CI 0.93 to 1.40, p=0.20, with an absolute prolonged time at 24 h of 2.1 h, p=0.5 . Mean times until readiness for discharge were 14.6 h [SD 9.7] vs 15.6 h [SD 11.3] for the investigational and placebo groups, respectively, p=0.9. Conclusions: Adding nebulized magnesium sulfate to combined nebulized bronchodilator and systemic steroid therapy is either very weakly or very rarely effective, or futile for benefitting pediatric patients with moderate or severe asthma.

OBJECTIVE: Differentiation capacity of human preadipocytes exhibits depot specific variation and is influenced by local cytokine production. The effect of obesity-induced insulin resistance on preadipocyte differentiation and its relation to proinflammatory cytokine release was investigated. RESEARCH DESIGN AND METHODS: Differentiation of preadipocytes obtained from subcutaneous & omental tissue biopsies isolated from obese patients undergoing weight reduction surgery and their cytokines release were compared between insulin sensitive and insulin resistant subjects. RESULTS: Compared to subcutaneous-derived cells, omental cells expanded from the same patients showed diminished differentiation, marked by increased proinflammatory cytokines secretion. When subjects were dichotomized into insulin sensitive & insulin resistant depending on their HOMA-IR, preadipocytes derived from insulin sensitive subjects exhibited a greater ability to differentiate into larger adipocytes with more lipid droplets, marked by a lower expression and secretion of IL-6 and TNFα. Addition of these cytokines inhibited preadipocyte differentiation in insulin sensitive-derived cultures, suggesting that proinflammatory cytokines may underlie inhibition of differentiation seen in insulin resistant patients-derived cultures. CONCLUSIONS: These findings suggest that insulin-resistance is associated with impaired preadipocyte differentiation and increased proinflammatory cytokines release. Understanding mechanisms underlying impaired preadipocyte differentiation associated with insulin resistance may help future treatment strategies.

Previous studies have shown that low intensity pulsed ultrasound (LIPUS) can prevent orthodontically induced teeth root resorption (OITRR) in human. Also, stem cells have been used to treat different types of bone defects. Severe OITRR is still untreatable problem in orthodontics. The aim of this study was to evaluate the effect of local injection of osteogenically induced gingival stem cells (OIGSCs) and LIPUS on periodontal ligament (PDL) during tooth movement that induces OITRR in beagle dogs. We hypothesized that local injection of OIGSCs and LIPUS can enhance PDL metabolism and hence enhances the reparative effect of OITRR. Seven adolescent beagle dogs were used and their third and fourth premolars were moved orthodontically. Gingival stem cells were isolated, characterized by flowcytometry and were differentiated into osteoblast -like cells using osteogenic medium to produce osteogenically induced gingival cells (OIGCs). OIGSCs were then re-injected into the alveolar bone in the proximity of the roots of the orthodontically moved teeth. Premolars were randomly divided into five groups. 1) Negative control (OITRR only); 2) Local injection of BMP; 3) OIGSCs; 4) LIPUS and 5) LIPUS +OIGSCs. In LIPUS groups, premolars were treated for four weeks. Animals were then euthanized and tissue blocks were processed for histological and histomorphometric analysis. Cementum thickness, PDL thickness and PDL cell number were counted using Metamorph software. Measurements were made at three levels of the tested roots. Level 1 is the coronal level 9towards the crown); level 2 (middle level of the root) and level 3 (apical, towards the apex of the roots). Variables were compared between groups by Wilcoxon Signed Ranks Test using SPSS statistical package. Results showed that LIPUS, BMP2 and LIPUS+OIGCs significantly increased cementum thickness compared to control group in the apical area of the teeth roots (P<0.05). There was statistically significant increases in PDL thickness and PDL cell count in all groups compared to control group (P<0.05). The combined LIPUS+OIGCs showed the highest cell count between al the groups. Conclusion: LIPUS + OIGCs showed the highest PDL regeneration potential and may be used in clinical cases with severe OITRR.

Cdc25C is a member of the dual specific protein phosphatase family capable of dephosphorylating both phospho-Tyr and phospho-Ser/Thr residues. In vertebrates, there are three isoforms of the Cdc25 enzyme (Cdc25A, Cdc25B and Cdc25C). The N-terminal (regulatory domain) region of these three enzymes is highly divergent (~20-25% identity) while the C-terminal (catalytic domain) region is more conserved (~60% identity). The Cdc25C isoform is of particular importance because it dephosphorylates Cdc2, which is part of the Cdc2/Cyclin B complex, allowing the cell to transition into mitosis. We want to gain further insight into how Cdc25C interacts with its substrate Cdc2. One way to do this is to utilize the ability of E. coli to heterologously express recombinant Cdc25c. This will allow us to obtain large amounts of the protein for in vitro characterization. First, several E. coli cell lines were tested (each with unique properties; e.g. expression of toxic protein, expression of disulfide rich proteins) for their ability to overexpress Cdc25C. The cell line and expression condition having the highest yield of full-length protein was chosen and purified using a single immobilized metal affinity chromatography (IMAC) column that will bind a 6xHis affinity tag fused to the N-terminus of Cdc25C or a tandem approach that utilizes two affinity tags (an N-terminal 6xHis affinity tag and a C-terminal StrepII tag). The pure protein will then be subjected to a variety of chromatography techniques such as UV-Vis spectroscopy and circular dichrosim (CD) to characterize structural changes the protein undergoes when binding to its zinc cofactor. Site directed mutagenesis will be used to create mutations in the zinc binding region of Cdc25C and these proteins will also be characterized for their ability to bind or not to bind the zinc cofactor.

Abstract Nanobiotechnology has been recently widely applied in multidisciplinary fields including pharmaceutical applications. The last decade has witnessed an increase research interests focused on the biosynthesis of metal nanoparticles from fungi as a natural sources and as bionanofactories. However, silver nanoparticles (AgNPs) are of great importance particularly in medical therapy applications and can be used as antimicrobial agent. The objective of this study was to biosynthesize silver nanoparticles from the soil fungus Curvularia tuberculata and to examine their efficacy against five strains of pathogenic bacteria namely; Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus using agar well diffusion technique. The synergistic efficacy of biosynthesized silver nanoparticles in a combination with commercially used antibiotic Gentamycin against the selected bacteria was also examined. The biosynthesized silver nanoparticles from fungal free-cell filtrate were characterized by using UV-Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). UV-Vis spectrophotometer analysis revealed a peak at 420 nm indicating the synthesis of silver nanoparticles. FTIR analysis verified the detection of protein capping of silver nanoparticles while SEM micrographs showed that the synthesized silver nanoparticles are dispersed and mostly having spherical shape within the size range between 10-50 nm. The biosynthesized silver nanoparticles possessed a varied growth inhibition activity ranged between 12- 28 mm diam inhibition zones at different concentrations against the tested pathogenic bacterial strains. A remarkable increase of bacterial growth inhibition (25.5-35.5 mm diam) was detected when a combination of silver nanoparticles and Gentamycin was used. A significant increase in fold area of antibacterial efficacy was observed when AgNPs in combination with Gentamycin was applied as compared with the efficacy of Gentamycin alone. The biosynthesized AgNPs did not show any toxicity against human blood. The synthesized silver nanoparticles by the fungus C.tuberculata is promising to be used as an antimicrobial agent in medical therapy due to their broad spectrum efficacy against pathogenic bacteria. Nevertheless, further studies are needed to investigate the activity of silver nanoparticles as antibacterial agent in vivo.

Worldwide 1.24 million deaths occur annually due to road traffic injuries. Additionally, 20-50 million are injured or disabled. Similarly, within the Gulf Cooperation Council countries injury and mortality due to road traffic injury are high. In the State of Qatar, for example, the road traffic fatality rate for the year 2010 is estimated to be 14 per 100,000. Young adult males are disproportionately affected. Road safety risk factor laws namely, seat belt use, driving under the influence of alcohol, helmet use and speed management exist in Qatar but enforcement is lax. An additional new risk factor causing distraction while driving and causing road traffic injury and fatalities is the use of mobile phone whilst driving a vehicle. Currently, in Qatar baseline national data pertaining to seat belt and mobile phone use among drivers is not available. We have thus conducted an observational study to estimate the prevalence of these two variables among vehicle drivers in Doha. Additionally, we will examine and present the associations between phone and seat belt use, and variables such as the gender of the driver, type of vehicle and the time of the day during which the observations were made. Furthermore, policy implications of the study findings will be discussed. Acknowledgement: This work is supported by the Biomedical Research Program at Weill Cornell Medical College in Qatar, a program funded by Qatar Foundation.

One of the key objectives of the surveillance of communicable disease (CDs) is the identification of areas for research and healthcare system improvement. In a recent paper published in Qatar Medical Journal (http://dx.doi.org/10.5339/qmj.2014.9) is shown the delay in diagnosis of CDs of cases reported at The Cuban Hospital (TCH) during 2012 and 2013. Examples of delay in diagnosis was for tuberculosis 61.7 days, acute hepatitis 18.5 days, typhoid fever 17 days, food poisoning 9.5 days, measles 8.0 days and meningitis 3.8 days. These are diseases that require immediate reporting because of their public health importance and some of them are highly transmissible (e.g. tuberculosis and measles). Additional evaluation of patients with tuberculosis admitted at The Cuban Hospital (many reported in Hamad General Hospital and referred to TCH because of bed crisis) from January 2013 to June 2014 (105 patients) show diagnosis delay of 49.5 days (standard deviation 51.9 years) with a maximum figure of 365 days. In patients with positive smear (highly infectious) the delay was 49.7 days (SD 53.5 days) (minimum 2 days, maximum 365 days). The delay was superior in cases with pulmonary tuberculosis (51.3 days (SD 52 days)) than in non pulmonary (36.1 days (SD 51.9 days). Delay in diagnosis of CDs is significant with regard to not only disease prognosis at the individual level but also transmission within the community. The delay could be divided in ¨patient delay and system delay¨. Patient delay refers to the time between onset of symptom and the first contact with a healthcare professional. The system delay refers to the time between this first contact to the diagnosis or confirmation of the disease. The knowledge of the two components of the delay is essential to identify actions to minimize the delay and reduce the probability of disease transmission at the community. In summary data from the surveillance of CDs suggest the need of research to identify the causes of diagnostic delay and subsequently implement actions its reduction, which will contribute to the prevention and control of CDs in Qatar.

Human endogenous retroviruses (HERVs) have been classified into three different classes I, II, III. Betaretrovirus-like sequences, including HERV-K, belong to the class II and contains ten groups, termed HML1-10. It has been reported that an HML-10 sequence, termed HERV-K(C4), is inserted within the gene coding for the human complement C4 in an antisense orientation (Tassabehij et al. 1994). The expression of the HERV-K(C4) RNA has been hypothesized to possibly modulate C4 expression as well as to act as a possible mechanism of defense against retroviral infections (Schneider et al 2001). Recently, it has been demonstrated that low HERV-K(C4) copy number is associated with type 1 diabetes, raising the possibility that this retroviral element contributes to functional protection against this autoimmune disease (Mason et al. 2014). Given the correlation between lower HERV-K(C4) copy number and expression, it has also been proposed that HERV-K(C4) may influence C4 RNA processing and/or have an impact on other endogenous retroviral sequences (Mason et al. 2014). To have more insights into this hypothesis, we identified, lassified and characterized the HML-10 sequences that are present in the human genome GRCh37/hg19 assembly. We used the model-based software Retrotector (Sperberg et al 2007) to search HML-10 sequences in the human genome assembly GRCh37/hg19 and identified 10 HML-10 proviruses in chromosomes 1, 6, 16, 19 and Y. These proviruses were confirmed to belong to the HERV-K (HML-10) group and shown to have the Rec sequence. Out of the 10 identified HML-10s, 5 sequences have both LTRs, 4 have only 3'-LTR and 1 sequence is without LTRs. Age estimation analysis performed on the 5 sequences with both LTRs indicated that they inserted into the human genome >25 Mya ago. Five sequences conserved the primer binding site (PBS) region, all recognizing the Lys tRNA. Translational analysis showed that all proviral sequences have multiple stop codons that preclude their production of functional proteins. Phylogenetic analyses were performed with complete DNA sequences as well as with the Pol amino acid sequences and will be presented. Overall, we have identified and characterized 10 HML-10 sequences in the human genome GRCh37/hg19 assembly. Precise knowledge of all HML-10 sequences will allow further investigation of their physiological and pathological role in autoimmune diseases (Yu & Whitacre 2004), particularly their possible involvement in type 1 diabetes, giving new insights into their understanding. References 1. Tassabehij et al. NAR 22, 5211-17 (1994). 2. Mason et al. Diabetes 63, 1789-95 (2014). 3. Sperber et al. NAR 35, 4964-76 (2007). 4. Yu & Whitacre Trends in immunology 25, 694-99 (2004).