Qatar Foundation Annual Research Forum Volume 2012 Issue 1

Objectives: Human papillomavirus (HPV) is the most commonly known sexually transmitted agent. To date, few reports are available on the distribution of most prevalent and variant types of HPV in Arab women. Therefore, the aim of this study is to determine the age-specific distribution of HPV types among Arab women being subjected to routine pap smear tests in the State of Qatar. Methods: 1100 pap smears have been collected in ThinPrep vials (BD SurePath™) from Arab women seeking routine gynecological care at the Women's Hospital, Hamad Medical Corporation (HMC) in Doha, Qatar. All the samples were transported to the laboratory in an icebox container. Viral DNA from ThinPrep samples was extracted by QIAamp MinElute virus spin kit according to manufacturer's instructions and was screened for HPV DNA by real-time PCR using L1 HPV specific (GP5+/6+) primers. The type-specific distribution of the viruses was determined by HPV high and low risk typing RT-PCR kits (Sacace Biotechnology, Italy) and PCR-based sequencing. Real-time PCR amplification was carried out in ABI 7500 real-time PCR (Applied Biosystems). Results: Based on the collected data, HPV DNA was detected in 125 women (11.36%), and 15 different HPV genotypes were detected, comprising high-risk and low-risk genotypes. The prevalence of HPV infection was seen in 54.5% Qatari and 45.5% non-Qatari women. With regard to age, 33.5% of all HPVs were found in women 30-39 years of age, 24.8% in women 40-49 years of age, 15.8% in women 50-59 years of age, 5.5% in women over 60 years and 20.4% in women less than 30 years old. Implications and Impact: The study shows that the prevalence of HPV infection in Arab women in Qatar is among the highest in the Arab world compared with previous reports. However, more extensive population-based studies should be undertaken before implementing HPV vaccination.

Background and Objective: The State of Qatar has achieved maternal, neonatal and perinatal survival rates which are comparable to many high income countries, both from the West and East. Our study aims to analyze fetal and perinatal determinants of Qatar's neonatal mortality rate (NMR) during 2011. Methodology: A PEARL Study (Perinatal Neonatal Outcomes Research Study in the Arabian Gulf), a joint collaborative research project between Hamad Medical Corporation (HMC) Qatar and University of Gloucestershire United Kingdom, is Qatar's prospective national perinatal epidemiological Study funded by Qatar National Research Fund. The study is quantifying maternal, neonatal and perinatal mortality, morbidities and their correlates by establishing a national neonatal perinatal registry for Qatar called Q-Peri-Reg. Data on live births and neonatal mortality were collected from all public and private maternity facilities in Qatar during 2011. Data on fetal and perinatal determinants was ascertained from maternal obstetric and delivery room record on predesigned performas. Univariate and multivariate regression analysis was done using Epi Info and SPSS-20. Results: Qatar's NMR during 2011 was 4.9. The incidence of low birth weight in Qatar is 11% and the incidence of preterm deliveries 10.7%. 10% of the babies required delivery room resuscitation. The relative risk of neonatal mortality was higher and statistically significant with fetal growth (p<0.001), fetal weight at birth (p<0.001), fetal gestation at birth (p<0.001), APGAR score at 1 and 5 minute (p<0.001) and the need for delivery room resuscitation (p<0.001). The RR of neonatal mortality increased (Table 1) with decreasing birth weight (p<0.001) and gestational age (p<0.001). Conclusion: Further improvement in Qatar's neonatal mortality is possible by addressing the high incidence of low birth weight and preterm deliveries. Better maternal nutrition, improved antenatal care, birth spacing and best obstetric and neonatal practices at delivery are likely to be most helpful.

Background: The AGE reader is a non-invasive device that measures tissue accumulation of advanced glycation endproducts (AGEs) through skin auto-fluorescence (AF) and gives prediction of cardiovascular risk. For the risk prediction, the AGE reader uses a single reference curve, for both females and males, obtained from Caucasian subjects. Based on these reference curves, clinical cut-offs for a low, medium and high AF have been made. This study examines whether these reference values can be used for ethnic populations seen in Qatar. Furthermore, we assess whether gender and smoking affect skin AF in this particular population. Methods: Skin AF was measured in 200 Arabs, 99 South Asians and 35 Filipinos. Using multivariate linear regression analysis and adjusting for the covariates age and the presence of type 2 diabetes, we assessed whether ethnicity, smoking and gender were associated with skin AF. Results: The Arabs and the Filipinos had a significant higher skin AF then the South Asian population (0.272 (95% CI: 0.138, 0.406), p <0.001 and 0.354 (95% CI: 0.147- 0.561), p=0.001 respectively). This is equivalent to a horizontal shift of 14.6% and 19.0%, respectively. Also, skin AF was significantly higher in women compared to men (0.432 (95% CI: 0.307, 0.558), p <0.001). Smoking was positively associated with skin AF (0.21 (95% CI: -0.01, 0.41), p=0.056), with an increasing effect of number of pack-years smoked on AF (p=0.024). Conclusions: The results of this study suggest that the existing reference values should be expanded for ethnicity, gender and smoking. These results also indicate that the use of the AGE reader in clinical settings should be used with caution, since the clinical cut-off points are dependent on various factors such as ethnicity that still need to be studied in greater detail.

Autism spectrum disorders (ASD) are a group of neuro-developmental disabilities common in childhood characterized by impairments in social interaction, communication skills, and patterns of activities. Background: The symptoms of ASDs typically are present before age 3 years and often are accompanied by abnormalities in cognitive functioning, learning, attention, and sensory processing. A formal diagnosis is generally not made until the age of five. The vast majority of cases of autism are idiopathic, the best scientific evidence available to us today points toward a potential for various combinations of factors, namely multiple genetic components and certain environmental and socio-economic factors. Timing of exposure during the child's development (before, during or after birth) may also play a role in the development or final presentation of the disorder. Objectives: To estimate the effect of certain socio-economic and other risk factors on the prevalence of autism in Qatar. Methods: The target population for this study are children diagnosed with autism attending the Shafallah Center for children with special needs. Clinical evaluation is conducted by a developmental psychologist, and/or pediatrician, it includes a medical, developmental, and behavioral history; a standard physical and neurological examination, In addition, the Autism Diagnostic Interview (ADI-R), and Autism Diagnostic Observation Schedule-G (ADOS-G) will be administered. Results: Preliminary analysis of 171 subjects showed the highest prevalence among age group 7-14 years (61%). Male/female ratio was 82%/18%, which is around 5/1. Other factors like consanguinity, education, and family income found to have an effect on the prevalence of the disease in Qatar. Conclusions: Obtaining a reliable estimate is important in planning for providing the best health care and educational services needed to improve the overall outcome of autism in Qatar.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells (HSCs). However, the number of HSCs in UBC remains limited and the attempts to amplify them in-vitro remain of poor efficiency. Several publications document amplification of HSC/progenitors with endothelial or mesenchymal cells, but the lack of homogeneity in culture conditions or HSC qualification impairs direct comparison of these results. Therefore, we compared the possible HSCs amplification using placental mesenchymal progenitors and Akt-activated umbilical vein endothelial cells. After HSCs isolation from UCB (defined as Lin-CD45-CD34+CD38-CD90+) on confluent feeder layer, the number of total cells and HSCs were monitored over 21 days. We demonstrate important cellular expansion on both niches. Most of the expanded cells were differentiated when characterized by flow cytometry. Only endothelial cells could trigger HSCs amplification with a pick at 14 days. Those amplified HSCs were able to differentiate in all cell lineages as attested by colony forming assays. Mesenchymal progenitors mainly triggered the amplification of CD38+ cells previously defined as precursors. A competitive assay demonstrated that HSCs had an in-vitro preference to interact with endothelial cells. Cytokines and transcriptomic analysis of our feeders indicate the mechanisms involved in HSC amplification and differentiation in both cases.

Background and Objectives: In the past 40 years Oman has witnessed remarkable social and economic growth, which is best reflected in the well-organized and efficient healthcare system. It has been anticipated that a change in disease pattern would be brought about by improvement in the quality of life, comprehensive healthcare facilities and successful control of communicable diseases. Although genetic and congenital disorders are increasingly observed in medical practice, there were no convincing available data to support these observations. The study was performed over the years 2010-2012 in order to assess the population needs for genetic services. Methods: A population-based study was conducted with detailed assessment and interviews of more than 3000 Omani mothers. Randomization was performed by counting the number of nationals in each geographical area and places of residence, and interviewing one mother per 600 Omani nationals in all geographic locations throughout the country. The collected data were verified with available hospital records and reports and analyzed. Results and Conclusions The population-based study revealed that 10% of Omani mothers reported congenital and genetic disorders causing morbidity in their offspring. Congenital disorders with mental disability accounted for over half of the reported morbidity, and a third of these reports had familiar recurrence. Morbidity and mortality from congenital and genetic causes that have been derived from this study reflect the situation in traditional Muslim community with high rate of inbreeding where communicable diseases were successfully controlled, and prevention measures are still in a preparatory phase. The current study confirmed that congenital and genetic conditions are the major contributors to childhood morbidity and mortality, and handicap prevalence in the Sultanate of Oman, indicating the need to prioritize future healthcare and planning, in view of their significant financial, social and research relevance.

Background: Prostate cancer is the most common malignancy in many industrialized countries and the second cause of cancer-related death in Europe and the United States. The incidence of the disease has been increasing in Arab populations. Large databases focused on genetic susceptibility to prostate cancer have been accumulated from population studies of different ancestries, including Europeans and African-Americans. Arab populations, however, have been only rarely studied. Genetic susceptibility to prostate cancer differs significantly across ethnicities. It would be of interest to identify the common alleles associated with prostate cancer risk in Arab population. Methods: With Affymetrix Genome-Wide Human SNP Array 6.0, we conducted a genome-wide association study (GWAS) in which 534,781 single nucleotide polymorphisms (SNPs) were genotyped in a Tunisian cohort of 92 cases with prostate cancer and 131 age-matched controls. Then we extended the study to evaluate promising associations of 11 SNPs, identified by GWAS, in a cohort of individuals of Arab ancestry living in Qatar and Saudi Arabia (155 cases and 182 controls) using TaqMan® SNP Genotyping Assays. Results: We identified 3 consecutive regions significantly associated with prostate cancer risk on chromosome 9, 17 and 22, including 18 SNPs (P=8.52×10-5 to P=2.18×10-6) in the Tunisian population. Three previous reported common loci associated with prostate cancer at 17q21 containing STAT3 gene in the Caucasian population were confirmed. Two novel chromosomal regions associated with prostate cancer contain candidate susceptibility genes: SMARCA2, LOC646851and SUN2 were revealed. Conclusion: Our findings, identifying novel GWAS prostate cancer susceptibilty loci, indicate that prostate cancer genetic risk factors could be ethnic specific.

Background: Cytogenetics of solid tumors in general is negatively affected by culturing artifacts such as preferential clonal expansion and introduction of chromosomal rearrangements, thus making it difficult to discriminate between primary changes and secondary events. Considering that chromosomal instability is a hallmark of cancer even at the precursor cancer level, it is of a great importance to devise a method of comprehensive chromosome analysis of cancer and cancer precursor cells that can bypass culturing problems as much as possible and that can provide informative karyograms of primary tumors at non-mitosis stages of the cell cycle. Consequently, we have developed a novel approach using chemically induced premature chromosome condensation (PCC) method and combined it with the technique of whole genome chromosome painting assay (M-FISH), in which chromosomal constitution of cancer cells could be detected independent of mitosis and without culture artifact within just three hours after receiving the biopsies. Objectives: 1. To perform karyotype analysis; 2. To define stage specificity; 3. To detect hall-mark(s) of cancer of different origins; 4. To improve therapy regimen by elucidating sensitivity of tumors and patients' lymphocytes to radiation and/or cytostatic drugs. Methods: Chemically-induced PCC method was applied on primary tumors (such as, cervical-, head and neck-, breast- and prostate-cancer) to generate metaphases. Afterwards, these preparations underwent M-FISH. Results: This unique/novel combined assay can give for the first time the possibility to assess genetic instability by detecting spontaneously occurring chromosomal instability (structural and numerical) in different types of primary tumors, as well as adjacent normal cells. It may open up the possibility to detect biomarker(s) for specific type of tumors, that can be used as screening tests. Furthermore, the sensitivity of specific types of tumors as well as patient's normal lymphocytes to cytostatic drugs and/or radiation can be determined. Conclusion: The unique combined state of the art assay we developed can lead to detect biomarker(s) for specific type of cancer. Moreover, for the first time therapy regimen can be individually designed based upon detecting tumors as well as patient's lymphocytes sensitivity.

Objective: To evaluate semen parameters and measure serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) concentrations before and 7 days after packed red cell transfusion (PCTx) in adults with sickle cell disease (SCD). Methods: This prospective study investigated 18 young adults with transfusion-dependent SCD, aged 20.7 +/- 2.88 years, with full pubertal development (Tanner's stage 5) (euogonadal), and capacity to ejaculate. Their serum ferritin levels were 1488 +/- 557ng/ml. Basal serum concentrations of FSH, LH, T and IGF-I were evaluated before and 7 days after packed red cell transfusion (PCTx). We studied the effect of PCTx on semen parameters and the endocrine functions in these 18 patients with SCD. Results: Following PCTx, a significant increase of Hb from 8.5 +/- 1.17 g/dl to 10.5 +/- 0.4 g/dl was associated with increased testosterone (12.3 +/- 1.24 nmol/L to 14.23 +/- 1.22nmol/L and gonadotropin concentrations. Total sperm count increased significantly from 87.4 +/- 24.6 million/ml to 146.2 +/- 51.25 million/ml total progressive sperm motility (TPM) from 40.8 +/- 11.1 million/ml to 93.4 +/- 38.3 million/ml, and rapid progressive sperm motility progressive motility (RPM) increased from 29.26 +/- 8.75 million/ml to 67.4 +/- 29 million/ml. After PCTx the total sperm count, TPM and RPM were significantly higher in the ETx group versus the TTx group. Before and after PCTx, testosterone concentrations were correlated significantly with sperm total count, volume, TPM and RPM (r= 0.53, 0.55, 0.42 and 0.38 respectively, p= 0.01). Before and after PCTx, hemoglobin concentrations were correlated significantly with sperm count, TPM, RPM and percentage of sperms with normal morphology (r= 0.60, 0.69, 0.66 and 0.86 respectively, p <0.001) Conclusion: Our study suggests that in males with SCD, blood transfusion associated with significant acute enhancement of sperm parameters and with an increased concentration of testosterone, LH and FSH. Improvements of sperm parameters were significantly higher in the exchange transfusion (ETx) group versus the top-up (TTx) group. These 'acute' effects on spermatogenesis are reached with an unknown mechanism(s) and suggest a number of pathways that need further human and/or experimental studies.

Obesity has increased at an alarming rate over the past three decades. It is likely to be caused by increased caloric intake combined with genetic predisposing factors and is a major risk factor for type 2 diabetes (T2D) and cardiovascular disorders. Obesity is linked to chronic inflammation, which was proposed as a cause for insulin resistance and T2D. However, the molecular mechanism of obesity driven T2D still lacks knowledge and thus is inaccessible to therapeutic intervention. In our research, we are exploring the molecular mechanism underpinning this association and aims to identify novel therapeutic targets for the rapidly growing population of obese individuals living with diabetes. The obese state creates a microenvironment within the adipose tissue that is susceptible to the accumulation of reactive oxygen species (ROS). Accumulated ROS causes oxidation of macromolecules such as DNA. Oxidative DNA damage encompasses a variety of DNA lesions such as DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) including non-telomeric and telomeric DSBs. Broken or dysfunctional telomeres have been hypothesized as a cause of cellular senescence, a state of irreversible cell cycle arrest observed in aging illnesses and even in obesity with accumulation of senescent adipocytes. A major player in detecting and defending against accumulation of oxidative DNA damage is PARP-1. PARP-1 activation has been shown to be associated to diabetes and obesity. We are exploring the role of PARP-1 in ROS-induced senescent and inflamed adipocytes by using a combination of biochemical, molecular and cell biological assays in primary human and mice adipocytes treated with hydrogen peroxide (H₂O₂) as a source of ROS.

Stromal interaction molecule 1 (STIM1), the ER calcium sensor, couples to Orai1, the calcium channel, and mediates store-operated calcium entry (SOCE). STIM1 localizes to the ER membrane. Following Ca²+ store depletion, STIM1 forms puncta that localize to the cortical ER and binds Orai1 to allow Ca²+ influx. The egg's competency to activate at fertilization is dependent on its ability to generate a fertilization-specific Ca2+ transient. This is achieved through remodeling of its calcium signaling machinery during oocyte maturation. Oocyte maturation is driven by kinase cascade including MOS- mitogen-activated protein kinase (MAPK) - maturation promoting factor (MPF). Coordinately, SOCE inactivates during maturation. This inactivation is due to internalizing of Orai1 and the inability of STIM1 to form puncta. Here we show that MPF phosphorylates STIM1. Through molecular and pharmacological manipulation we are able to differentially activate the kinases along the kinase cascade that drives oocyte maturation. STIM1 phosphorylation in these studies was monitored through a mobility shift on SDS-PAGE. When MPF is activated a mobility shift is observed. However, this is not the case when only Mos or the MAPK cascade were active. Our next step is to determine the role of MPF phosphorylation of STIM1, because previous studies have argued that phosphorylation does not inhibit STIM1 clustering during meiosis. Our data argue that MPF is the primary kinase that phosphorylates STIM1 during meiosis.

Background: The use of the serum prostate-specific antigen (PSA) test for prostate cancer screening has resulted in a diagnostic dilemma: PSA is not prostate cancer specific and could be found in the normal prostate at equal or higher levels than is found in prostate cancer. Prostate cancer gene 3 (PCA3) encodes a prostate-specific mRNA with a median 66-fold up-regulation compared to adjacent benign tissue. In contrast, PSA gene expression is similar in cancerous and benign cells; PSA mRNA levels may therefore be used to normalize for the amount of prostate-specific ribonucleic acid (RNA) in molecular test samples. This report describes the characterization of a prototype quantitative PCA3-based test for whole urine. Methods: First-catch urine specimens were collected after digital rectal examination. The total RNA was isolated with Trizol, amplified, and quantified by use of a real-time PCR method. PSA mRNA concentrations were used to normalize PCA3 signals and confirm the yield of prostate-specific RNA. PCA3 score is calculated by one thousand times of PCA3/PSA mRNA ratio. ROC curve analysis is applied to determine the cutoff value of PCA3 score according to prostate biopsy results. Results: We have collected urine samples of 163 patients from uro-oncology clinic at Hamad Hospital. Due to the mixture of the blood cells and prostate cells in the urine, we failed to obtain RNA with proper quality for 29 samples. The specimen informative rate (fraction of specimens yielding proper RNA for analysis) was 82.2%. From ROC curve with the known diagnostic data, the cut off of PCA3 score is 1232 with a sensitivity of 70% and specificity of 68%. Conclusion: The established PCA3 assay could increase specificity to the current prostate cancer diagnosis. Comparing to the commercial kit, it provides equivalently useful information and it is much cheaper and faster. The challenge is to avoid the interference from the blood cells in the urine.

Background & Objectives: Charcot neuroarthropathy (CN) is a devastating complication of diabetes. It has two-fold higher rate of major amputation compared to those without CN. Unfortunately, to date, there is no pathological marker or diagnostic criterion for it and diagnosis relies on pattern recognition and clinical intuition. Not surprisingly, the diagnosis can be missed up to 95% of the time and the average diagnostic delay has been estimated at almost 7 months. Recent studies reported asymmetric plantar temperature differences secondary to inflammation as a hallmark for the diagnosis and treatment response of Charcot foot syndrome. However, little attention has been given to temperature response to activity. Methods: Fifteen individuals with acute CN and 17 age-matched non-CN participants with type 2 diabetes and peripheral neuropathy were recruited. All participants walked for two predefined paths of 50 and 150 steps. A thermal image was acquired at baseline after acclimatization and immediately after each walking trial. The plantar temperature (PT) response as a function of number of steps was examined using a validated wearable sensor technology. A custom image processing toolbox was designed to characterize the plantar temperature response to activity. Results: During initial activity, the PT was reduced in all participants, but the temperature drop for the non-affected foot was 1.9 times greater than the affected side in CN group (p=0.04). Interestingly, the PT in CN was sharply increased after 50 steps for both feet, while no difference was observed in non-CN between 50 and 200 steps. Conclusions: The observed differential thermal response to walking initiation between Charcot and non-Charcot feet warrants future investigation to provide further insight into the correlation between activity dosing and thermal response. It may also lead to a valuable insight into identifying an "inflammatory trigger" that may ultimately provide an early warning sign or increased sensitivity for subsequent unilateral or bilateral CN development or clinical expression of foot ulcer. These results also support that managing modulating duration of continuous steps and or prolonged standing during daily activity could be helpful for reducing the trauma in patients with CN or patients at risk of diabetic foot ulcer.

Background and Objectives: Oncogene induced senescence is a tumor suppressor mechanism that limits progression of pre-malignant lesions. Identifying the molecular mechanisms by which cells can escape senescence may provide novel cancer therapeutic and preventive targets. Two tumor suppressors, p53 and Rb, are involved in senescence, but their specific roles are still unclear. Our aim is to investigate the role of the Rb and p53 pathways in cellular senescence, to identify mechanisms of cellular escape from senescence, and to evaluate human tumor samples for disruption of the identified pathways. Methods: We are using a transgenic mouse with pineal-cell specific cyclin D1 expression (Irbp-Cyclin D1 mouse). These mice develop premalignant pineal hyperplasia with features of cellular senescence, and tumors progress only when the p53 or the RB pathway is disrupted. Results: We found that p53 was activated early in the senescence response, and led to cell cycle exit. RB activation occurred days later, and coincided with the senescence phenotype that included formation of heterochromatin foci. Disruption of either pathway alone was sufficient for pineal tumor progression. Using genetically engineered mice with regulatable expression of p53, we are now investigating the specific role of p53 in the induction versus maintenance of cell cycle exit. In addition, we are using primary cultures of Irbp-Cyclin D1 pineal cells to examine the effects of turning the p53 and RB pathways "on" and "off" during cellular senescence, to dissect their roles during induction and maintenance of senescence. We are also analyzing premalignant versus malignant tumors from these transgenic mice, to identify recurring genetic lesions that may be important targets for tumor prevention. Finally, we are evaluating human tumor samples for genetic lesions that overlap with the identified pathways. Conclusion: Our preliminary results suggest that the p53 and RB pathways have distinct and complementary effects in the senescence response, where the p53 pathway is important for induction of senescence, while the RB pathway may be responsible for its maintenance. Studies are currently ongoing to verify these observations. These findings will have direct implications on novel approaches to cancer therapy and prevention, by activating cellular senescence and tumor suppression.

Background and Objectives: The prevalence of type 2 diabetes (T2D) in Qatar is the highest in the world. It is estimated that about one quarter of the T2D patients in Qatar are still undiagnosed. We set out to examine determinants of pre-diabetes (PD) or undiagnosed T2D (UT2D). Furthermore, we examined risk factors for glucose regulation in patients with known T2D. Methods: We examined 178 patients with known T2D and 196 controls. Anthropometrics and HbA1c were measured. Socio-demographic (age, gender, ethnicity and educational level) and health information were assessed through questionnaires. Results: Twenty-six (13.3%) and twelve (6.1%) participants in the control group were PD and UT2D, respectively. Control participants from South Asian descent were at a greater risk of being PD or UT2D than Arab controls (Odds Ratio: 5.27 (95% confidence interval: 2.10, 13.22). Being obese was also associated with an increased risk of PD or UT2D (Odds Ratio: 2.94 (95% confidence interval: 1.31, 6.59). Of control participants from South Asian descent, 38% were actually PD or UT2D. In patients with known T2D, insulin use and a longer duration of T2D were both related to higher HbA1c levels (both p<0.0001). No socio-demographic factors were associated with glucose control in T2D. Conclusions: In a nation with a large South Asian immigrant population, we found a strong increased risk of being PD or UT2D in these subjects. Focus should be on the prevention and early identification in South Asian individuals. Larger studies are necessary to further examine the determinants of T2D and glucose regulation in Qatar.

Many tumors are regulated by complex interactions with cellular components of the microenvironment including mesenchymal stem cells (MSC) or endothelial cells (ECs). While the role of receptor-ligand interaction at the cell surface is well documented, direct cell-to-cell contact has not been clearly established. Recently, tunneling nanotubes (TnTs) have been shown to support cell-to-cell transfer of organelles, various plasma membrane components and cytoplasmic molecules in several cell lines. We thus investigated the formation of TnTs between stromal cells and cancer cells supported by them. We demonstrate that TnTs occur between different cancer and stromal cell lines. TnTs formation seemed to be dependent of large membrane adhesion. We show that intercellular transfers of cytoplasmic content can occur by TnTs. However, we demonstrate that the exchange of mitochondria occurs preferentially between endothelial cells and cancer cells and contributes to chemoresistance. Our results point out the role of direct endothelial to cancer cell exchange, which emphasize our hypothesis that this angiogenesis-independent role of the endothelium plays a role in the constitution of the metastatic niche.

Objectives: To investigate the incidence, characteristics and patterns of football injuries at club level in Qatar. Design: Prospective cohort study. Methods: Data were prospectively collected from the first division football league clubs in Qatar, in accordance with the international consensus statement on football injury epidemiology. An injury was defined as any physical complaint sustained during football activity resulting in the inability to participate fully in the next training or match. Individual injuries and exposure of each player were recorded by the medical staff of each team over one season. Results: A total of 217 injuries were recorded, with an injury rate during matches of 14.5/1000 h (95% CI: 11.6-18.0) compared with 4.4/1000 h during training sessions (95% CI: 3.7-5.2). More than one third of all injuries were muscle strains (36.4%). Hamstring strains (54.4% of all muscle strains) exhibited a higher incidence than all other injury types (p < 0.001). The thigh was the most frequent injury location (41.9%, p < 0.001). Re-injuries (15% of total injuries) were mainly comprised of muscle strains associated with a higher severity compared with new injuries. Conclusions: Despite the different environmental, social and cultural setting, our findings are comparable with previous data from a European club football, confirming the previous finding at national team level that there are no regional peculiarities of football injuries in this part of the Asiatic continent. The relatively high overuse injury incidence rate and the high recurrence rate for (severe) thigh muscle strains, especially during games, warrants prevention strategies.

Eight consanguineous Arab families with novel autosomal recessive disorders were mapped with illumina 700K SNP. All relevant positional candidate genes were screened for pathogenic mutations. None were identified. Multiple homozygosity intervals were obtained for each family since no significant LOD scores were possible. Whole exome sequencing was on ABI SOLiD4 for 1 affected individual from each family. Mapping and annotation was on LifeScope software. Data validation was done manually for each linkage interval, by visual inspection of read depth and bead number coverage. On average 30,000 sequence variations were detected in each sample including novel variants, known polymorphisms & exome sequencing errors. For each chromosome with a linkage interval, data was isolated and filtered by exportation to Excel spreadsheets and visual inspection to exclude non-linkage interval data. The number of variants in the linkage intervals for each family was between 400 and 1300. Homozygous sequence variations within the linkage intervals were between 50 and 300 with 15-30 novel variants. Determination if a variant was homozygous or heterozygous, novel or annotated was done manually upon visual inspection of data on Excel spreadsheets. For each novel variant it was manually determined if it were exonic, splice site specific or intronic. For each annotated variation it was manually determined if it is associated to a disease phenotype relevant to the family disease. Minor genotype frequency was investigated for annotated variants if they represent disease states. All novel exonic variants were tested in silico with PolyPhen and Sift Protein Modeling software to access the effect on protein function. All damaging variants (novel or annotated exonic, and splice site) were validated by Sanger sequencing and tested for co-segregation to disease. An identical approach is essential to access pathogenic effects of insertion/deletion variants within each linkage interval. This approach is tedious, involves a tremendous amount of manual work and is prone to oversight errors. Software tools development for automating next-generation sequencing data analysis is essential to eliminate manual work and identify pathogenic mutations among the plethora of existing variants. Such automation is applicable in cases without linkage intervals to limit the number of variants under consideration.

Background and Objectives: There is a high prevalence of obesity and its co-morbidities within the Arab population, especially in Qatar. Furthermore, the younger age of onset of obesity and its preponderance amongst females have seen an increase in type 2 diabetes and cardiovascular disease in this cohort. Adipose tissue dysfunction preceding frank obesity may underlie the increased risk of metabolic syndrome (MeS). Therefore aims of this study were to characterize the relationship between components of MeS and adipokines in a Qatari population. Methods: Non-diabetic subjects were recruited from the general Qatari population and patients awaiting weight reduction surgery (Al-Emadi Hospital). Lipid profiling and liver function were determined by conventional techniques (HMC). Insulin resistance was measured by HOMA. Adipokines were measured by ELISA. Results: In the whole study population (n=56, 13 males/43 females, 30.5+/-7.5 years old, BMI of 37.5+/-11kg/m-2) significant positive correlations between plasma leptin and BMI were confirmed. However, after controlling for BMI, partial correlations showed that leptin was associated negatively with all measures of blood pressure (all p≤0.01), triglycerides, total- and LDL-cholesterol (all p≤0.01), with liver enzymes (SGPT, AST and Billirubin, p=0.009, p≤0.01 and p=0.02 respectively), and positively with HDL cholesterol (p=0.05). Furthermore, leptin, was also negatively correlated with fasting blood glucose and HOMA (p=0.03 and p=0.01 respectively). In order to investigate this further, the population was dichotomized into age-matched normal weight (n=14) and obese (n=42) sub-groups. The obese group had significantly higher SBP (p=0.006), triglycerides (p=0.03), leptin (p≤0.001) and lower HDL (p≤0.001). Obesity was also associated with deteriorating liver functions manifested as elevation in SGPT (p =0.02), ALP (p= 0.01) and AST (p =0.02). Conclusions: Leptin was elevated in obesity. After correcting for BMI, an inverse relationship between leptin and risk factors for MeS was apparent, suggesting a protective role for leptin in this population, perhaps through its vasodilatory capability. However, when dichotomized into lean and obese groups, chronic elevation in leptin appeared to be associated with increased risk of MeS and deterioration of liver function, as previously described. These novel data are currently being confirmed in a larger ethnicity matched cohort.

A global survey of cancer has shown that lung cancer is the most common cause of the new cancer cases and cancer deaths in both men and women worldiwide. In Qatar a recent retrospective study based on a cohort of patient registry of Al Amal Cancer Hospital from 1991-2006, showed that lung cancer and lumph node cancer are the major cancers in men (5.9%, incident rate per 100,000) while in women breast cancer had the highest incidence. To study the mechanisms of lung cancer development we recently developed a transgenic mouse model overexpressing CRT under the control of Tie2-promotor. The main phenotype of these mice is the development of metastatic lung adenocarcinoma similar to human patients. The objectives of the current study was to identify the molecular pathways involved in the metastatic behavior of lung adenocarcinoma. To examine our objectives we used anchorage independent and adherent culture of lung adenocarcinoma cells isolated from mouse and human cell lines. Using microarray and bioinformatic analysis we have identified several pathways and genes that are altered in mouse cells. The microarray analysis demonstrates that 90 genes are up-regulated and 66 genes are down regulated under anchorage-independent condition compared to adherent conditions. The bioinformatic analyses demonstrate that the up-regulated genes are implicated in cellular development, cellular growth and proliferation pathways. These pathways include: VEGF signaling, bladder cancer signaling, VEGF family ligand-receptor interaction and Ephrin-receptor signaling. The bioinformatic analyses also demonstrate that the downregulated genes were implicated in cell cycle regulation and cell-to-cell signaling and interaction. The analysis of these genes by canonical pathawy demonstrate that these genes are involved in the regulation of very relevant pathaway implicated in cancer essentially : Hematopoeisis from multipotent stem cells, TGF-β signaling and tight junction signaling. We are currently examining these pathways in human adenocarcinoma cell line. In conclusion, our data illustrate that although calreticulin is an endoplasmic reticulum calcium binding chaperone, it is involved in the process of tumor development and metastasis. Further work is needed to decipher the molecular mechanism of involvement of calreticulin in tumor development. This research was funded by QNRF grants UREP09-084-3-016 and NPRP4-043-3-016.

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.