Qatar Foundation Annual Research Forum Volume 2013 Issue 1

Background and Aim: The prevalence of type-2 diabetes (T2D) has doubled in the last three decades and is still rising at an alarming rate, thereby posing a major challenge to global health. MicroRNAs (miRNAs) are a class of short RNAs, which play an important role in regulating physiological processes in diabetes. These small RNAs post-transcriptionally suppress mRNA target expression and, therefore, modulation of specific miRNAs may prove to be a promising strategy to treat diabetes. However, the potential roles of the different microRNAs in the eitiology of diabetes and diabetes-related complications are not yet completely understood. In the present study, we aimed to explore the role of miR221/ 222 in diabetes. Materials and Methods: Mouse microvascular endothelial cells (MMECs) were cultured for 48 hours under conditions designed to mimic the mileu of type 2 diabetic mice: normal glucose (NG=11mM) and high glucose (HG=40mM). The levels of miRNA221/222 expression were then analysed by real-time PCR. MMECs were transfected with miR221/222 inhibitors and mir221/222 mimics and then cells were exposed to normal/ high glucose in the presence or absence of metformin. Expression of miRNA 221/222 were again analysed and compared. The effects of miR 221/222 expression on eNOS, phospho-eNOS and eNOS monomer/dimer protein levels were determined through western blotting. Results: We found that exposure to high levels of glucose, which mimics hyperglycaemia conditions, induced expression of miR-221 and miR-222. Treatment of metformin partially restored this HG-induced high expression of miRNA. Moreover, metformin showed an additive effect with miR221/222 inhibitors to inhibit miR 221/222 expression in hyperglycaemic condition. Furthermore, we observed that protein levels of total eNOS and phospho-eNOS decreases in HG in comparison with NG. The eNOS/P-eNOS protein levels were restored upon inhibition of miR221/222, thus indicating correlation of these micro-RNAs with eNOS signalling. Results were inversely validated by using miR mimics (overexpression). Conclusion: These findings suggest that miR221/222 may offer a new therapeutic strategy for treatment of endothelial dysfunction in diabetic patients or may work as a therapeutic modulator. The project is supported by UREP 13-116-3-024

Background: Expanding adipose tissue in obesity requires effective angiogenesis and vasoreactivity to combat hypoxia and its consequences, such as insulin resistance and type-2 diabetes. While recent evidence suggests that the adipose tissue is highly angiogenic and inflammed, the tissue arteriolar vasoreactivity has been less investigated. As a consequence of the inflammation the omental adipose tissue (OAT) also synthesizes greater levels of vasoconstrictive molecules, such as cytokines and catecholamines, compared to the sub-cutaneous (SAT) depot, and these are likely to impact on the maintenance of vascular tone leading to greater susceptibility to hypoxia. This study compared the contractile responses of OAT and SAT arterioles from insulin-resistant (IR) and insulin-sensitive (IS) morbidly obese non-diabetic Qatari patients. Methods: Segments of arterioles (ID ~240-250 µm) isolated from SAT and OAT obtained from obese non-diabetic Qatari patients (age ~29 years, BMI ~40kg.m-2), undergoing bariatric weight reduction surgery, were mounted on a dual wire myograph (510A) and investigated for noradrenergic responsiveness. Cumulative concentration-response curves were constructed for noradrenaline (10-9 -10-5 M). Curves were also constructed for potassium chloride (KCl, 1-70 mM). Results: OM arterioles from IR patients were significantly less sensitive to NA compared with SAT arterioles from same patients (log EC50 -5.9±0.2 vs. -6.5±0.1, p<0.05). Maximum NA contractile response was also attenuated in OAT compared with SAT vessels (p<0.05) from these patients. On the other hand, KCl curves were comparable for OAT and SAT vessels from the same patients. In addition, no differences in noradrenergic sensitivity were observed between OAT and SAT vessels from IS patients. Conclusions: The data suggest that insulin resistance selectively alters noradrenergic responsiveness of OAT arterioles compared with SAT vessels from morbidly obese non-diabetic Qataris. Differences in adrenoceptor density/function may underlie these depot-specific responses. Studies on the disease specific differences need further investigation.

Purpose The first stage of this QNRF funded study includes a series of key informants' interviews with stakeholders in the PHC sector in Lebanon and Qatar to formulate an understanding of the recruitment and retention strategies of health human resources (HHR) and understand the obstacles and challenges that they face. This abstract reports on the findings obtained in Lebanon, as data collection remains ongoing in Qatar. Participants & Methods Study utilized a qualitative design involving semi-structured key informant interviews with stakeholders in the PHC sector. An initial list of key stakeholders was acquired from a review of public and private information sources while ensuring maximum variability across institutions, disciplines and geographical locations. Overall, 22 key informants participated in the study. They included decision and policy makers in the PHC sector, managers/directors of PHCCs, human resources coordinators in PHCCs, physicians and nurses, and academicians. Analysis Thematic and content analysis, using Nvivo 8, was conducted on the data collected from the key informant interviews. After coding the responses into similar concepts, axial coding allowed for the emergence of five comprehensive themes: perception of PHC, PHC services, recruitment of HHR in PHC, retention of HHR in PHC, and recruitment and retention in rural areas. Results Responses of stakeholders with regards to the definition of PHC revealed a lack of a unified understanding of the concept. With regards to services offered at PHCCs, there was a consensus that there is a deficiency in various services, most notably was mental health, as well as various preventive functions such as vaccination, women's health, and health behavior modification. Thematic analysis identified a number of impediments to the recruitment of HHR, mainly related to shortage in the overall supply of HHR, of qualified HHR, and HHR gender imbalances. Despite recruitment strategies in place, factors including financial constraints and poor leadership/management hinder the effectiveness of recruitment efforts. Although stakeholders report an acceptable retention of HHR, they relate turnover to poor working environment and lack of professional development. There was consensus that challenges faced are more pronounced in PHCCs of rural areas. Conclusions The study findings reveal that the current status of recruitment and retention within the PHC sector is not conducive to a solid and stable workforce. There is an evident need for the establishment of a unified contextualized definition of PHC to be applied across all PHCCs operating nationally. Moreover, the adoption of a system's thinking approach is crucial for PHC capacity building, in which the existent structure is better supported by qualified personnel. Extensive efforts need to be exerted towards directing health care professionals to the PHC field especially in rural areas, while concurrently enhancing the working conditions within PHCCs. Accordingly, essential services may be more adequately provided to the community. Of particular importance is the integration of mental health services into community care. Decision and policy makers are urged to reflect upon the recommendations developed in order to not only stabilize the PHC workforce but to also ensure the longevity of its services.

Sickle Cell Anaemia is an, autosomal, genetically-inherited, blood disorder, arising due to a point mutation in the bases coding for the sixth amino-acid of the β-chain of haemoglobin. The effects of the mutation begin to play role at the event of translation; during which the mutated haemoglobin (HbS) is synthesised. Here a hydrophobic protein residue (valine) is incorporated at the position of a hydrophilic residue (glutamic acid) in the growing protein chain. However, its orientation imitates that of the hydrophilic residue as otherwise seen in the wild type haemoglobin (HbA). Due to this change the hydrophobic side-chain of the amino-acid (valine) is prevented from being buried within the hydrophobic core of the protein and remains exposed to the surface. The resulting HbS thus displays a hydrophobic and unstable character with a tendency to polymerise with other HbS molecules causing the Red Blood Cells (RBC) to take a characteristic sickle shape. Through this research initiative, an attempt was made to unfold the protein to an extent that was sufficient to expose its hydrophobic core, thereby allowing it to engulf the side-chain through the formation of hydrophobic interactions. Thus the resultant modified protein would display an overall stable character, mimicking the wild-type HbA in spite of its mutational status. As a preliminary analysis, partial unfolding and refolding experiments were carried out on HbA molecules to determine whether the quaternary structure of haemoglobin would be retained. These experiments were monitored through Circular Dichroism (CD) Spectrophotometry. Partial unfolding was targeted by treating the protein with different concentrations of a mild denaturant (dimethyl sulfoxide). Unfolding was arrested at different stages of time (12, 24, 36, 48, 60, 72 hours) by the introduction of an organic solvent (chloroform) that induces precipitation of the protein. The modified protein was then tested for changes in hydrophobic character and stability by Reverse Phase Chromatography using C18 columns. Tests for solubility and aggregation were also performed spectrophotometrically. All tests were carried out under both oxygenated and deoxygenated conditions. It was observed that modified haemoglobin molecules arrested at 36 and 48 hours displayed a change in structural conformation. However, CD spectrophotometric analysis confirmed that they did not refold to resemble the wild-type HbA . Chromatographic results showed that the modified protein developed an overall neutral character, while spectrophotometric analysis proved that the molecules were insoluble in potassium phosphate buffer (pH 7) under both oxygenated and deoxygenated conditions. The study proved that partial unfolding up to 36 to 48 hours was sufficient to expose the hydrophobic core to trigger interactions that causes the haemoglobin molecule to attain an overall neutral and stable character. However, further analysis is ongoing to control the refolding of the protein to more closely match its native state. The study is also experimenting with other models of denaturants as well as more refined methods to arrest unfolding in order to improve solubility of the protein.

Autoinflammatory disorders are a group of Mendelian disorders characterized by seemingly unprovoked inflammatory bouts without high-titer autoantibodies or antigen-specific T-cells and are probably due to defects in the innate immunity. We here report on a 4-year old Arabic child with the clinical presentation of an autoinflammatory disorder, namely Pyogenic Arthritis, Pyoderma Gangrenosum and Acne (PAPA) syndrome. The presentation includes abscess formation after immunization and recurrent mono-articular acute arthritis in various joints that responded favorably to systemic glucocorticosteroids, albeit without acne or pyoderma gangrenosum. The mutation analysis of the child identified a novel de novo mutation in PSTPIP1, the gene responsible for PAPA syndrome. We recommend that the diagnosis of PAPA syndrome should be entertained in the differential diagnosis of patients with recurrent sterile pyogenic arthritis prior to the development of pyoderma gangrenosum or acne in order to initiate a timely management of the disorder.

The Yamanaka factors (Oct4, sox2, Klf4, cMyc) used to produce induced pluripotent stem cells (iPSC) have become a staple of stem cell biology and ChIP-seq studies have led to an understanding of the genomic landscape of how these as well as other recently discovered factors transduce their effects to form iPSC. A conundrum that previously existed was that the Sox family has high homology and yet various members drive completely different cell fates. My lab in collaboration with Larry Stanton&#39;s lab at the Genome Institute of Singapore unraveled part of the mystery by identifying and validating a single residue within the hmg domain of the Sox family responsible for a specific interaction with Oct4 (pou5F1 domain) which then decides specific cell fates. In addition a novel genomic motif was discovered which has a single base difference between the Oct4 and Sox binding sites. Using this knowledge we were able to transform the function of pluripotent Sox2 into an endodermal TF and endodermal Sox17 into a pluripotent TF. In fact the mutated Sox17 TF was even more efficient in forming iPSC (compared to Sox2) which was not expected. Recently we investigated the C-terminal activation domains of these Sox family members and discovered that they play a major role in the level of activation of iPSC. Conversion of Sox17 into a Pluripotency Reprogramming Factor by re-engineering its Association with Oct4 on DNA. Ralf Jauch, Irene Aksoy, Andrew Paul Hutchins, Calista Keow Leng Ng, Xian Feng Tian, Jiaxuan Chen, Paaventhan Palasingam, Paul Robson, Lawrence W. Stanton and Prasanna R Kolatkar. Stem Cells. 2011. Jun;29(6):940-51. Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm. Aksoy I, Jauch R, Chen J, Dyla M, Divakar U, Bogu GK, Teo R, Leng Ng CK, Herath W, Lili S, Hutchins AP, Robson P, Kolatkar PR, Stanton LW. EMBO J. 2013. 32(7): 938-53. Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming. Aksoy, I., Jauch, R., Eras, V., Bin, A.C-W., Chen, J., Divakar, U., Ng, C. K-L., Kolatkar, P.R., Stanton, L.W. Stem Cells. 2013.

Background. Chronic myelogenous leukemia (CML) is the most common myeloproliferative disease accounting for ~15% to 20% of all cases of leukemia (1-1.5/100.000 cases per year). It originates from a pluripotent bone marrow stem cell in which a t(9;22) results in the production of BCR/ABL fusion protein which has a constitutive tyrosine kinase activity and deregulates signal transduction pathways. Phosphorylation of key residues is required for the full transforming activity of BCR/ABL; for this reason much attention has been focused on the role of phosphatases, natural regulators of the tyrosine kinase signaling. We have previously reported that protein tyrosine phosphatase receptor type ? (PTPRG) is a tumor suppressor gene which interacts with BCR/ABL, inhibits downstream signaling events and is downregulated in CML. Whitin the QNRF research project NPRP 4-157-3-052 we analyzed the expression levels of PTPRG gene in 32 CML patients at diagnosis and following TKI treatment aiming at the evaluation of the clinical impact of PTPRG dowregulation in CML. Methods: Thirtytwo patients diagnosed with CML in chronic phase and 13 untreated patients diagnosed with philadelphia-negative myelod disorders as control group were included in the study. The study was approved by the Local Ethics Committee, and informed consent in accordance with declaration of Helsinki was obtained from each patient. The expression level of the PTPRG gene was evaluated by a sybr-green absolute quantification RT-PCR assay in 2 samples from each patient, taken at diagnosis and following TKI treatment using the beta-Actin (ACTB) housekeeping gene for normalization. Results were expressed as PTPRG/ACTB ratio and were validated using predesigned TaqMan quantitative RT-PCR assays for PTPRG and ABL1 genes. BCR-ABL1 transcript was quantified by realtime RT-PCR according to the European Leukemia Net guidelines. Statistical analysis and comparisons were performed using the SPSS-software. Results: PTPRG transcript was undetectable in 11/32 (34,3%) CML samples at diagnosis and the median levels of PTPRG mRNA were significantly lower in CML samples at diagnosis compared to the non-CML control group (0,44%, range 0-0,37 vs 6,29%, range 0,09-52; p=0.02). On the contrary, PTPRG mRNA was detected at variable levels, ranging from 0.17 to 30%, in 29/32 follow up samples, taken at different time points of treatment. Differences in PTPRG gene expression levels between CML samples before and after treatment were statistically significant (p=0,027). Quantitative RT-PCR for PTPRG has been also set up at the Hematology center, NCCCR, Doha, Qatar. Preliminary results showed that mean levels of PTPRG mRNA were comparable to the italian group of patients. No statistically significant correlation was observed between PTPRG levels and clinical/biological factors. Interestingly, 2 of the 3 patients showing higher level of PTPRG mRNA at diagnosis than in the follow up sample showed resistance to TKI treatment. Conclusions: We found a down regulation of PTPRG in a high percentage of CML patients and a recovery of its expression upon treatment with TKIs. Deregulated expression of PTPRG phosphatase underline its role as a tumor suppressor gene in CML and highlights its potential use as a new bio-marker of disease potentially usable in association with BCR/ABL1.

Background &amp; objectives: Vascular complications account for much of the morbidity of obesity. The proportion of Qatari population that is overweight or obese is one of the highest in the world. With this comes the increased risk of blood vessel dysfunction and predisposition to type 2 diabetes and hypertension. The vascular endothelium is often the first target of the negative impact of obesity. It is however unclear how the heterogeneity in the metabolic status of obese individuals (ie metabolically healthy [MHO] vs. Pathologically obese [PO]) will impact on endothelial function, particularly in a relatively young obese population, as in Qatar. This study investigated endothelium-dependent relaxation of small arteries embedded in the visceral (omental) and subcutaneous adipose tissues in morbid obesity with varying metabolic status. Methods: Arteries were isolated from abdominal omental (OM) and subcutaneous (SC) fat collected from consented Qatari patients undergoing bariatric surgery for weight reduction. The arteries (normalized luminal diameter ~250 &#181;m for SC and ~ 240 &#181;m for OM) were cut into segments (~2 mm) and mounted on a dual wire Myograph (510A) for measurement of isometric tension. Cumulative concentration-response curves were constructed for acetylcholine (1- 30000 nM, the classical endothelium-dependent relaxant) in the absence or presence of Nω-Nitro-L-arginine methyl ester (L-NAME,100 &#181;M, nitric oxide [NO] synthase inhibitor) on initial tone generated with noradrenaline (5 &#181;M). Relaxation to sodium nitroprusside (SNP, an NO donor) was also recorded. Results: There were no differences in age (~32 years), blood glucose (~5.6 mmol/L) and body mass index (BMI , ~ 43.4 Kg.m-2) between the MHO and PO patients . Insulin levels were 3 vs 19 &#181;U/ml for MHO vs PO patients and their indices of insulin resistance (HOMA) were 1 vs 5 respectively. In general, relaxation to Ach was significantly attenuated in OM vessels (Emax 44&#177;8 %) compared with SC vessels (Emax 78&#177;4 %, p&lt;0.01) from same patients. In contrast, relaxation to SNP was greater in the OM compared with the SC vessels. When Ach relaxation of the OM vessels were separated according to the patients&#39; metabolic status, the MHO patients had significantly improved result compared with PO patients. On the other hand, relaxation of SC vessels from both groups of patients were comparable. In both vessel types, L-NAME caused a right-ward shift in the Ach curves. Conclusions: These results suggest that the metabolic status of obese Qatari patients has bearing on the physiology of their microvascular endothelium. The data also demonstrate that early changes in endothelial vasomotor function are depot-specific, being more marked in OM compared with SC vessels of pathologically obese patients.

Abstract The use of herbal and nutrition supplements is widespread. It has been reported that about 80% of the world's population use herbal medicines [1, 2, 3, 4]. Very few supplement use studies have been conducted in the Gulf Cooperation Council (GCC) and neighboring nations (5,6). Supplement use data among young adults is scarce. In one study aimed at determining the use of supplements among athletes in Qatar (5), over 60% of the study participants reported using vitamin supplements. Data regarding attitudes on the use of supplements among young adults is almost non-existent. The data on their perceptions about alternative medicine practitioners is also inadequate. We thus conducted a survey in which we examined the a) prevalence of supplement use among college students in Qatar, and b) their perceptions about the use of supplements and alternative medicine practitioners. We have previously presented our study findings regarding the use of supplements (7). At this meeting we will present our study findings concerning the perceptions of college students about the a) effectiveness and safety of supplements and b) alternative medicine practitioners. References 1. Eisenberg, DM, Davis, RB, Ettner, SL et al. Trends in alternative medicine use in the United States, 1990-1997: results of a follow-up national survey. JAMA 280: 1569-1575, 1998. 2. Farnsworth, NR, Akerele, O, Bingel, AS., Socjata, DD, Eno, Z. Medicinal plants in therapy. Bull World Health Organization 1985: 63: 965-981. 3. Gesler, WM. Therapeutic landscape medical issues in light of the new cultural geography. Soc.Sci. Med. 1992; 34: 735-746. 4. Rafferty, AP, McGee, HB, Miller, CE, Reyes, M. Prevalence of complementary and alternative medicine use: state-specific estimates from the 2001 Behavioural Risk Factor Surveillance System. Am. J. Public Health 92: 1598-1600, 2002. 5. Knez WL and Peake JM, The prevalence of vitamin supplementation in ultraendurance triathletes. Int J Sport Nutr Exerc Metab. 2010 Dec;20(6):507-14. 6. Abu-Irmaileh, BE, Affi, F.U. Herbal medicine in Jordan with special emphasis on commonly used herbs. J. Ethnopharmacology 89: 193-197, 2003. 7. Mamtani R, MacRae B, Mahfoud Z, Cheema S , El Hajj M, Lopez T and Lowenfels A. Use of herbal and nutrition supplements among college students in Qatar. American Society of Clinical Nutrition, 2013, Dubai. Acknowledgement: This work is supported by the Biomedical Research Program at Weill Cornell Medical College in Qatar, a program funded by Qatar Foundation.