Qatar Foundation Annual Research Forum Volume 2013 Issue 1

Asperger syndrome is one of the Autism Spectrum Disorders (ASD's), characterized by significant difficulties in social interaction and nonverbal communication, alongside restricted and repetitive patterns of behavior and interests. ASD's have a strong and complex genetic basis that cannot be distinguished by the clinical presentation. A South African family with an affected father and three affected sons all with characteristics of Asperger syndrome including repetitive routine physical gestures and Sensory Processing Disorder was studied for the possible identification of the responsible genetic factor(s). Due to the significant proof of heritability and the extreme heterogeneity of Asperger syndrome, next generation sequencing was performed on all members of this family to extrapolate monoallelic variations in the affected father that have been inherited by all three of the affected sons probably through an autosomal dominant pattern of inheritance. These variations, validated by Sanger sequencing, were analyzed, prioritizing significant changes in the encoded protein. Variants that segregate with the affected individuals include one deletion in C17orf80, an unidentified protein expressed in the brain, (c.1745_1748delGTAA), three missense mutations that change highly conserved amino acids in PARK2, which codes for a component of a multiprotein E3 ubiquitin ligase complex that targets proteins for degradation also known to cause juvenile Parkinson disease (c.110 C>T, p.Pro37Leu), FAT1, member of a large cadherin family required for cell-cell association and actin organization, (c.2563 C>A p.Gly855Arg), and OR4C6, an olfactory receptor protein coding gene, (c.293 A>C p.Gln98Pro) and one nonsense mutation introducing a premature stop codon in HYAL4, a hyaluronidase that intracellularly degrades hyaluronan, one of the major glycosaminoglycans in the extracellular matrix (c.628 C>T p.Arg210STOP). The direct link of these previously reported variants to Asperger syndrome is yet unknown, however some of these genes such as PARK2, HYAL4 and FAT1 have previously been reported to be in involved in brain function and development, indicating a possible role in the onset of Asperger syndrome.

This study evaluated the effectiveness of paravertebral block as alternative anesthetic technique for extracorporeal shock wave lithotripsy (ESWL) procedure. Fifty patients with renal stones, aged 20 to 60 years, were randomly allocated into two groups; twenty five patients in group P; received unilateral paravertebral block from T8 through L1 with injection of 5ml 0.5%bupivacaine and 25 patients in group L; received local infiltration by bupivacaine 0.25%(2mg/kg) into the 30 cm2 area after localizing the stones site, 10 minutes before the session. 10 mm visual analogue scale (VAS) was used to evaluate pain every 10 minutes during the session. At the end of the procedure, total doses of rescue analgesia, the number of shockwaves, their power and the total duration of shockwave treatment were recorded. After completion of the procedure the patient was assessed for pain and nausea in the post anesthesia Care Unit (PACU) using the Visual Analog Scale. Patient's satisfaction and time needed to discharge patients to home also were recorded. Time to do the anesthetic technique was significantly higher (p<0.001) in group-P than group-L, it was 12.7±2.3 minutes versus 6.9±1.9 minutes respectively, intraoperative rescue analgesia by fentanyl was lesser (P<0.001) in group-P than group-L, 26.7 ± 6.32mcg versus 78.6±5.41mcg, respectively, also time interval between ends of the procedure till discharge to home was significantly higher (P<0.001) in group-P than group-L, it was 99±17 minuets versus 133±31minuits respectively. VAS was not significant difference between both groups either intraoperative or postoperative in first hour. Patient's satisfaction was significantly higher (P<0.05) in group-P than group-L, it was 8.8±1.1 versus 6.1±0.6, respectively. Adverse events were lesser, but not significant in group-P than in group-L. Two patients (8%) in group-L and one patient (4%) in the group-P experienced episodes of postoperative nausea and vomiting (PONV). Paravertebral block is effective alternative anesthesia for outpatient lithotripsy; multiple level paravertebral blocks provide an optimal anesthetic condition, with acceptable adverse events for ESWL. And providing proper analgesia during the procedure and in first hour after finishing of the procedure, early discharge to home and providing better patient's satisfactions.

Abstract: Background: While promising, cationic lipid-mediated gene delivery can still benefit from improvements in lipid design and lipid-DNA (lipoplex) formulation. The putative mechanism of cellular lipoplex uptake is believed to occur by endocytosis, where the key influential factors are lipoplex size and morphology; lamellar and inverted hexagonal. Lamellar lipoplexes offer superior protection to the DNA cargo, while the inverted hexagonal phase best facilitates endosomal escape. Ideally, the initial lipoplex packaging would have the lamellar phase upon uptake, followed by a phase transition to hexagonal, facilitating cargo release into the cytosol. The cationic lipid structure defines its molecular packing parameter, S, which in turn controls the lipid phase transition. A molar weighted average packing parameter (Smix) for the overall cationic and neutral co-lipid mixture within a lipoplex formulation is predictive of a lamellar (S&lt;1) or hexagonal (S&gt;1) phase lipoplex. Objectives: Pyridinium-based cationic lipids represent a class of non-viral vectors that have shown promise in gene delivery. The objective of this study was to test the influence of lipid shape on lipoplex phase structure through small-angle x-ray diffraction (SAXD), and correlate shape with transfection efficiency. Lipoplexes co-formulated with pyridinium lipid, (16:0)(11:1) having a calculated shape parameter, S, of 1.08 and the commercial cationic vector, EPC (S=0.94) were predicted to undergo a packing transition from lamellar to hexagonal as the ratio of (16:0)(11:1) / EPC is increased. A fixed amount of neutral co-lipid was employed (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE: S=1.01; or cholesterol, Chol: S=1.20). Given that cholesterol is a higher-S lipid than DOPE, the lipoplex phase transition from lamellar to hexagonal was anticipated to occur at a lower ratio of (16:0)(11:1) to EPC. Methods: Liposomes were prepared from pyridinium-based cationic lipids in combination with EPC and co-lipid, DOPE or cholesterol. Lipoplexes were then formulated by incubating the liposomes with plasmid DNA at various N/P (+/-) molar charge ratios, and subsequently characterized by gel retardation, DNAse I degradation, biocompatibility and β-galactosidase (β-gal) transfection assays using Chinese Hamster Ovarian (CHO-K1) cells. Lastly, lipoplexes at N/P molar charge ratio 3 (only) were analyzed by SAXS at the synchrotron in Grenoble, France. Results: The SAXD results revealed that the (16:0)(11:1)/EPC/DOPE-DNA lipoplex formulations underwent a lamellar to hexagonal packing transition when the (16:0)(11:1)/EPC molar ratio was increased from 1:2 to 1:1, where the Smix increased from 1.01 to 1.04. However, (16:0)(11:1)/EPC/Chol-DNA lipoplex formulations underwent a lamellar to hexagonal transition when the (16:0)(11:1)/EPC molar ratio increased from 1:1 to 2:1; when Smix increased from 1.11 to 1.13. For both DOPE and Chol containing lipoplexes, the greatest transfection was found at (16:0)(11:1) to EPC ratios below 2:1, at an N/P molar charge ratio of 3. Conclusion: The lamellar to hexagonal packing transition, as determined by SAXD, for the lipid-DNA lipoplexes composed of the (16:0)(11:1)/EPC mixture occurred as predicted by our Smix calculations when DOPE was employed as co-lipid. The same was not observed when cholesterol was employed co-lipid. Finally, superior lipoplex transfection correlated with lamellar packing.

Introduction: Mild Brain Injury or concussion is frequently observed during contact sports such as football and soccer. If it remains undetected, a repeat concussion can lead to long term consequences because of the vulnerable brain tissue damage. Balance Error Scoring System (BESS) test is a widely used test to detect concussion. It requires the athlete with suspected concussion to maintain his/her position in three different stances, that are, both legs, single leg and tandem, and has a total score ranging from 0 to 30. The recommended way of performing this test is to do it barefoot in clinical or semi-clinical settings. Such conditions are however difficult to achieve during an ongoing match, and athletes would like to be near the field with their cleats on - situations often found on soccer fields in eastern countries. Objective: Therefore, we aimed to assess the reliability of BESS test in different field conditions. This research is in line with Qatar National Research Strategy 2012 pillars, H.E.1.9 (prevention of brain Injury) and H.E 1.10 (control of sports injuries). Methods: This study was conducted under the auspices of McGill Sports Emergency Medicine Clinic. Athletes from soccer and football teams were approached on the field during practice games. After informed consents, they performed BESS test in three conditions, that were, barefoot, on turf with cleats and on hard surface with cleats. Each athlete was rated by three observers independently of each other. We computed mean difference in total BESS scores with 95% confidence intervals (95%CI). Comparison of total BESS scores under different conditions as well as inter-observer reliability was assessed using intraclass correlation coefficient (ICC).. Results: We recruited 49 athletes from football (n=39) and soccer (n=10) teams in this study. Thirty nine of them were male, 10 were females. Average age was 21.1 years (standard deviation [SD]=1.9). We found that total BESS scores were significantly different (P&lt;0.001) between barefoot and the two conditions with cleats-on: 2.2 (95%CI=1.6, 2.8) for turf and 2.0 (95%CI=1.4, 2.6) for hard surface. Concordances of barefoot with turf (ICC=0.47, P=0.02) and hard surface (ICC=0.51, P=0.01) conditions were moderate. A moderate to high inter-observer reliability (0.60≥ICC≤0.75) was observed for BESS test under three conditions. Conclusion: These findings show that BESS test has a fair reliability under different conditions, and may be useful in screening concussion on the field. However, cut-off of BESS should be reduced by 1 to 2 points if it is applied on the field with cleats.

Diabetic nephropathy (DN), a serious complication of diabetes, is characterized by hyperfiltration, hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. In renal hypertrophy, tubules increase in size and accumulate extracellular matrix and are also associated with alterations in renal sodium handling as well as hypertension; processes linked by involvement of the arachidonic acid (AA) metabolites 20-HETE and EETs. This study aims to determine the specific AA-metabolizing CYP450 isoforms present in proximal tubules (PT) that are altered by high glucose (HG) in cultured PTs, and in an animal model of diabetes. It intends to investigate the effects of alterations in CYP isoforms and/or AA-metabolite levels in DN. This work will investigate the mechanism of PTs injury and the effect of inhibition of AA-metabolites in vitro and will also get insight onto the cross-talk between CYP450 isoforms and other sources of Reactive Oxygen Species (ROS). Immunohistochemistry, hypertrophy, apoptosis, fibrosis, ROS generation, 20-HETE and EET formation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. To assess the significance of our in vitro findings, in vivo experiments were performed. Type 1 diabetic rats were used to assess the levels of different cytochromes as well as the levels of injury in these rats. In this study, we demonstrate that rats with streptozotocin-induced diabetes develop renal hypertrophy and increased fibronectin expression concomitant with an increase in CYP4A expression and a decrease in CYP2C expression. These observations were paralled by an alteration in 20-HETE and EETs productions. These results were also paralleled by an increase in reactive oxygen species (ROS) production and NADPH oxidase activity. Treatment of diabetic rats with HET0016, selective inhibitor of CYP 4A, prevented all these changes. In contrast, treatment of diabetic rats with MsPPOH, a potent inhibitor of EETs formation worsens the injury seen in the kidneys of the diabetic rats. Our results indicate that hyperglycemia in diabetes has a significant effect on the expression of AA-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites; this pathway is through an oxidative stress-dependant mechanism.

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.

Introduction: Diabetes mellitus (DM) is a disease with severe complications and major health/economic impacts. It is a leading cause of morbidity and mortality worldwide, with an estimated 346 million adults being affected in year 2011. World Health Organization (WHO) projects indicated that diabetes death will increase by two thirds between 2008 and 2030. The WHO estimated that 80% of the populations of developing countries rely on traditional medicines, mostly plant drugs, for their primary health care needs. Diabetes is an example of a disease that has been treated with plant medicines. Our study evaluated ethanolic and aqueous extracts of selected Sudanese plants that are traditionally used to treat diabetes; these are: Ambrosia maritima, Ammi visnaga, Acacia senegal, Sesamum indicum, Nigella sativa and Foeniculum vulgare. The plants extracts were tested for their glycogen phosphorylase inhibition, toxicity and antioxidant activity. Materials & Methods: Ethanolic and aqueous extracts prepared from leaves of Ambrosia maritime, fruits of Foeniculum vulgare and Ammi visnaga, exudates of Acacia Senegal, and seeds of Sesamum indicum and Nigella sativa were investigated. The antioxidant properties of the extracts were tested using (DPPH) photometric and Iron Chelating Assays. The enzymatic inhibition of glycogen phosphorylase (GP) activity was monitored using multiskan spectrum (Thermo-Scientific). GP activity was measured in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate. Brine Shrimp Lethality Test was also used to determine plants toxicity. Results and Discussion: Free radicals are formed in diabetes by glucose oxidation, nonenzymatic glycation of proteins and subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals can lead to damage of cellular organelles and enzymes, increased lipid peroxidation and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus. In this study all plant extracts with exception of Acacia senegal exhibited significant antioxidant activity in DPPH free radical scavenging assay. This may support the traditional usage of these plants to improve complications that caused by diabetes mellitus. Nigella sativa aqueous extract showed no toxicity on Brine shrimp Lethality Test, while its ethanolic extract was toxic. All other extracts are toxic and ethanolic extracts of Foeniculum vulgare and Ammi visnaga exhibit the highest toxicity. Results of this study did not show any significant inhibition of glycogen phosphorylase, but extracts of these plants may act on one of other enzymatic reactions that involved in carbohydrate metabolism and improved glucose homeostasis. Conclusion and Recommondation: Changes in oxidative stress and effects of antioxidants in diabetes management should be considered, and hopefully, further research into the pathophysiology of oxidative stress and the role of antioxidant therapy will lead to appropriately-designed clinical trials in which the promise of antioxidant therapy will be realized. Extraction processes and usage doses should be monitored. Further work is underway to test the plant extracts on diabetes-induced mice. Key words: Diabetes mellitus, medicinal plants, antioxidant activity, glycogen phosphorylase, brine shrimp

Rationale Despite improvements in oral health care during the last decade, caries and other tooth structure defects still represent major global public health concerns. To date, no index has satisfied records of different tooth structure defects that could be present in the same mouth and in the same tooth. Available indices provide data on some defects separately. Exposing decision makers to deficient information leaves them unaware of the high levels of untreated defects. Objective Our long term objective is to develop a method for valid and reliable evaluation of tooth structure defects. Such a method would have a broader utility in the implementation of cost effective preventive, non-operative and therapeutic measures to sustain oral health. Specific aims of the current study was to describe: 1) Different tooth structure defects in a random sample for the target population ( Qataris and Egyptians); 2) determine the most prevalent defect(s) in this population and 3)identify the most frequently affected teeth subject to these defects Materials and methods We carried out a cross sectional study to address the stated objectives. A random sample was recruited from 2 subpopulations ( Qatar and Egypt). Each study unit received thorough clinical assessment and each tooth was scored accordingly (table 1 shows clinical evaluation criteria). Data was entered in the data base and statistical methods were used. The association between condition and nationality was performed using the Chi square test/ Fischer's exact test. Results All individuals(93) showed evidence of tooth structure defect(s). Table 2 shows the frequency and percent distribution according to teeth structure defects (fig a, b). Apparently, decay is the most common defect in this target population (87%) followed by filled teeth (57%). The least common defects are abrasion and abfraction. A remarkable finding was that hypoplasia and hypo calcification are common in this population. These 2 conditions render teeth susceptible to caries. Among Qataris, the most common defect was decay followed by missed teeth(wisdom probably due to age) which is a similar pattern observed in Egyptians . Qataris are less likely to experience fractured teeth in comparison to Egyptians (odds ratio = 0.06 , p<0.001). Qataris are four times more likely to experience hypoplasia in comparison to Egyptians ( odds ratio 3.7 and the p value is 0.07). However, Qataris are 1.4 times more likely to experience hypo calcification compared to Egyptians but the odds ratio is not significant (p value 0.5). In this population it was common to see that teeth have more than one tooth structure defect which was not captured in the previous indices. Molars and premolars were most susceptible to decay whereas anterior teeth showed hypo calcification in Qataris whereas for Egyptians, molars and premolars were mostly decayed (tables 3 and 4) Conclusions To our knowledge, this is the first attempt to demonstrate the need to develop a thorough evaluation method that captures all possible tooth structure defects likely to occur in the same mouth and within the same tooth. The results urges the development of a valid and reliable index.

Background and objectives: A high prevalence of insulin resistance (IR) amongst normal weight Qatari individuals may contribute to progression of type II diabetes and account for the current epidemic. Factors contributing to this increased risk of IR and its associated co-morbidities, especially in the absence of obesity, are still under investigation. Therefore the objectives of this pilot study were to characterize components of IR in normal weight Qatari individuals and to investigate the potential molecular mechanisms underlying this phenotype. Methods: Non-diabetic lean/overweight Qatari subjects were recruited and anthropometric measures including body weight (kg), height (m) and blood pressure recorded, along with determination of systemic lipids, glucose, insulin and adipokines. Subjects were dichotomized into IR and IS groups based on their HOMA index (fasting plasma glucose < 6.8 mmol/l and insulin levels < 6.5 miU/ml). The expression of 84 most abundantly expressed and characterized miRNA species was profiled in peripheral blood samples. Target genes of miRNA let7 were determined using Human let-7a Targets PCR Array. Results: When subjects (29+/-6.8 years old, BMI of 23+/-4.9 kg/m-2) were stratified into two groups based on their HOMA index (IS 1.3 (1-1.6) and IR 2.2 (1.6-2.7), IR (66%) was only associated with higher insulin levels {(IS 5.3 (5.2-5.5) vs IR 9.5 (7.7-12.1) u/ml, p <0.01)} with no significant differences in blood pressure, lipids profile or adipokines levels. Among 84 profiled miRNA species, the expression of 7 miRNA varied significantly between the two groups; among these were four members of let7 family (g/b/c/f). Three potential target genes of let7 exhibited significant variable expression between the two groups, including dual specificity protein phosphatase 1 (DUSP1). Conclusions: Prevalence of IR among young, lean/overweight Qatari individuals is alarmingly high (66%). This study has revealed let7 miRNA as a potential target mediating this phenotype. This miRNA has been shown previously to play an essential role in adipogenesis. The targets of let7 with an ability to regulate adipogenesis is currently being investigated to confer functionality.

Hematological Malignancies (e.g., leukemia and lymphoma) are among the top 5 causes of cancer death in the Middle Eastern Countries, including Qatar, Kuwait and Saudi Arabia. The monoclonal antibody rituximab, directed at the CD20 antigen, has become an essential drug for the treatment of Non Hodgkin Lymphoma (NHL). Although transient B cells depletion frequently occurs after rituximab treatment, it usually resolves after 6-9 months. Nevertheless, high frequency of non-neutropenic infections and persistent hypogammaglobulinaemia during follow-up period have been recently reported. However, impaired humoral response to the recall and primary antigens was found in NHL patients during (or few months after), rituximab treatment. Influenza vaccination is generally recommended in lymphoma patients, but few data are available about the activity of this vaccine after rituximab-containing regimens (RCR). It is presently unclear whether patients treated with RCR regain normal immunocompetence after achievement of complete remission. We presently combined data of 3 sequential studies conduced at our Institutions assessing the humoral response to seasonal influenza vaccination (2008/2009, 2009/2010 and 2011/2012 seasons; RIT-01, RIT-02, and RIT-03 studies, respectively) in NHL patients in complete remission (CR) for at least 6 months after treatment with rituximab-containing regimens (RCR). Response was evaluated by hemagglutinin inhibition assay 3 or 4 weeks after vaccination. The following (inactivated) vaccine formulations were used: virosomal vaccine (RIT-01; N=31), MF-59 adjuvanted vaccine (RIT-02; N=14), and intradermal vaccine (RIT-03; N=22). Data were compared with those from age-matched cohorts of healthy volunteers (HV). In the RIT-03 study, cancer patients who received chemotherapy without rituximab (CWR) more than 6 months before study entry were also evaluated. In the RIT-02 study, patients also received two doses of pandemic H1N1 vaccine. To determine early transcriptional changes predictive of immunoresponsiveness and to determine differences in innate immunity activation among patients treated with RCR, HV, and patients treated with CWR, PBMC were collected just before and 1 day after vaccination (RIT-03 study). Whole-genome gene expression analysis of these samples using microarray analysis is currently ongoing. We found that the intradermal vaccination is associated with dramatic transcriptomic changes in PBMC, already detectable 24 hours after vaccination. These changes underlie modulation of innate response (eg, interferon stimulated genes). Changes after influenza vaccination differ among CWR, CRC, and healthy volunteers. Overall, we found that patients treated with RCR have a significant lack of humoral response to both recall and na&#239;ve influenza antigens as compared with HV and with cancer patients treated with CWR. This impairment persisted even long time (&gt; 6 years) after last rituximab administration and was associated with depletion of CD27+ memory B cells and hypogammaglobulinemia. Therefore, patients previously treated with RCR should be strictly monitored during influenza epidemic season. Intradermal vaccination seems to induce a stronger response as compared with the other two formulations.

Background: Traumatic brain injury (TBI) and related death rates vary worldwide. Objectives: To evaluate the incidence, causes and outcome of TBI in Qatar. Method: A retrospective review of all TBIs admitted to the trauma center between January 2008 and December 2011 was performed. Patients' demographics, mechanism of injury, morbidity, and mortality were analyzed. Results: A total of 1665 patients with TBI were admitted, the majority were males (92%) with a mean age of 28±16 years. The common mechanism of injury was motor vehicle crashes (MVCs)and falls from height (51% and 35%, respectively). TBI was incidentally higher in young adults (34%) and middle age group (21%). The most frequent injuries were brain contusion (40%) followed with subarachnoid (25%), subdural (24%), and epidural hemorrhage (18%). The mortality rate was 11% among TBI patients. Mortality rates were 8% and 12% among adolescents and young adults, respectively. The highest mortality rate was observed in elderly patients (35%). Head AIS, ISS and age were independent predictors for mortality. Conclusion: In Qatar,TBI is reported in around 27% of all the trauma admissions;mostly due to MVCs and is associated with high mortality. Elderly are the most vulnerable group. particularly in the older group. Public awareness and injury prevention campaigns should target young population.

Objectives: Dizziness is a relatively common medical complaint that that rarely requires hospitalization for work-up and management. Whether gender related differences exist in the etiologies and outcome of patients hospitalized with dizziness is unknown. The aim of this study was to compare women and men presenting with dizziness in a real-world population. Methods: Retrospective analysis of all patients hospitalized with dizziness in Qatar to the cardiology service from 1991 through 2010 was made. Patients were divided into two groups according to gender. Clinical characteristics, management and outcomes were analyzed. Results: During the 20-years period, 1578 patients were hospitalized with dizziness; 404 women (25.7%) and 1173 men (74.3%). Women had significantly more prevalence of hypertension (46.9% vs. 31.1%, P=0.001) and diabetes mellitus (39.8% vs. 31.1% P=0.001) compared to men. Cardiac arrhythmia was the most common underlying diagnosis and was significantly more common in women than men (40% vs. 28.4%; P= 0.001), whereas acute coronary syndromes were significantly less common in women (13.6 vs. 25.9%; P= 0.001). The in-hospital mortality rate was significantly higher in women with dizziness compared to men (5.7% vs. 3.2%; P=0.02) [table]. Conclusions: Our study demonstrates that women hospitalized with dizziness have worse in-hospital outcome and different underlying etiologies compared to men. Further prospective research is warranted to confirm our observations in other registries.

Failure to offer evidence based structured diabetes educational program impacts negatively on the effectiveness of diabetes management and leads to increased morbidity and mortality. We assessed the efficacy of a nurse led group based structured diabetes education in improving glycemic and metabolic parameters among Arab type 2 diabetic patients compared to usual care Methods This was a parallel group randomized trial in adult Arab type 2 diabetic patients living in Qatar. Subjects were randomized to nurse led structured diabetes educational program ,or to usual care .The primary outcome was the improvement in HbA1c and other metabolic parameters including lipid profile ,albumin/creatinine ratio ,blood pressure and body mass index.. Patients were invited to attend four 2-hour sessions of self-efficacy improvement education .Outcomes were assessed at base line and 12 months later. The primary analysis was an intention to treat. Results Participation of the intervention was shown after 12months to have led to a statistically significant improvements in HbA1c(-0.55 m mol /L ,p=0.012),F.B.S(-0.92 m mol/L ,p=0.022),B.M.I(-1.70,p=0.001) and albumin/creatinine ratio(-3.09,p<0.001) but not in the control arm. conclusion Inclusion in the Nurse led intervention by adult Arab type 2 diabetic subjects was shown 12-months post intervention to have led to enhanced glycemic and metabolic parameters including body mass index and blood pressure .The success of this trial justify the feasibility and generalization of this intervention to neighboring Arabian Gulf countries who have similar Arab population with identical cultural beliefs and practices.

Facilitated LMA fiberoptic intubation while keeping on ventilation in ischemic heart patient with unexpected difficult airway Authors: Mahmoud Abdalla MD. Hesham Ewila,MD. Hany Osman, MD. Abdulrashed Pattah MD. Institution: HMC, heart hospital, DOHA QATAR Difficult tracheal intubation remains an important cause of mortality and morbidity during general anesthesia, especially in ischemic cardiac patients where hypoxia rapidly compromise myocardial function and may induce dysrrhythmias. We report 68 years old male was admitted to Qatar heart center for elective CABG with poor left ventricular function EF 30 -35 % .No signs of difficult intubation were appreciated preoperatively. Intraoperatively LMA was inserted due to unexpected difficult airway after failing of 3 optimized trials of intubation. Endotracheal fiberoptic intubation through LMA also failed as patient rapidly desaturated. We attached T piece to the distal end of LMA, keeping patient ventilated through the side port of T piece, fiberoptic intubation achieved through pre cut plastic venous cap 0.5 inch which was attached to distal end of LMA. Endotracheal tube with ID 6.5 mm was inserted over the fiberscope then exchanged over a ventilating bougie to 8.5 mm tube. No significant changes in heart rate, blood pressure or oxygen saturation were appreciated throughout the procedure time. This maneuver allowed us to secure the airway while keeping on ventilation without compromising the poorly reserved cardiac function or exposing the patient to hypoxia or hypercarbia. Key Words: fiberoptic bronchoscope, unexpected difficult intubation, laryngeal mask airway. Ischemic heart disease

Household spices comprising, chili, Kashmiri chili hot, Kashmiri chili mild, basil, oregano, ginger, curry, cumin, turmeric, tandoori masala, garam masala, black pepper, garlic and coriander, were collected from local markets in Doha, Qatar, during 2012, and were surveyed for the presence of potentially harmful mycoflora and for contamination with aflatoxins B1, B2, G1, and G2 by high-performance liquid chromatography (HPLC). Among the tested spice samples, chili powder showed the highest presence of fungal propagules, while ginger, curry and garlic samples did not present any fungal contamination. A total of 120 isolates, mostly belonging to Aspergillus and Penicillium genera, were collected and 33 representative species were identified by amplification and sequencing of the internal transcribed spacer (ITS) region. Aspergillus flavus, Aspergillus nomius and Aspergillus niger were the most dominant. Thirty-seven Aspergillus strains were screened for their potential to produce aflatoxins using biochemical and molecular tools. Upon these methods, only 9 A. flavus strains showed both fluorescence and amplification with all the three primers targeting aflP, aflM and aflR genes. Aflatoxins were detected in five spices (black pepper, chili, tandoori masala. turmeric and garam masala), and with the exception of garam masala, the tested samples of turmeric, black pepper, tandoori masala and chili powder exceeded B1 and ⁄or total aflatoxin maximum levels. Our results demonstrate the potential for mycotoxin biosynthesis by fungi contaminating imported spice products.

Phospholipids in cells are mainly synthesized in the cytoplasmic leaflet of the endoplasmic reticulum (ER) and the newly made polar lipids must flip-flop rapidly across biological membranes to sustain cellular life. But this 'flipping' is energetically costly as well as its translocation rate is low. As a solution to this, cells have membrane proteins that function as lipid transporters - 'flippases', proteins that facilitates the rapid, bi-directional, energy-independent flip-flop of phospholipids between the cytosolic and lumenal leaflets of the ER membrane. Flippases have been known to play a key role in membrane stability as well as in the mechanism by which cells avoid being killed by macrophages. Candidate flippases have been implicated in human diseases that include intrahepatic cholestasis, angelman syndrome, autism, tangier disease, macular dystrophy and adrenoleukodystrophy. Establishing the primary function of candidate flippases and how they contribute to cell function and human disease is becoming a central issue in biology. Although the flippases that operate at the plasma membrane of eukaryotes at the expense of ATP hydrolysis resulting in unidirectional lipid flipping have been identified, the ATP-independent bi-directional flippases that translocate lipids in specialized compartments such as the ER have not yet been identified at their molecular level. The objective of the current study is to identify ER flippases in yeast Saccharomyces cerevisiae using a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC). Yeast cells were grown in synthetic medium supplemented with either 'light' or 'heavy' lysine. Proteins extracted from unlabeled (light) cells were further fractionated by velocity sedimentation in a glycerol gradient and flippase activity of each fraction was quantified by a phospholipid reconstitution-based procedure. An aliquot of labeled (heavy) extract (containing equal amount of protein by weight) was added to each light fraction. The mixed fractions were then subjected to in-gel digestion followed by quantitative proteomic analysis using mass spectrometry. The data obtained were processed using MaxQuant followed by Spearman correlation analysis for identification of proteins with enrichment profiles matching that of the activity profile. The potential flippase candidates were tested for their activity using genomically tagged yeast strains.