Qatar Foundation Annual Research Conference Proceedings Volume 2014 Issue 1

Background Bacterial superantigens (SAGs) are potent T cell stimulatory molecules and comprise a large family of disease-associated proteins. Superantigens bind to APCs on the outside of the MHC class II molecule and to T cells via the external face of the T cell receptor (TCR). This enables them to activate up to 20% of resting T cells, whilst conventional antigen presentation results in the activation of 0.001- 0.0001% of the T cell population. These biological properties of superantigens make them very attractive for use in immunotherapy. Objectives 1.Codon optimization, for maximum expression in E. coli and synthesis of four superantigens genes 2.Cloning, Overexpression, purification and molecular characterization of four superantigens Methods The four superantigens codon-optimized genes were produced by Geneart GmbH Company (Regensburg, Germany). Additionally, we introduced several new restriction enzyme sites into the gene, e.g. NdeI and HindIII sites were placed at the 5' and 3' ends of the gene, respectively to facilitate the cloning. Each gene was excised from pGA/sAg by digestion with NdeI and HindIII and inserted into similarly digested pET28a DNA using T4 DNA ligase. The newly formed construct of each gene was transformed into competent cells of E. coli BL21 (DE3) RIL. The expression of each superantigen was done by induction using IPTG. The induced cells were harvested by centrifugation and the total proteins were produced by sonication. The expression analysis was performed using SDS-PAGE. Novel SAGs (TSST, SPE, SEA and SEB) were tested in vitro for their superantigenicity and anticancer activity using peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers and prepared by Ficoll-Paque gradient centrifugation.Superantigenicity was assessed by monitoring the SAG-induced release of PBMCs cytokines. PBMC were treated in the presence or absence of SAGs for 24 h and the release of IL-1β, IL-8, IL-10, TNFα and IFNγ measured by Luminex assay. Furthermore, 3 different colon cancer cell lines were treated with SAGs in the presence or absence of PBMC for 48 h. Inhibition of cancer cell proliferation was measured by MTT assay. Results In this study, four synthetic genes coding for four different superantigens, SEA, SEB, TSST-1 and SPEA were codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work showed that in all cases the recombinant superantigens were expressed to 40% of the total host protein. We managed to optimize the condition to obtain 20% of the recombinant superantigens in a soluble form. The purification of the soluble His-tagged recombinant superantigens have been achieved in a single step by Ni2+ charged column chromatography. Superantigenicity assay by measuring the released interferon gama showed that the four recombinant superantigens are active with superantigenicity functions. Conclusion The successful cloning and expression of four codon optimised synthetic superantigens genes will provide the recombinant protein which will be modified for production of safer superantigen for cancer immunotherapy.

Background: Healthcare in Qatar is undergoing a period of major reform, driven by a strong economy and vision for a world-class healthcare system. One area identified as a potential contributor to developing a world-class healthcare system is interprofessional education (IPE), with the goal of facilitating collaborative practice among healthcare workers. Several key steps have been taken towards developing IPE in Qatar, such as the formation of the Qatar Interprofessional Health Council (QIHC), the development of an IPE program for undergraduate healthcare students, the development of a set of shared core competencies, the receipt of substantial buy-in from leaders across the healthcare system, and recent approval of funding to develop a post-licensure healthcare IPE program. In order to improve IPE in Qatar, it is important to better understand the facilitators and barriers to interprofessional collaboration in Qatar. This study seeks to do so by qualitatively exploring facilitators and barriers to interprofessional collaboration in Qatar from the perspective of healthcare professionals. This research can assist in improving healthcare in Qatar by increasing understanding about how healthcare workers give meaning to interprofessional education and collaboration. Objectives: The purpose of this paper-presentation is to report on finding from a qualitative study that explored different facilitators and barriers of interprofessional practice in Qatar. Method: 10 healthcare professionals who work in Qatar were interviewed using semi-structured, open-ended interviews organized by the theoretical approach of phenomenology. The interviews focused on exploring barriers, facilitators, and what is working well in terms of interprofessional practice for healthcare professionals in Qatar. Findings and Implications: Different factors associated with interprofessional collaborations will be discussed. In doing so, this research adds to the literature on IPE by shedding light on interprofessional collaboration and education in the Middle East. Furthermore, this study identifies barriers for healthcare workers to work collaboratively in healthcare settings in Qatar. Addressing such barriers, and building on what is working well, will facilitate Qatar in reaching one of the Qatar National Vision 2030 goals of improving Qatar's health and wellness.

Background & Objectives The state of Qatar has witnessed significant lifestyle changes due to rapid urbanization, the introduction of labour-saving devices and the availability of high-caloric density food. This has impacted on the daily lifestyle and health habits of young adults leading to significant increases in non-communicable diseases (WHO, 2014). This study explored the risk factors associated with such diseases amongst young adults in Qatar. Methods A representative sample of 732 males and females (aged 18-25 years) from Qatar University took part in this cross-sectional, mixed-method design study. Physical Activity (PA) and dietary habits were assessed using a validated questionnaire. Total energy expenditure per week was calculated based on the metabolic equivalent values of each activity reported by the participant (Al-Nakeeb et al., 2012). Body Mass Index (BMI) was calculated according to the International Obesity Task Force criteria and using the age and gender-specific BMI classification established by Cole et al. (2000). Results The percentage of overweight/obesity in males and females was 39.5% and 38.5% respectively. It was evident that there was a significant increase in the percentage of students classified as overweight/obese from year 1 to year 4. Meanwhile, there was a decline in the level of PA and an increase in sedentary time during that period. Whilst health was reported to be the main reason for participation in PA/sport, lack of available time was singled out as the main barrier to engagement in an active lifestyle. Ironically, students reported more than 4 hours of TV/DVD viewing and internet use per day. Conclusions The adoption of healthier lifestyles amongst the Qatari population, including an increase in PA and a reduction in overweight/obesity are major objectives cited in Qatar Vision (2030). This study has revealed a high prevalence of overweight/obesity amongst male and female university students with regressive trends in their lifestyle and health habits. The findings reveal a worrying picture of young people's lifestyle that ought to be a cause for concern for policy makers and health professionals. Undoubtedly, there is an urgent need to seriously consider putting in place intervention strategies concerning behaviour modification and the built environment in order to reverse these trends. Such strategies could have major implications on the health and well-being of young people at this critical age developmentally and on the future welfare of the wider community in the long run. References *WHO (2014) World Health Statistics - 2014. WHO Press: Switzerland. *Al-Nakeeb, Y., Lyons, M., Collins, P., Al-Nuaim, A., Al-Hazzaa, H., Duncan, M. and Nevill, A. (2012) Obesity, physical activity and sedentary behavior amongst British and Saudi youth: A cross-cultural study. Intl J Environ Res Public Health, 9, 1409-1506. *Cole T.J. et al. (2000) Establishing a standard definition for child overweight and obesity worldwide: International survey. Br. Med. J., 320 (7244): 1240-1243. *General Secretariat for Development Planning - Qatar National Development Strategy 2011-2016, Towards Qatar National Vision 2030. Qatar: Gulf Publishing and Printing Company, Doha.

Protein misfolding and amyloid formation is an underlying pathological hallmark in a number of prevalent diseases of protein aggregation, including Parkinson's disease, Alzheimer's disease and Type 2 diabetes (T2D). The expansion in the prevalence of T2D, including in Qatar, where a high percentage of the population is affected by diabetes, poses considerable risks to individuals, the healthcare system and the economy. Epidemiological studies reveal that up to 95% of all patients with T2D are shown to have pancreatic amyloid deposits, as detected in post-mortem studies. Most importantly, the severity of the disease appears to correlate with the degree of the deposition of IAPP aggregates. The misfolding of IAPP followed by the aggregation has been shown to be highly cytotoxic and to play a key role in the death of β-cells in T2D. Thus, the modulation of the aggregation process, promoting the proper folding of IAPP, may be considered as an attractive avenue for a therapeutic intervention. Indeed, molecular chaperones have been shown to suppress the misfolding and to prevent amyloid formation. The mechanism of how Hsc70 inhibits hIAPP fibril formation is complex and is not yet fully understood. It remains unclear how specific domains of Hsc70 function independently or in cohort to produce the observed inhibition of fibril formation of hIAPP. To address these questions, we used the power of in vitro methods to dissect out the relative contribution of the different Hsc70 structural domains, by investigating the effect of a series of deletion mutants of Hsc70 on hIAPP fibril formation. We also investigated in further detail the mode and mechanism of interaction between Hsc70 and hIAPP. The results indicate that Hsc70 can bind to hIAPP and delay fibril formation even in the absence of the ATPase domain, but interaction of hIAPP with the substrate- binding domain is strongly influenced by the C-terminal lid region. Determining the molecular interplay between hIAPP and molecular chaperone Hsc70 will provide the basis for designing modulators of IAPP aggregation.

Background: Sickle Cell Anaemia (SCA) is a genetically-inherited blood disorder caused by the occurrence of a point mutation in the bases coding for the sixth amino-acid of the β-chain of haemoglobin. The presence of Fetal Haemoglobin (HbF) in blood is known to show a number of beneficial effects in improving the conditions of SCA. Hence, clinical symptoms of SCA arise only after HbF levels drop. HbF is synthesized by the HBG1 (gamma-globin) gene. From birth there is a gradual shift from fetal to adult haemoglobin triggered by the downregulation of the HBG1 gene and the upregulation of the HBB gene. Rationale: Hydroyxurea has been widely evaluated in the treatment of SCA for its ability to induce synthesis of HbF by reactivating the expression of the HBG1 gene. It functions by repressing the activity of various transcription factors involved in the downregulation of the HBG1 gene during transition to adult haemoglobin. However, hydroxyurea has proven to be potentially toxic in a number of cases and may also carry a possibility of long term carcinogenicity. Therefore it is administered under restriction and may not be used in the treatment of pregnant women and children. In this study, an attempt is made to identify and characterise the altered gene expression pattern caused by hydroxyurea treatment through Differential Display Reverse Transcriptase PCR (DDRT-PCR) and Real Time PCR (Q-PCR). Further, the phenomenon depicting the repression of the transcription factors leading to the reactivation of HBG1 gene will be reproduced using techniques of RNA interference (RNAi) and antisense. Objective: To reproduce the altered genetic conditions produced by Hydroxyurea treatment for the in-vitro induction of HbF using molecular genetic techniques. Methods: Blood is obtained from SCA patients at diagnosis and after treatment with hydroxyurea. Erythroid Progenitor cells are purified from the collected blood using the EasySep™ Human progenitor cell enrichment kit. Cells are then cultured in a suitable medium and maintained for further analysis. Treatment with hydroxyurea is performed at varying doses at different intervals of time. The evaluation of gene expression is carried out through the generation of c-DNA through a reverse transcriptase PCR cycle followed by Q-PCR for identification and quantification. Results & Conclusion: Buffy coats containing mononuclear cells were purified from peripheral blood obtained from SCA patients through density-gradient centrifugation. Further, erythroid progenitor cells were successfully isolated and maintained in culture for variable periods of time. Currently, mRNA expression analysis is ongoing through the use of reverse transcriptase PCR protocols. If proven successful, this technique holds a promising future scope; as molecular genetic techniques could be used to reproduce the effects of hydroxyurea without the actual administration of the drug. Thus, the toxicity issues of the drug are avoided while also making it available to all classes of patients including pregnant women and children.

Background: Strokes account for over 8% of deaths in men and 12% of deaths in women in the UK and the total cost of Stroke to the National Health Service within the UK is estimated to be over £7 billion per year. Carotid artery disease can cause stroke, transient ischaemic attack (TIA) and amaurosis fugax. Surgical intervention to remove the carotid plaque is likely to be helpful in symptomatic patients with > 50% stenosis of the carotid artery. The aims of this study were to (i) investigate referral pattern of carotid artery disease for duplex vascular ultrasound in a local hospital in UK; (ii) investigate whether confocal microscopy can be used to obtain 3D images of the micro-structure of atherosclerotic carotid plaques; (iii) construct a blood flow model with a view to investigate how forces resulting from fluid flow interact with structural stability of carotid atherosclerotic plaque Methods: A one-year audit for the total number of carotid artery vascular ultrasound scans referred to the Medical Physics Department by all specialists was carried out between (January- December). Those with > 50% stenosis (using St Mary's criteria) were identified, including those with symptoms who underwent carotid endarterectomy (CEA) . Carotid plaques were collected from routine carotid endarterectomy surgery and examined using flow-modelling, bright-field and Laser-Scanning Confocal-Microscopy (LSCM) to generate 3D image datasets and visualisations of surgically removed carotid plaques. Results: Over a one-year period, the total number of carotid artery referrals by all specialists for ultrasound scanning at the Medical Physics Department was 1353; 63.3% (n=856) of whom were referred from the TIA Clinics by stroke specialists. Out of this total referral, there were 139 patients (10.3%) with > 50% carotid artery stenosis, 57 of whom were symptomatic and underwent surgical intervention (CEA). 3D imaging of carotid plaques, using LSCM showed that most specimens were predominately composed of lipid material, comprising necrotic core of amorphous debris and cholesterol clefts, with varying degrees of fibrous tissue present in all plaques. Regions of actual fibrous cap disruption and some ulceration were also seen. Fraying of the fibrous cap was notable with fibrous cap erosion and exposure of underlying necrotic core to lumen. Evidence of carotid plaque vulnerability as demonstrated by reduced fibrous cap thickness and large lipid-necrotic core with evidence of cracking was also seen in the 3D visualization. A blood flow simulation model shows how blood velocity changes could occur associated with reduction in lumen diameter caused by the plaque. The degree of carotid artery stenosis measurements obtained from these flow models were consistent with, and comparable to the degree of stenosis measurement recorded on the pre-operative vascular ultrasound reports of patients from whom these plaques were taken. Conclusions: The visualization of the internal 3-dimensional microstructure and geometry of the plaques could help in: (i) investigating key features that affect plaque structural stability; (ii) comparing 3D microstructure of the plaque with clinical imaging assessment and blood flow investigations; and (iii) developing markers to identify patients requiring clinical intervention.

Background & Objectives: Integration of Clinical Decision Support Systems (CDSS) in mobile and smart environments helps to improve the quality of life of people with health problems. CDSS are used to derive clinical conclusions from patient data, in order to automate and help the process of diagnosing and treating the patient. One way that CDSS can be implemented is to formalize a clinical guideline (document detailing best practices for diagnosing and treating patients) and use it on a knowledge base containing the patient's data. An interesting perspective is to use CDSS in a smart home (SH) setting, where data for the remote CDSS can be obtained from SH services and mobile devices using ambient and wearable sensors. However, in order to maintain minimum quality of service for CDSS decision support, the CDSS decision process must be deployed locally as a SH service an on mobile devices. An ideal example domain for this integration scenario is the diagnosis of sleep apnea. Sleep Apnea has several symptoms that include recurrent awakening, loud snoring, choking episodes, non-restorative sleep and daytime sleepiness. Usually, an individual with sleep apnea is not aware of having difficulty breathing, and is often recognized by others witnessing the individual during sleep apnea episode or is suspected because of the observed symptoms. Sensors could detect such episodes automatically without the need for a human intervention, or recognitions of said symptoms. The objectives are to illustrate the feasibility of CDSS as SH service and on mobile devices by using the Sleep Apnea CDSS. Methods: The sleep apnea CDSS decision process uses Semantic Web tools and rule-based reasoning, in order to formalize the current Canadian guideline for the recognition of sleep apnea. A total of 9 rules were derived. A patient dataset comprises health factors related to sleep apnea, including clinically relevant personal information, clinical measures and observations, and symptoms specific to sleep apnea. To validate the decision process of a CDSS integrated with smart homes, we implemented the Sleep Apnea CDSS decision process on an Android smartphone (Samsung Galaxy SIII). For this validation, we assume that we have received data from the patient diary application, the SH services or local smartphone monitoring services. We generated datasets containing clinical data (measurements), whereby fact values were created based on ranges encompassing both clinically normal situations as well as abnormal situations. We have 7 dataset configurations (1 to 7 days of data), with 10 generated datasets by configuration (70 datasets). Results: The validation shows promising results for using CDSS on mobile phone: loading data and rules (140-425 ms), executing rules (45-82 ms) and memory usage for the reasoning process (174-350 KB). Conclusions: The results show that a guideline compliant CDSS system can be implemented on currently available mobile devices. While this validation was limited in scope, as we did not tackle the precise derivation of sensor data into the used clinical facts, it shows nonetheless that with such set of inferred clinical facts, interesting and clinically relevant problems can be tackled.

Back ground Antibody Directed Enzyme Prodrug Therapy (ADEPT) is a technique which has been used in cancer treatment. This therapy consists of two steps the aim of which is to convert a prodrug to a powerful cytotoxic drug only in the vicinity of the tumor. The technique requires a bacterial enzyme, glucarpidase (former name: carboxypeptidase G2, CPG2). The use of glucarpidase in ADEPT and in detoxification of the cytotoxic methotrexate, MTX is needed to be done in several cycles. This however, hampered by the induced human antibody response to the glucarpidase. The technique therefore will benefit from novel glucarpidase which might avoid the human immune system. Objectives 1.Collections and screening soil samples for novel glucarpidase producing bacteria 2.Characterisation of the isolated bacteria 3.Cloning, overexpression, purification and the molecular characterisation of the novel glucarpidase Methods Isolation of novel glucarpidase producer(s) Over one hundred soil samples were collected from different fields in Qatar where folate rich fruit and vegetables are routinely cultivated. Screening of these samples was performed using folate as the sole carbon and/or nitrogen source. Several molecular techniques were used to characterize the isolated strains. Cloning of the novel glucarpidase gene The gene was cloned by production of genomic library of the novel strain chromosomal DNA and PCR techniques. The gene was subcloned in the overexpression vector pET28a and transformed into E. coli (DE3). The novel gene was expressed by induction using IPTG. The recombinant protein was purified using Ni2+ column. The activity assay was carried out using methotrexate as substrate. Results We successfully isolated two new glucarpidase producing strains, Stenotrophomonas Sp. AA1, and Pseudomonas Sp. AA2. The CPG2 gene from Stenotrophomonas Sp. was cloned and expressed in E. coli using pET28a vector. The SDS analysis showed that the gene was expressed in the soluble form to about 30% of the total protein. The glucarpidase gene was cloned so as to place a polyhistidine tag on the N-terminus of the enzyme. A one-step purification protocol was usually sufficient to obtain essentially pure protein for detailed enzyme assay and characterization. Previous studies indicate that the native form of glucarpidase is Zn2+-dependent. Our data indicate that the recombinant enzyme showed no activity towards the substrate methotraxate in absence of Zn2+ ions. Full activity was restored to the zinc-free enzyme by the addition of Zn2+ to the assay mixture. Therefore, the formation of active enzyme was shown to be dependent on Zn2+. Conclusion We successfully isolated two new glucarpidase producers. We managed to clone express and purify the new glucarpidase gene from one strain. Molecular studies on the novel glucarpidase have been carried out. The novel glucarpidase will pave the way for further application in the cancer drug detoxification and Antibody Directed Enzyme Prodrug Therapy techniques.

Background hypertrophic Cardiomayopathy is an inherited heart muscle disease with considerable heterogeneity at genetic and phenotypic levels and poor correlation between genotype and phenotype. Next generation sequencing could help in addressing this problem. Subjects and Methods The present study involved 144 unrelated consecutive index HCM patients enrolled in the BA HCM National Program in Egypt subjected to large scale high throughput targeted next generation of coding and exon flanking regions of over 100 genes involved in inherited cardiac conditions (ICC) including genes with a known role in HCM (Illumina HiSeq) Results Putative pathogenic variants were detected in 67 samples (67/144, 46%), mostly found in sarcomeric genes in the order of: MYBPC3 (38.8%), followed by MYH7 (33%), MYL3 (9%), TNNT2 (4.5%), TNNI3 (in 2 samples). TNNC1, MYLK2 and TPM1, ACTN2, TCAP, PLN, ACTN2, MYH6 showed a potentially pathogenic in 1 patient, each. Validation was performed by Sanger sequencing. Twenty six variants were novel (26/144, 18%) and were not found in any of the control series and by in silico analysis were scored as potentially pathogenic, hence were considered as variants of unknown significance and should be tested for coseggregation. An approximate of 10% (14/144) of the samples revealed likely pathogenic variants in genes not commonly known to be linked to HCM. Complex heterozygosity involving sarcomeric and non sarcomeric genes particularly ion channel genes was observed in almost a quarter of the samples in the present cohort (35/144, 24%), and homozygousity was found in nine patients(9/67, 9%). This could provide an explanation to the heterogeneity of the phenotype observed amongst different members within the same family and among unrelated patients having the same sarcomeric mutation. The studied cohort involved six HCM phenocopies 3 had mutations in PTPN11 and two cases had novel possibly pathogenic variants in RAF1. One Fabry case with a reported pathogenic mutation in GLA gene. Conclusion High throughput NGS has enabled detection of possibly/likely pathogenic variants in over 60% of samples studied. The detection of homozygous patients and complex heterozygosity in almost a quarter of the HCM patients provides a possible explanation to the phenotype heterogeneity observed in HCM.