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Qatar Foundation Annual Research Conference Proceedings Volume 2018 Issue 2
- Conference date: 19-20 Mar 2018
- Location: Qatar National Convention Center (QNCC), Doha, Qatar
- Volume number: 2018
- Published: 15 March 2018
1 - 50 of 82 results
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Automated Classification of Diabetic Retinopathy Severity: A Deep Learning Approach
Authors: Rawan AlSaad, Sabri Boughorbel and Somaya Al-MaadeedBackground: Diabetic retinopathy (DR) is a damage to the retina caused by complications of diabetes and is the fastest growing cause of blindness. It is a major concern for the Qatari population, affecting about 40% of diabetic patients in Qatar. Automated DR classification techniques with high accuracy have a strong potential to help doctors in early diagnosis of DR and quickly routing patients who need medical interventions to a specialist. Most of the previous work utilized traditional machine learning techniques for the task of DR severity classification. Such techniques are based on “feature-engineering”, which involves computing explicit features designed by domain experts, resulting in models capable of detecting specific regions of DR damage or predicting the classification of DR severity. Recently, deep learning has emerged as an efficient technique that avoids such engineering. It shifts the burden of feature engineering to the design of general-purpose learning system which allows an algorithm to learn by itself the most important predictive features from the raw images, given a large dataset of labeled examples. Objectives: In this work, we studied deep learning techniques described in recent literature and combined it with our own ideas to develop a deep convolutional neural networks (ConvNets) architecture for the task of diagnosing diabetic retinopathy and classifying its severity from retina images. In addition, we explored the impact of the following parameters on the performance of the model: 1) number of fully connected layers, 2) number of units within each fully connected layer, and 3) batch size (number of training examples which will be forward/backward propagated through the network in one pass). Methodology: We trained the ConvNets model on the publicly available retina images dataset from the Kaggle competition for diabetic retinopathy detection. The dataset included labels with information about the presence of DR in each of the images, rated by a clinician on a scale from 0 to 4 (0: No DR, 1: Mild, 2: Moderate, 3: Severe, 4: Proliferative DR). The model was implemented using Theano, Lasagne, and cuDNN libraries and trained on two Amazon EC2 p2.xlarge instances (NVIDIA GPU K40). We used the same evaluation metric of the Kaggle competition, which is the Cohen's quadratic weighted Kappa function. In our case, Kappa is described as being an agreement between two raters: the agreement between the scores assigned by human rater (labels) and the predicted scores. Results: On the dataset of 30,262 training images and 4864 testing images, the model achieved a Kappa of 0.72. Our experimental results demonstrated that the number as well as the size of the fully connected layers does not have a significant impact on the model's performance. Moreover, it indicated that increasing the batch size does not necessarily speed up the convergence of the gradient computations. Conclusion: We have shown that convolutional neural networks have the potential to be trained to identify the features of Diabetic Retinopathy in retina images. Given the many recent advances in deep learning, we hope our work will open the door for many new examples demonstrating the power of deep learning to help solving important problems in medical imaging and healthcare. Keywords: deep learning, machine learning, diabetes, diabetic retinopathy, medical imaging.
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Hyperinsulinemia is associated with adipocyte hypertrophy and mitochondrial dysfunction
Insulin resistance is often associated with hypertrophy of adipocytes. Furthermore, hyperinsulinaemia may mediatechanges in cellular mitochondrial biogenesis and function. Therefore, the current study investigated the hypothesis that lower mitochondrial mass and mitochondrial uncoupling (whereby the electron transport is not used to drive ATP synthesis) is a feature of hypertrophied adipocytes.Methods. Adipose tissue (sub-cutaneous and omental) and blood was obtained from morbidly obese patients (BMI ≥ 40 kg/m2, age 29 ± 2.8 years), undergoing bariatric weight reduction surgery. Anthropometric data was recorded. The blood was used for systemic determination of glucose and insulin by commercial methods. The adipose tissue was separated into the adipocyte and stromal vascular fractions by collagenase digestion. Paraffinembedded adipose tissue was stained with Hematoxylin and Eosin and Image J software was used to determine cell size.Total RNA was isolated using Tri-reagent and the transcriptomeanalyzed using Affymetrix whole transcriptome arrays. The ArrayAnalysis.org statistics module using the Limma package of R/Bioconductor was used for the statistical comparison between High insulin subjects vs. Low insulin group. Genes were considered to be differentially expressed when their absolute log2 fold change (FC) > = 1, and p-value < = 0.05.Indices of mitochondrial function was examined in an in vitro model using 3T3-F442A murine differentiated adipocytes.Cellular lipid content and mitochondrial membrane potential(DΨm) were determined by confocal microscopy with 40 μMBodipy 493/503, a neutral lipid dye, and TMRE (30 nM).NADH/flavoprotein autofluorescence was measured in ‘multitracking’ mode, in which fluorescence was excitedalternately at 351 nm (signal measured at 435-475 nm; NADH)and at 458 (emission measured at >505 nm; flavoprotein). Theresting level of each coenzyme was expressed as a function ofthe maximally oxidized (with the uncoupler FCCP, 1 μM) andmaximally reduced signal (with cyanide, 1 mM). Oxygenconsumption was measured using a Clarke electrode and ATP generation using a luciferase assay.Results.The patient population was dichotomized into hyperinsulinaemic(>7.0 μU/ml) and normoinsulinaemic ( < 6.5 μU/ml) groups. The groups were matched for age and BMI. The hyperinsulianemicgroup, compared to those with normoinsulinaemia, had significantly greater numbers of hypertrophied adipocytes (p = 0.04). Pathway analysis showed that genes involved in the mitochondrial biogenesis (e.g: NRF1, GABPA, TFAM, POLRMT, MTERF and SP1) were comparable between the two groups. However several genes related the electron transport chain and ATP synthesis were significantly different between the hyperinsulinaemic and normoinsulinaemic groups (See table below). Expression of genes related to the electron transport and ATP synthesisComplex ? NDUFA4, ND3,ND6Complex IISDHA, SDHB, SDHC. Complex IIIUQCRH, UQCRFS1.Complex IVCOX5A, COX6A, COX7A. Complex VATP5A, ATP5E, ATP5HAdenine Nucleotide TranslocaterSLC25A4 In adipocytes characterized as having >40% intracellular lipid (hypertrophied), compared to those with < 20% (normal adipocyte), lipid, the rate of oxygen consumption increased(380%). DΨm was significantly reduced in cells with high,compared to those with low, intracellular lipid. Assessment ofredox state in lipid-engorged cells showed that flavoproteinswere more oxidized than NADH, consistent with a shift to b-oxidation as a dominant metabolic pathway. Resting levels ofboth coenzymes were significantly oxidized in lipid-engorgedcells compared to those with < span class = »bumpedFont15
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Association of levels of polybrominated diphenyl ethers in two fat compartments with increased risk of insulin resistance in obese individuals
Authors: Mohamed Elrayess, Murad Helaleh, Ilhame Diboun, Nada Altamimi and Aishah LatiffMurad Helaleh1, Ilhame Diboun2, Nada Altamimi1, Aishah Latiff1, Mohamed Elrayess1* 1Toxicology and Multipurpose Lab, Anti Doping Laboratory Qatar, Sports City, Doha, Qatar. 2 Department of Economics, Mathematics and Statistics, Birkbeck, University of London, London WC1E 7HX, UK. Corresponding author: [email protected] diphenyl ethers (PBDEs) represent a class of widely utilized flame retardants [1]. With over 200 congeners that vary by the extent of halogenations, various PBDEs can leak freely into the environment [2, 3]. Despite cessation of their manufacturing, concerns of their bioaccumulation remain [4-6] due to their stability in products manufactured before the ban and recycled materials [1]. With their high lipophilicity, PBDEs tend to accumulate in adipose tissue, potentially altering the function of this endocrine organ by increasing lipolysis and decreasing glucose oxidation, causing increased risk of metabolic disease including obesity, insulin resistance and type 2 diabetes [7]. Exposure to PBDE-47, for example, during the early post-natal period was shown to induce disturbance in glucose metabolism causing insulin resistance in susceptible mice [8]. In this study, levels of various PBDEs were assessed in subcutaneous and omental adipose tissues from 33 obese and morbidly obese patients (11 insulin sensitive and 22 insulin resistant) and their correlation with mediators of metabolic disease were established. Our results suggested that out of 22 detectable PBDEs in subcutaneous and omental adipose tissues, PBDE99, 28, 47 and 126 were significantly higher in insulin resistant individuals compared to their insulin sensitive counterparts. When considering PBDEs congeners, penta congeners as a group were also higher in insulin resistant individuals compared to insulin sensitive counterparts, while no significant differences were detected in mono, tri, tertra, hexa, hepta and octa congeners between the two studied groups. This data suggest that accumulation of various PBDEs in human adipose tissues obtained from obese individuals is associated with increased risk of insulin resistance. Further investigation of the functional relevance of these associations is currently underway. This study is partly funded by QNRF's grant number NPRP6-235-1-048. References 1. Birnbaum, L.S. and D.F. Staskal, Brominated flame retardants: cause for concern? Environ Health Perspect, 2004. 112(1): p. 9-17. 2. Chen, D. and R.C. Hale, A global review of polybrominated diphenyl ether flame retardant contamination in birds. Environ Int, 2010. 36(7): p. 800-11. 3. Ernest, S.R., et al., Effects of chronic exposure to an environmentally relevant mixture of brominated flame retardants on the reproductive and thyroid system in adult male rats. Toxicol Sci, 2012. 127(2): p. 496-507. 4. Kelly, B.C., et al., Bioaccumulation behaviour of polybrominated diphenyl ethers (PBDEs) in a Canadian Arctic marine food web. Sci Total Environ, 2008. 401(1-3): p. 60-72. 5. Mercado-Feliciano, M. and R.M. Bigsby, Hydroxylated metabolites of the polybrominated diphenyl ether mixture DE-71 are weak estrogen receptor-alpha ligands. Environ Health Perspect, 2008. 116(10): p. 1315-21. 6. Sjodin, A., D.G. Patterson, Jr., and A. Bergman, A review on human exposure to brominated flame retardants–particularly polybrominated diphenyl ethers. Environ Int, 2003. 29(6): p. 829-39. 7. Hoppe, A.A. and G.B. Carey, Polybrominated diphenyl ethers as endocrine disruptors of adipocyte metabolism. Obesity (Silver Spring), 2007. 15(12): p. 2942-50. 8. McIntyre, R.L., et al., Polybrominated diphenyl ether congener, BDE-47, impairs insulin sensitivity in mice with liver-specific Pten deficiency. BMC Obes, 2015. 2: p. 3.
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Effect of C1q Tumor Necrosis FactorRelated Protein 11 Ctrp11 On Macrophages
More LessBettahi I1-2, Ramanjaneya M1-2, Jerobin J1-2, Sivaraman SK1, Rasha Alsiddig3, Mohammad RM4, Skarulis MC1-2 and Abou-Samra AB2-3. 1Translational Research Institute, Academic Health System, 2Department of Medicine, 3Qatar Metabolic Institute, Hamad Medical Corporation, Doha, Qatar, 4Karmanos Cancer Center, Wayne State University. Background: Obesity is a major risk factor for cardiovascular disease, increasing the risk of hypertension, hyperglycemia, and dyslipidemia, recognized as the metabolic syndrome. Adiponectin paralogs designate as C1q/TNF-related protein (CTRPs) have recently emerged as key regulators of metabolism and is a core component in the interrelationship between insulin resistance, adiposity, and inflammation. CTRP-11 functions as an adipose stroma-derived regulator of adipogenesis. Aim and objectives: The aim of this study is to characterize the anti-inflammatory potential of the CTRP-11. Methods: Cell lines differentiation: Human THP1 cells were maintained in RPMI-1640 supplement with 10%FBS. Cells were seeded in 24 well and stimulated with PMA for 48 hours to differentiate them to macrophages and rested for 24 hrs. For all stimulation experiments, cells were grown for overnight in serum-free media. Cells were then treated with CTRP11 for 24 hours and then re-stimulated with LPS for 6 hrs. Cells were collected for RNA extraction and genes expression using Real-Time PCR. FACS analysis: After differentiation and incubation with LPS and LPS/CTRP11, Cells were stained with human anti-CD206 and anti-CD163. Following incubation, cells were washed, and fluorescence compared to unstained. All data were analyzed using FlowJo software. Results: Our preliminary data provide that CTRP11 decreased both IL-6, TNF-α and NF-kb expression of mRNA level in THP-1 macrophages activated with lipopolysaccharide (LPS) (p < 0.005), compared to (LPS) alone. THP-1 macrophages showed and increased in M2 polarization marker CD206 and CD163 after incubation with LPS/CTRP compared to LPS alone. Conclusion: Future studies are in progress to further assess the effects of adiponectin paralogs (CTRP11) on cytokine excretion by ELISA on Human macrophages M1 and M2. Abbreviation: LPS: lipopolysaccharide; PMA: phorbol 12-myristate 13-acetate.
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Viscoelastic behaviour of Bovine Myocardiam and valvular tissue: Applications in Bio prosthetic heart valve with enhanced properties
Cardiovascular diseases are among the major causes of morbity and mortality. Particularly, the prevalence of heart valve disease(damaged heart valve leaflet) is one of the most common ailment. In elderly people, every year 30,000 patients are treated with heart valve replacement surgeries in the developed countries. Mechanical and bioprosthetic heart valves are commonly used in heart valve surgeries. While mechanical valves require the patient to be on blood thinning agent, rest of the life, bioprosthetic valves have only limited life span. Adequate knowledge of the biomechanics of nano-microscale structure and the viscoelastic properties of the native (bovine) heart valves can pave the ways for enhancing the strength of bio-prosthetic heart valves. In present work, we measured the viscoelastic and structural properties of the native bovine myocardiam and valvular tissue of heart valve. These heart valves were taken from bovine hearts and cryopreserved as necessary. The rheological and viscoelastic properties of heart valves were investigated by fixing in formaldehyde and phosphate buffer solutions (PBS). The samples were then tested in SEM (Scanning Electron Microscope) to investigate the microstructure of valve leaflets. Using Dynamic Shear Rheometer, the critical parameters such as modulus of elasticity, storage modulus, loss modulus, complex modulus, complex viscosity and the oscillatory shear properties were thoroughly investigated. Results show that the rheological properties vary with different chemical fixation effects. Chemical fixations like formaldehyde fixation were improving the rheological properties of heart valves. However, there is no significant influence of different time periods of fixations on mechanical properties. The complex modulus as well as the compression and storage moduli of the sample fixed with formaldehyde showed the satisfactory values after the fixation. It was indicating the mechanical strength has improved in terms of its structure, as much as solid/rigid as before the fixation. Also, the viscosity of fresh valve was higher, showing that the aldehyde fixation alters the mechanical property of the heart valve. Through the creep tests, it was investigated that the fixation of the heart valves did affect the viscoelasticity and mechanical properties of the heart valves. The valves become stiffer when they were fixed with formaldehyde. The reactions were less by a whole order of magnitude. However, by fixing the sample in formaldehyde we observed that the aortic valve had much more strength than the fresh sample. Another proof of how the aldehyde fixation drastically affected the stiffness of the valve was in the fact that the extension of the aldehyde-fixed valve under a stress of 5 kPa was less than that of the fresh valve at 0.5 kPa. These outcomes provide significant insights into the correlations between the microstructure and mechanics of the heart valves and their macro scale behaviors under various conditions. These results were modeled using Computer Aided Engineered software. The software can help scientists in evaluating the performance of tissue engineered heart valves with natural heart valves.
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Overexpression of Calreticulin in endothelial cells changes insulin trascytosis
Authors: Tatiana Carneiro Lobo, Rajja Dalloul and Nasrin MesaeliCarneiro-Lobo T.C., Dalloul R.S.D., Mesaeli N. Department of Biochemistry, Weill Cornell Medical College in Qatar, Doha, Qatar. Introduction: The incidence of diabetes and obesity has been rising world wide in the past few decades. In the Gulf Cooperation Council (GCC) a rapid increase in the prevalence of diabetes has been reposted in the past few years. In Qatar the incidence of diabetes is 16.7% in the Qatari population that is almost 3 times higher than the incidence of diabetes in UK. Type 2 diabetes mellitus is a growing public health problem and a major cause of cardiovascular disease in the United State. Type 2 diabetes is associated with systemic insulin resistance, which promotes hyperglycemia, and it has been proposed that these metabolic abnormalities account for increased cardiovascular risk. Endothelial dysfunction contributes to the pathogenesis and clinical expression of atherosclerosis and has been linked to type 2 diabetes mellitus and insulin resistance. Transport of insulin across the microvasculature is necessary to reach its target organs and is rate limiting in insulin action. The two possible mechanism of this movement is either through leaky tight junction in vascular endothelial cell layer or via transcytosis through these endothelial cells. Therefore, we hypothesized that overexpression of calreticulin would reduce insulin transport to the target tissue due to onset of endothelial dysfunction. Methods and Results: To test our hypothesis, we used genetic approaches to overexpress calreticulin (CRT). For in vitro experiments CRT was overexpressed using a Lentiviral-CRT-RFP to infect Human Umbilical Vein Endothelial Cells (HUVEC). For ex vivo studies we used endothelial cels isolated from our endothelial specific CRT overexpressing transgenic mice (ECCRT+). We examined the uptake of fluorescently labeled insulin in the endothelial cells. CRT-HUVEC and WT-HUVEC were incubated with 25 μg of FITC-insulin for 30 mins and 60 mins. We found that CRT-overexpressing cells reduce the transcytosis of insulin through the endothelial cells compared to control. The primary endothelial cells were isolated using columns containing PECAM (CD31) coated beads. Cells were then characterized by detection of CD31 (PECAM) and Von Willebrand factor (vWF) expression by immunofluorescence and western blot. After confirming the endothelial identity of our primary cells (ECCRT+) they were incubated with Alexa-647 insulin for 30 min or 60 min. Confocal microscopy was carried out to examine insulin uptake and transcytosis. Our data illustrate a reduction in insulin trancytosis in ECCRT+ endothelial cells as compared with WT cells. To evaluate how Calreticulin affects insulin trancytosis in vivo in whole animal, ECCRT+ mice and WT-mice were used. Alexaflour-647 insulin (1.2 μg/L) was injected via tail vein of ECCRT+ mouse and WT-mouse. To label the surface of endothelial cells, 20 μg/L FITC-isolectin was injected via tail vein following insulin injection. 30 mins after the injection mice were euthanized and tissue (lung, liver, heart and retina) were isolated, fixed and embedded for cryosections. Tissue sections were then mounted on slides and examined by confocal microscopy. We observed a lower concentration of insulin in cell membrane of endothelial cells of vascular wall in lung, liver, heart and retina of ECCRT+ mouse than WT-mice. Finally, we examined the leakiness of the vascular wall in our ECCRT+ mouse model. In these experiemnts we injected 2.5 mg/ml FITC-Dextran in the tail vein for 30 min. After euthaniasia, mice tissue was imaged under a Ziess flourescnet microscope to evaluate leakage of the flourescet signal in the tissue. Fluorescent images from the lung, liver and heart were collected and illustrated an increase in the leakiness of vascular wall of the small vessels in the ECCRT+ mice as compared to the WT mice. Conclusion: Taken together, our results illustrate that increased calreticulin expression in endothelial cells affect endothelial cell function, increase the vascular permeability and reduce the transcytosis of insulin through the endothelial cell of vasculature. The impaired insulin export from the vascular wall to the target tissue could contribute to the onset of insulin resistance and diabetes in the ECCRT+ mice which we have observed in our other studies on these mice. This is the first report to link calreticulin expression level and insulin transport in intact animal. Acknowledgment: This research was made possible by a grant by QNRF, NPRP07-208-3-046.
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Electrospun polymer nanocomposite scaffolds containing metal oxide nanoparticles for diabetic wound healing
More LessRobin Augustinea, Noorunnisa Khanama, Hany Elsayed Mareib, Sabu Thomasc, Ala-Eddin Al Moustafad, Anwarul Hasana a College of Engineering, Qatar university Doha, Qatar bBiomedical Research Centre, Qatar university Doha, Qatar cInternational and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam – 686 560, Kerala, India. dCollege of Medicine, Qatar university Doha, Qatar Abstract It is very important to treat diabetic foot injuries at early stage since even minor wounds can turn into serious foot ulcers which can lead to the amputation of the entire foot if not treated early. The management of diabetic foot ulcers requires the use of appropriate wound dressings to provide a moist wound environment and protect from infection. Large number of polymeric materials has been tried for wound coverage applications with many successful outcomes, but the search for an ideal wound dressing material which can enhance diabetic wound healing is still continuing. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), commonly known as PHBV has got a lot of attention in biomedical applications due to its biocompatibility and biodegradability Electrospinning is a robust technique that can produce highly porous membranes composed of nano or submicron fibers from polymer solutions. Formation of active blood vessel network through an implanted wound dressing is one of the most important issues in the treatment of diabetic wounds. Ability of metal oxide nanostructures to promote angiogenesis and wound healing is already established. Thus, in the present work, electrospun PHBV wound dressings containing metal oxide nanoparticles were fabricated and characterized. Our results demonstrated that metal oxide nanoparticles in the wound dressings enhanced human cell adhesion and their migration. Further, angiogenic property of the membranes was enhanced as evident from chicken chorioallantoic membrane assay and leads to the enhancement of diabetic wound healing. Present study strongly suggest the potential application of metal oxide nanoparticles incorporated PHBV scaffolds in promoting angiogenesis and their effective use in diabetic wound healing.
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Electrospun polymer nanocomposite scaffolds containing metal oxide nanoparticles for diabetic wound healing
Background: Most of the evidence -so far- in support of the link between Transient Ischemic Attack (TIA) and the development of future stroke are -in fact- based on epidemiological rather than scientific evidence. Meanwhile, Current prediction models for long-term prognosis in TIA and stroke patients have limited validity as discrimination between patients at high risk and patients at low risk is relatively poor. Although the ABCD2 prediction model seems to be most adequate because of its applicability in clinical practice, the American Heart Association pointed out that validated prediction models for long term cardiovascular risk in patients with ischemic stroke or TIA were not available. Furthermore, it has been suggested that all these stroke-based prediction models were derived from trial populations with certain inclusion and exclusion criteria. It could therefore be argued that the best fitted model for everyday practice will probably need to be derived from a consecutive stroke/TIA population without exclusion criteria. Improvements (such as stronger predictors of outcome) are therefore needed. Aims: This study investigates the prevalence of microalbuminuria in patients who have suffered a TIA for the first time. The aim is to identify a subgroup of TIA patients who are at higher risk of developing future cerebrovascular events. Methods: This is an observational cohort study with two arms (in Qatar and UK). The Qatari arm involved 56 patients with acute TIAs or small stroke (TIAs with tissue evidence of infarction), attending a local hospital in Qatar; whereas, the UK arm involved 74 first time TIA patients, attending a TIA Clinic at a local hospital in the UK. Microalbuminuria was first measured by calculating the Albumin/Creatinine Ratio (ACR). Urine samples (with and without microalbuminuria) were then examined using SDS-PAGE electrophoresis. Protein bands were identified by their rate of migration in comparison with standard molecular weight (MW) markers. Results: The Qatari arm of the study recruited 56 patients (mean age 55.50 years); 35 Males: 21 Females), attending a Stroke/TIA Clinic in Qatar. 19 (33.92%) TIA patients showed evidence of microalbuminuria (30-300 mg/24 hr) and 37 (66.08%) showed normal albuminuria ( < 30 mg/24 hr). The UK arm of the study recruited 74 first TIA patients (mean age 74.75 years; age range 52-91); 38 Males: 36 Females). 25 (33.78%) TIA patients showed evidence of microalbuminuria and 49 (66.22%) showed normal albuminuria. Out of the 25 TIA patients with microalbuminuria, 92% of them have a blood pressure of more than 140 mmHg. SDS-PAGE electrophoresis of urine samples obtained from patients recruited (for both arms of the study) revealed a similar pattern of 3 major protein bands. Band (A) is heavily stained, representing albumin (MW = 70 kDa) in patients with micro-albuminuria, and less intensity in patients with normal-albuminuria. Band (B) was shown in all urine samples of patients with microalbuminuria, but not in patients with normal-albuminuria. Its position on the gel is roughly equidistant between bands A and C. Band (C), denoting a slower migration band, representing Tamm-Horsfall protein. Tamm-Horsfall protein is suspected since not only is it the most abundant protein in urine after albumin, but also its position is in concordance with the relative molecular weight of Tamm-Horsfall protein (between 85 and 110 kDa). The intensity of this band is generally not as great as band A. Other bands (MW < 50 kDa and < 40 kDa) were also seen; they may represent degradation products of albumin due to storage. Discussion: Microalbuminuria has been proven to be a predictor of cardiovascular disease and mortality in both diabetic and non-diabetic patients, and it is a reflection of an overall vascular damage. This study aims to identify a subgroup of TIA patients who are at higher risk of developing future cerebrovascular events. Our data confirmed that microalbuminuria is very common in TIA patients as one third of participants (in both arms of the study) presented with microalbuminuria, most of whom have a history of high blood pressure. However, it is important to highlight here that participants in the Qatari cohort are much younger and their microalbuminuria is more severe than the UK cohort. This might be due to the severity of their condition, the presence of co-morbidity, ethnicity and genetic susceptibility. With such a high prevalence of microalbuminuria (in first time TIA patients), such a prognostic marker cannot be ignored in TIA Clinics. There is now a case for microalbuminuria to be considered for possible adding it to the current ABCD2 predictive model, taking into account the fact that microalbuminuria can now be easily measured using a urine dip stick testing. The clinical applications of our findings may include an aggressive approach to antihypertensive management. Conclusion: Microalbuminuria following TIA is likely to be an added marker of risk and could indicate a more aggressive approach to hypertension management or the choice of ACE inhibitors angiotensin 2 receptor blockers over other antihypertensive drugs. The novelty of this project stems from the fact that it investigates two predictive makers; one biochemical (microalbuminuria, presented in this study) and one physiological (auto-regulation of cerebral blood flow, still being analyzed). No previous research has combined the two predictive markers together in a cohort group of first time TIA patients. These two predictive parameters will provide us with more evidence to predict further stroke and whether current intervention is sufficient enough to prevent future stroke. If this is proven to be correct, both predictors will add more validity to the current conventional risk models predictors. If there is such a link between these two measures then changes to the regulation of blood flow in the brain can be predicted by a simple urine test. This will help predict how well patients will recover after TIA. Furthermore, if microalbuminuria is associated with impaired cerebral auto-regulation (following TIA) then one might take more caution with anti-hypertensive treatment early after TIA. By identifying impairment in auto-regulation this study may provide an important reason for caution which may otherwise be overlooked. «This study was made possible by grant NPRP 6-565-3-141 from Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the author[s].»
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Effects Of Gestational Diabetes Mellitus On Hematopoietic Stem cell quality and IL6 Amount
More LessGestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy (1), Maternal glucose and lipid metabolism changes substantially during normal pregnancy, In women with GDM, fasting insulin and fasting glucose concentrations are elevated. Also, women with GDM have a lower insulin sensitivity compared to controls and the difference is most evident before and during early pregnancy (2). Gestational diabetes mellitus has proven it»s effects on many variables of pregnancy and so it»s effect on umbilical cord blood (UCB), which is increasingly used as an alternative source of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) for bone marrow transplantation and other therapeutic uses, And for successful outcomes of cord blood transplantation certain criteria are used in UCB banks include the TNC count, percentage of CD34, CFU and the volume of blood (3) As many variables that may affect the quality of hematopoietic stem cells are still under study and research, And gestational diabetes mellitus is a disease expected to increase by 20 fold, AS regard it»s effect on hematopoietic stem cell and IL6 it»s found that «The proportion of CD34+ CD45 HSPCs among the nucleated cells was significantly higher in the cord blood samples of neonates born to mothers with GDM compared to neonates born to non-diabetic mothers.»(4) Also, it»s found that IL-6 concentrations are increased in women with current GDM (2). Another study reported that: the CD16+CD56 − NK cells was increased, while IL-2 was lower in maternal blood with GDM. The placental extravillous layer of the gestational diabetic mothers showed high levels of IL-4, IL-6, IL-10, IL-17, and IFN- and low levels of IL-1 and IL8, whereas the placental villous layer contained high levels of IL-17 and IFN(5). In this study we provide atrial for knowing whether the cord blood unit of gestational diabetic mother fulfilling the appropiate criteria for banking, As in previous studies all what had discovered is minimal non concolusive data about whether GDM unit fulfilling the normal criteria for banking or not.
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Generation of induced pluripotent stem cells from insulin resistant Qatari patients
Insulin resistance (IR) is a precursor and accelerating factor for Type 2 diabetes (T2D), the greatest health challenge facing Qatar and the world today. Psoriasis, is an immune-mediated, chronic skin disorder that can aggregate in families, because of its strong genetic predisposition. It has been found that patients with psoriasis for more than two years, regardless the severity of the disease, are at a very high risk of developing insulin resistance and diabetes. Although several studies highlighted the link between IR and skin disorders, no reports studied the relationship between IR, T2D, and epidermal dysfunction using induced pluripotent stem cells (iPSCs). Therefore, our aim in this study was to generate patient-specific hiPSCs from IR Qatari patients (associated with psoriasis or T2D) and differentiate them into insulin target cells. Blood samples were collected from Qatari individuals with a family history of IR associated with T2D or psoriasis as well as from healthy individuals. The Ficoll-Paque density gradient method was used to separate the peripheral blood mononuclear cells (PBMCs). The isolated PBMCs were cultured in vitro for 5 days in StemPro-34 culture medium before transduction. PBMCs were reporgammed using Sendai viral vectors encoding four pluripotency factors including OCT4, SOX2, C-MYC, and KLF4. After 20-30 days of the transduction, several undifferentiated colonies of high morphological quality (defined border and high nuclear to cytoplasmic ratio) were manually picked up and transferred to new matrigel-coated plates to establish different clones. Several hiPSC clones were established from each individual (sample) and only three clones were maintained after extensive characterization of all clones. The generated hiPSC clones were characterized using different techniques including immunostaining, RT-PCR, Western blotting, alkaline phosphatase assay, embroid body (EB) formation, karyotyping, and hPSC ScoreCard assay. H1-human embryonic stem cell (H1-hiESC) line was used as a positive control in all experiments. Similar to hESCs, hiPSC clones expressed pluripotency markers, such as OCT4, SOX2, NANOG, KLF4, C-MYC, SSEA4, TRA-60, TRA-81, REX-1, DPPA4, and TERT at mRNA and protein levels. All hiPSC lines showed standard hESC morphology, normal karyotype and stained positive for alkalaine phosphatase. Only the hiPSC lines that showed similar characteristics as those of hESCs were maintained and expanded in culture. To confirm the pluripotent ability of the generated hiPSCs, we used EB technique to differentiate patient-specific hiPSCs into three germ layers in vitro. Immunostaining and RT-PCR analyses showed that these hiPSCs can differentiate into endodermal (SOX17+/FOXA2+), mesodermal (BRACHYURY+) and ecodermal (NESTIN+) lineages. To further validate the multilineage differentiation potential of the generated hiPSCs, we used the hPSC ScoreCard assay in vitro. These findings indicate that hiPSCs generated in this study are pluripotent, fully reprogrammed stem cells having the ability to differentiate into any cell type of the body. Thus, we will differentiate patient-specific hiPSCs into insulin target cells carrying the genetic background of the patients to identify signaling mechanism involved in the inherited form of IR and to understand the genetic link between IR, T2D, and psoriasis. To our knowledge, this is the first study to generate hiPSCs from diabetic and psoriatic patients in the MENA region.
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Obesity is associated with the upregulation of TLR4 protein but not associated with polymorphisms of TLR4D299G TLR4T399I
Authors: Mariam Alwakeel, Afnan O. Galash, Amina Saleh, FadelMooza Alkhanji and Nasser M. RizkTitle: Obesity is associated with the upregulation of TLR4 protein but not associated with polymorphisms of TLR4D299G TLR4T399I.Background: Toll-like-receptor 4 (TLR4) is a pathogen-specific receptor, expressed in white blood cells, adipocytes, and other metabolic cells. Lipopolysaccharides (LPS) and free fatty acids (FFA) can act upon TLR4 and activate systematic inflammation that may lead to obesity and metabolic syndrome (MS).Aim: The aim of this research was to investigate the association of TLR4 polymorphisms of TLR4D299G TLR4T399I, with obesity and metabolic syndrome components in young adult female Arab subjects.Methodology: A prospective cross-sectional study was performed on female students from Qatar-University. The subjects were classified according to BMI classifications by WHO category into two groups: “obese; n = 69” and “non-obese; n = 136”. Anthropometric measurements included weight (kg), height(m), waist circumference (WC) were assessed, and the body mass index (BMI) was calculated. Several biochemical assays were done to determine glucose and lipid profile. Plasma concentration of Interlukin-10 (IL-10) was measured using ELISA assay. Plasma concentration of IL-6, MCP-1, leptin, and insulin was measured using the multiplex Luminex assay. Also, fresh blood samples were used to measure TLR4 protein expression using flow cytometry assay.Results: TLR4D299G/T399I carriers had no significant association with obesity indicators; body mass index (BMI), body fat percentage (%BF), and waist circumference (WC). Haplotype analysisof TLR4 D299G/T399I showed that GT carriers of TLR4 D299G/T399I had a significant association with increased risk of insulin resistance with (odds ratio = 4.73), 95% CI (1.19- 18.90), (P-value = 0.016). Increased level of leptin was associated with obesity phenotype by BMI, WC, and %BF. In addition, up-regulation of TLR4 protein increased significantly in obese subjects by 1.2 fold over the non-obese, with (P-value = 0.006). Conclusions: TLR4 D299G/T399I polymorphism is associated with increased insulin resistance, a core component of metabolic syndromes but not with obesity. In addition, the up-regulation of TLR4 in obese subjects may be related to insulin resistance with increased leptin suggesting that increased free fatty acids may be a possible link to act as a ligand for TLR4.
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The role of the autophagy pathway in phagosome formation in macrophages interacting with adipocytes
More LessIntroduction The interactions between macrophages and inflamed white adipose tissue (WAT) have been implicated to play a role in several diseases including diabetes mellitus type 2, obesity, and breast cancer. Macrophages degrade dead adipocytes through a process known as “exophagy”, which is extracellular lysosomal hydrolysis. They form crown-like structures (CLSs), encircling dead or dying adipocytes. The macrophages create a tight seal on the adipocytes and their lysosomal contents are secreted into these seals or compartments which are known as lysosomal synapses. This project was investigating the way that macrophages digest dead adipocytes, specifically if the autophagosome protein marker LC3 is recruited to the apoptotic cell phagosome in CLS macrophages. Autophagy and phagocytosis have traditionally been thought of as completely separate processes, but recent studies have shown that there are some autophagy proteins which are involved in the phagocytosis of apoptotic cells (Florey et al, 2012). These autophagy proteins can lipidate microtubule-associated protein light chain 3 (LC3) onto phagosomes in a way that is independent of the autophagosome. This recruitment of autophagy proteins LC3 and Beclin1 to phagosomes has been called LC3-associated phagocytosis, or LAP, and ends in the acidification and lysosome fusion with phagosomes. Even though LAP was originally only thought to be used for the phagocytosis of pathogens, autophagy is also being shown to play a role in apoptotic cell engulfment, with some research showing that autophagy proteins play a role within phagocytes (such as macrophages) for regulating the degradation of apoptotic cells. Methods The adipocytes used in this model system were differentiated 3T3 L1 cells. In order to induce apoptosis in the adipocytes, they were incubated with 25 nM TNF-a for 24 hours, or on one occasion exposed to 365 nm UV radiation for 1 hour. The macrophages that were used in this project were from the J774 cell line and also the stable cell line J774 GFP LC3. In order to replicate the in vivo setting of macrophages surrounding a dead adipocyte, the in vitro CLS cell culture model was carried out based on preliminary work that had been already established in the Maxfield lab. All fluorescence microscopy experiments were carried out using the confocal microscope Zeiss LSM 880 equipped with the high resolution detector, Airyscan. After the adipocytes were incubated with TNF-a for 24 hours to induce apoptosis, they were labeled with NHS-Alexa633, and the macrophages were trypsanized in order to add them to the adipocytes. Several different time points were used for this experiment, starting every 30 min from 30 min post addition to 210 minutes. Macrophages were added to each dish containing adipocytes and the dishes were incubated for the specified times. At the end of the incubation time, the macrophages and adipocytes were fixed by adding PFA for 10 min, and then the PFA was washed off and blocking IF solution was added for 1 hour. Primary antibody diluted in BSA was added after the blocking step. The dishes were then left in the cold room overnight. The following day the secondary antibody was added, and left for 60 min at room temperature. This was then washed off and the dishes were left in PBS until imaging with the confocal microscope Zeiss LSM 880 equipped with the high resolution detector, Airyscan. In order to have a control for the experiment, previous researchers had used zymosan, since it is known to induce LAP, and that LC3 is translocated to the zymosan containing phagosome. We therefore had dishes with macrophages and zymosan instead of the apoptotic adipocytes. There were also dishes solely with macrophages imaged however these were also simply a control and not leading to any results. Results The results obtained from the confocal imaging of the macrophages with apoptotic adipocytes were consistent in showing no recruitment of LC3 to the macrophage-adipocyte contact sites. The adipocytes were labeled in blue with the NHS-Alexa633, the macrophages and LC3 with the Alexa546, and the plasma membrane of the macrophages were labeled in green with the Alexa488. Discussion The results of the study showed that the hypothesis was rejected, in that LC3 is not recruited to the phagosome in the digestion of dead adipocytes by macrophages, and therefore the process is not a form of LC3- associated phagocytosis. Although past research has shown that the ideal time to image LC3 being recruited would be at 120-150 minutes, images taken both before and after those times yielded no results in this case. Because this experiment had never been done before by the lab or by any other researchers, there was no reason to either specifically refute or support the fact that LC3 would be involved and the process would be LAP for CLS macrophages with dead adipocytes. In the future, the search for regulatory molecules that act in this process may lead to improved strategies to prevent or treat type 2 diabetes, metabolic disorders and certain types of cancers.
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The Role of Soluble Adhesion Molecules in Type 2 Diabetes Mellitus
By Ola AljammalBackground: Diabetes mellitus (DM) is a chronic metabolic disorder that is steadily increasing worldwide and is a leading cause of morbidity and mortality. As reported by the World Health Organization (WHO), 346 million people are currently suffering from diabetes worldwide, and the number of deaths related to diabetes is expected to double by 2030. This highlights the importance of continued research and the need for novel methods to both prevent and treat this pandemic. Endothelial dysfunction plays a central role in the pathogenesis of diabetes mellitus and its vascular complications. The damage to the endothelium results in the release of soluble forms of adhesion molecules into the circulation and their concentrations are thought to correlate with endothelial cell activation or dysfunction. Aims: To determine the serum levels of soluble vascular cellular adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in type 2 diabetes mellitus (T2DM) and their association with macrovascular and microvascular complications. Methods: A total of 20 blood samples were collected form diabetic patients at the Hamad Medical Corporation (HMC) between 2010 and 2011. The control blood samples were obtained from non-diabetic female students at Qatar University in Fall 2017. Serum concentrations of sICAM-1 and sVCAM-1 level were determined using magnetic bead multiplex assay. Results: The levels of sVCAM-1 (P- value 0.000005) and sICAM-1 (P- value 0.04) were both significantly higher in T2DM patients compared to the control group. There was a further increase in the levels of sVCAM-1 and sICAM-1 in diabetic patients with complications. There was a positive but not significant correlation between hyperglycemia, sVCAM-1 and sICAM-1 levels. Conclusion: T2DM is associated with high levels of sVCAM-1 and sICAM-1. Hyperglycemia is a major factor causing endothelial dysfunction in T2DM and the release of soluble adhesion molecules.
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The Effect of Protein Supplementation on Body Muscle Mass and Fat Mass in Qataris PostBariatric Surgery: A Randomized Controlled Trial RCT
Authors: Fahad Hanna, Sahar Dahawi Al-Shamari, MOHAMED Aly ElSherif and Wahiba WahidBackground and objectives: Obesity is a chronic medical condition characterized by an accumulation of excess fat in the body that may lead to negative health consequences. Bariatric surgery has been shown to be the most effective type of interventions to achieve and sustain significant weight loss in morbidly obese people. The objective of this study was to examine the effectiveness of protein supplementation in reducing the risk of developing protein malnutrition and low muscle mass, in post-bariatric patients in Qatar. Methodology: This study is a double-blinded randomized control trial. Recruitment of participants began in early 2017 following the ethical approval of the trial (HMC IRB approval no. 16433/16). The intervention group received protein supplement that contain 20 g of protein while the placebo group received zero protein supplement. All participants were followed up for 1 month post-surgery. Randomization was done on a weekly basis within blocks of 8 or 10 patients. Independent Sample-T Test and Paired Sample-T Test were performed to assess the effect of the intervention. Results: The mean weight loss in the control group was 9.6 kg, while the intervention group mean weight loss was 10.7 kg (p = 0.03). Change in muscle mass percentage was +0.50% in the placebo group, and +2.3% in the intervention group (P = 0.149). Fat percentage change in the placebo group was − 1.6% and − 2.6% in the intervention group (P = 0.153). The percentage change in Albumin in the placebo group was 2.76% and 9.71% in the intervention group (P = 0.031). Conclusion: Our study has confirmed findings from multiple studies that protein supplementation in post-bariatric surgery patients is a successful intervention for healthy and balanced weight loss. This is yet another endorsement that surgery alone cannot put an end to obesity and must be combined with well-structured nutritional education so patients do not go back to their old habits and put the weight back on.
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Evaluation of Antidiabetic and Antihypertensive Potential of Some Traditional Desert Food Plants of Qatar Using in Vitro Assays
More LessNumerous studies have shown that food plants, the source of basic nutritional components may also possess an additional bioactive therapeutic properties associated with the prevention of diseases such as type 2 diabetes and its related complication, hypertension. The incidence of type 2 diabetes in Qatar is one of the highest in the world. In the present study, four edible plants from Qatar were selected to analyze the phenolic bioactives and their potential health benefits with relevance to managing type 2 diabetes and hypertension using in vitro enzyme assays. High antioxidant activity along with high total soluble phenolics associated with high in vitro enzyme inhibitory α-glucosidase, moderate α-amylase and ACE inhibitory effects was shown by aqueous extracts of Cynmorium coccineum. Malva parviflora and Glossonema edule had moderate antioxidant potential, total soluble phenolics and ACE inhibitory potential. Edible plants like Cynmorium coccineum that is also suggested to possess other medicinal properties has a high potential for diet-based solutions in combating, preventing and managing early stage of type 2 diabetes coupled to overall healthy life style and pharmacological management strategies. This study provides the biochemical rationale for further animal and clinical studies to understand the health benefits of edible plants of Qatar as dietary strategies for chronic disease management.
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Image Stitching System With Scanning Microscopy for Histopathological Applications
More LessHistopathological analysis of biopsy or surgical specimen is a common clinical practice for diagnostic purposes. Essentially, the process involves slicing the biopsy or surgical sample into very thin slices, placing them on glass slides and viewing them under microscopes. Predominantly, the placement, positioning, and view control is done manually by the pathologists in most of the clinics and hospitals because of which the diagnosis remains heavily dependents upon the experience and performance of the pathologist. Moreover, the slide scanning relies predominantly on the slide placement accuracy. A misaligned slide will create misaligned images which can either miss out information or have blank artifacts due to image frame placement methodology. In this paper, a simple ‘add-on’ system has been presented that can be used to scan single slide with moderate speed and produces the image on a Virtual reality headset to provide the submerged feeling. Most importantly, it utilizes advanced image stitching algorithms to align the frames from the captured video stream of the slide to produce a very accurate image with a very large size. The stitching is done using the standard feature-based algorithms which have been modified in this work by incorporating affine blending maps to combine the features into final image. It has been found that the image stitching algorithm provides the stitched image with less than 2% error for the given test images. Research Methodology: The proposed approach utilizes a standard clinical microscope with 10x, 40x, and 100x magnifications. The microscope has x-y movable stage which is moved by the pathologist using two vertical knobs on the right side. Another knob on the left side is used for fine focusing. The two knobs on the right are modified in this work by placement of two stepper motors on the knobs and making them fixed to a frame structure so that the knobs can be moved easily and the whole support structure moves with the stage. Since the stage is controlled by the system in two directions only, the focus must be adjusted initially by the user manually using the left side knob until a clear vision of the tissue slide is reached. The two stepper motors are driven by standard driver units and are controlled through an Arduino board. Slide image capture is done by a digital camera fitted with an Amscope adapter so that it could be attached to microscope's eyepiece. The camera is interfaced to the PC using USB link and provides Continuous Video when the scanning starts. Although, the same camera can be used for taking still images rather than video, the mechanical delays in stopping the scanner, shutter operation, and image storage rendered this method less practical than using videos. The high level diagram for the system is shown in Fig. 1. After obtaining the video frames, the designed image stitching (IS) algorithm is applied on them. The process of IS involves a series of steps which were applied on the consecutive images, such that one of the image is taken as a reference image (RI) whereas the other one is termed as the current image (CI). The resultant stitched image will be RI for the next consecutive image and then the whole stitching process is applied. The process remain continue for each set until a final stitched image has been obtained from them. Simulation Results In order to perform autonomous clinical analysis on the slide images, it is highly desirable to visualize all slide together (as WSI) especially when the shape and size of the particle is of great importance. The designed algorithm has been applied to Various Images (Frames) acquired from the video of the slide scan and the resultant overall WSI is shown in Fig. 2. Note that the black region shown in the Fig. 2 is produced due to the over-done y-displacement. As can be seen, the stitching is done quite smoothly in spite of the motorized shifts in the scan. In order to show the capability of alignment, the y- axis displacement was over-done by slipping more steps through the worm gear. The algorithm was able to stitch the x segments separately and the y-axis was stitched subsequently. In order to quantify the performance of the presented algorithm, another test was performed using one of the colorectal cancer image from Glas Database. One full image (401x521) was divide into 36 sub-images with 25% overlap in rows and columns between the adjacent sub images. After stitching, the resultant and the original images were compared to highlight the errors as shown in Fig. 3. The grayscale values of the error image were summed together to obtain the total error gray values (TE). This number was divided by the total number of pixels in the image to calculate the percentage error (PE). It was found that the PE for the shown image was 1.16% and was found to be less than 2% for most of the images. Figure 8 shows the test images and results from various steps in the algorithm.
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Nutritional Value of Kid's Meal offered by International Fast Food Restaurants in Qatar
More LessBackground Global food consumption patterns have dramatically changed in recent years. One common consumption pattern that is shared by many countries is the increasing expenditure on food away from home (FAFH). Convenience and accessibility seems to be the two major reasons why consumers seek for fast food. It is easy to get fast foods anywhere, everywhere and anytime. Consumption of fast-food in the world is becoming an increasingly important component of the food market as more of the working class chooses to dine out rather than prepare meals at home. Despite the importance of the fast-food sector, limited attempts have been made to study the consumption and expenditure behavior of consumers of fast-food. Fast food consumption is increasing among kids, who might lead to many health conditions, one of them is obesity. The nutritional content of kid's meal in Qatar has often been overlooked. The objectives of this study are to assess nutritional value of kid's meal offered by some international fast food restaurants in Qatar and to determine density of international fast food restaurant offering kid's meal by municipality. Methods 6 international fast food restaurants that offer kid's meals were randomly selected. The selected restaurants were McDonald's, Burger king, KFC, Hardee's, Papa john's, and DQ Grill & Chill. We assessed the number of branches of the restaurants using phone applications zomato and talabat. These applications allow the user to know the location and the numbers of branches in different municipalities. We used also the official restaurants websites to collect data of the nutrient content of kids’ meals offered by the 6-international restaurant in Qatar. Also, we used GPS to make sure of the location and the number of these restaurant. All the restaurant offered 4 combinations of kid's meals except Papa John's offered 4 meals. We selected from each restaurant chain 25% of the total branches available in Qatar. During our visit, we asked for nutrition fact of meals offered for kid's. Only McDonald's provide nutrition fact with meals. For other restaurants, we got the information from their official websites or management department. We calculated the density by dividing the number of international fast food over the total number of aged 1-9 years old multiplying by 1000. SPSS version 23 windows was used to analyze the data. We compared the differences in calories, fat, carbohydrate, protein, fiber and sodium content in kid's meals using the one-way ANOVA (at p < 0.005). Results McDonald»s has the highest number of outlets which represent 41 out of 133 international fast food outlets offering kids meals and the DQ grill and chill had the lowest number of outlet which represent 5 out of 133. The average nutrients content of 23 kid»s meal offered by international fast food restaurants in Qatar was 653.30 ± 31.5 calories for calories contents, 21.61 ± 10.24 g for protein content, 28.32 ± 9.07 g for fat content, 84.57 ± 19.22 g for CHO content, 4.40 ± 2.79 g for fiber content and 968 ± 372.31 mg for sodium content. The percentage of covering the RDA calories recommendation of children aged 1-9 years old by 23 kid»s meals offered by six international fast food in Qatar was 47.8 ± 0.02% for calories, 134.6 ± 0.13% for protein, 58.9 ± 0.04% for fat, 63.6 ± 0.03% for CHO, 20.2 ± 0.03% for fiber and 91.4 ± 0.08% for sodium. In general, international fast food kid's meals are rich in carbohydrates, fat, protein, sodium and low in fiber. Conclusion Nowadays children are eating foods prepared outside the home more frequently than ever, improving the nutritional quality of food items offered at fast-food restaurants can contribute to important gains in population health. In this study, we found that fast food meals are nutritionally inadequate and its regular consumption is associated with excess intake of calories, fat, sodium, protein, carbohydrate, and low fiber intake.
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The efficacy of Dapagliozin as a novel oral antihyperglycemic drug in the treatment of patients with type 2 diabetes mellitus
More LessBackground and Aims: Type 2 diabetes mellitus (T2DM) is a prevalent disease affecting millions of patients worldwide. The search for new antidiabetic drugs with innovative mechanisms continues. Dapagliflozin is a second agent in a new class of oral antihyperglycemic drugs; the sodium-glucose co-transporter 2 (SGLT2) inhibitors. Materials and Method: Aim: To evaluate the efficacy of new oral antihyperglycemic drug Dapagliflozin in the treatment of type 2 DM as monotherapy or combination with other hypoglycemic agents. All patients treated with Dapagliflozin in HGH since its introduction as nonformulary medication on 1st April 2013 until 30th April 2015 were included. Data regarding prescribed drugs were obtained from the pharmacy computerized system. Demographic information and laboratory results of patients were obtained from the patient»s electronic system (CERNER). Results: 81 patients were identified to receive Dapagliflazone during the study period, 71 % of them were males, 100 % were Qataries with mean age 57 ± 9 and mean A1c baseline 9 ± 1.4Dapagliflozin as add-on therapy was found to decrease A1c significantly after 6 months by − 0.8 (P = 0.006) and after 12 months by − 1.5 (P = 0.062) The fasting blood was significantly reduced at 6 months and 9 months (P = 0.001, P = 0.03 respectively) There was no significant association between different coadministered antidiabetic medication and reduction in A1c or FBG Conclusion: Dapagliflozin significantly reduced HbA1c level of type 2 diabetic patients in combination of other OHA or insulin within 6 to 12 months of treatment References: 1. Komoroski B, Vachharajani N, Feng Y, Li L, Kornhauser D, Pfister M. Dapagliflozin, a novel, selective SGLT2 inhibitor, improved glycemic control over 2 weeks in patients with type 2 diabetes mellitus. Clin. Pharmacol. Ther. 85(5), 513–519 (2009) 2. Rosenstock J, Vico M, Wei L, Salsali A, List JF. Effects of dapagliflozin, an SGLT2 inhibitor, on HbA1c, body weight, and hypoglycemia risk in patients with type2 diabetes inadequately controlled on pioglitazone monotherapy. Diabetes Care 35, 1473–1478 (2012).
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Wearable RealTime Heart Attack Detection and Warning System to Reduce Car Accidents in Qatar
Introduction Fatal car accidents have become an alarming issue all over the globe. A sudden medical condition such as a heart attack causes medical symptoms that lead a driver to lose consciousness while driving and consequently leads to a crash. Many studies have demonstrated the high correlation between the driver's sudden medical conditions and involving in a car crash [1][2]. Therefore, to reduce car crashes from the driver's sudden illness from heart-attack as well as save the driver's life in a timely manner, in this work, we discuss the development of a portable wearable system that can continuously monitor the driver for any early symptoms of heart attack and inform him before losing conciuous to stop the car as well as inform medical caregivers to save life. Background Myocardial infarction (MI) is the medical term for the medical condition commonly known as a heart attack, a serious medical emergency in which the blood supply to the heart is suddenly blocked, usually by a blood clot, leading to damage heart muscle [3]. A complete blockage of a coronary artery is a ‘STEMI’ heart attack (ST-elevation MI), whereas a partial blockage would be a ‘NSTEMI’ heart attack (a non-ST-elevationMI) [4]. The average, resting heart rhythm has a QRS-complex following a P-wave and followed by a T-wave, as illustrated in Figure 1(a). A STEMI heart attack will cause an elevation in the ST-complex (Figure 1(b)), whereas a NSTEMI heart attack would not signify ST elevation, but nonetheless can cause ST-segment depression or T-wave inversion (Figure 1(c)), which can be detected immediately by a real-time device to save the driver's life. Method The prototype system consists of two subsystems (Figure 2) that communicate wirelessly using Bluetooth low energy (BLE) technology: wearable sensor subsystem, and an intelligent heart attack detection and warning subsystem. Wearable Subsystem: The wearable chest-belt sub-system includes dry electrodes (reference and two electrodes for differential acquisition), analogue front end (AFE), power management module, and RFDuino microcontroller with BLE. This subsystem acquires the ECG signals from human body continuously and sends these raw measurements wirelessly using BLE technology to the intelligent subsystem. Reusable and smaller dimension dry electrodes (Cognionics, Inc) were embedded in a chest belt to be worn by a car driver. AD82832 AFE is an integrated signal conditioning block to extract, amplify (60 dB gain), and filter (0.48-41 Hz) ECG signal in the presence of noisy conditions. Lithium Polymer (LiPo) battery of 3.7 V (1000 mAH) with the Microchip MCP73831 charge controllers, and Texas instruments' TPS61200 voltage regulators to supply 3 V to the wearable system. The miniaturized ARM Cortex M0 RFDuino microcontroller digitizes the signal at 500 Hz sampling rate and transmits the acquired signal through built-in BLE to decision making subsystem. Intelligent Decision-making Subsystem: This subsystem will receive the ECG signals from the wearable subsystem continuously. It is capable of processing, analyzing the received ECG signals, and making the right decision using support vector machine (SVM) algorithm to classify the normal and abnormal ECG signal to detect heart attack symptoms. This subsystem was built around the single board computer, Raspberry Pi 3 (RPi3) along with SIM 908 GSM and GPS module for location information and alerting service. Multi-threaded python code was written for RPi3 to automatically acquire, buffer, baseline correction and digital smoothing and analyse the ECG data. SVM algorithm was implemented in RPi 3 and used for real-time abnormality detection using the trained model and classification was done using LIBSVM, an open source library [5]. 4-fold cross-validation was used to evaluate classification accuracy. SIM908 GSM+GPS shield attached on the RPi3 to provide car location (latitude, longitude) and to connect to the mobile network for generating an automatic call to medical emergency. This subsystem is designed to take power from the car battery using Cigarette Lighter Socket, which powers the system only when the car's engine is ON. To develop the intelligent program for decision-making subsystem, public MIT-BIH ST change database [6] was used to train a SVM model for normal, ST-elevated, and T-inverted ECG-beats with the time domain (TD), frequency domain (FD) and extended time-frequency domain (TFD) features extracted. The TD features mean, variance, skewness, kurtosis, and coefficient of variation and the FD features spectral flux, spectral entropy and spectral flatness were calculated to spot abnormalities in the ECG-beats. Three time-frequency (TF) distributions were also used in this study: Wigner-Ville Distribution (WVD), Spectrogram (SPEC), and Extended Modified B-Distribution (EMBD). Result and Discussion Recorded ECG Traces: It was clearly revealed from Fig. 5 that the ECG signal transmitted using the prototyped system is in clinical grade. Training SVM: Five hundred traces from each patient and total 2500 traces from MIT-BIH database having either normal or abnormal heart rhythm were segmented and averaged for each case (Figure 6 (A, B, & C)). The power spectral of the signal in Figure 6 (D, E & F) shows that the power spectral density peaks appear at different frequencies for normal and abnormal ECG signals. This reflects that the FD feature can help in classifying the ECG signals. However, TD, FD, and TFD features provide an insight on the signal while compensating for the noise or motion artefacts. Classification using SVM: Table 1 below summarizes the accuracy of the prototyped device. EMBD produces higher accuracy in classification of ECG signal. Conclusion This work shows the possibility to detect driver's heart attack reliably using the developed prototype system. SVM machine learning algorithm that was trained with a sufficiently high number of training data can classify STEMI or NSTEMI with approximately 97.4% and 96.3% accuracy respectively when the extended TF features (with EMBD distribution) were used for training and classification. The maximum current drawn by the wearable chest-belt subsystem during continuous acquisition is 9.3 mA, which ensures the life span of a 1000 mAh LiPo battery is 75 hours, once it is fully charged and therefore it can be expected that the device can run longer without requiring recharging daily.
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Efficacy and safety of once daily liraglutideversus twice daily exenatidein type 2diabetic patients in Qatar: an observational study
Authors: Rana Moustafa Al Adawi and Zainab Malik JassimBackground: Type 2 diabetes prevalence is strongly associated with the increase in obesity prevalence. Many of the current diabetic treatments cause further weight gain. Glucagon-like peptide-1 receptor agonists (liraglutide & exenatide) offer advantages of either keeping weight stable or even reducing weight while achieving good glycemic control. Objectives: To compare the glycemic effect of liraglutide versus exenatide over 26 and 52 weeks in uncontrolled type 2 diabetic patients.Methods: A retrospective observation study. Patients with type 2 diabetes who took liraglutide (1.8 mg subcutaneous [SC] once daily) or exenatide (10 mcg SC twice daily) for at least 1 year in addition to their anti-diabetic medications were eligible. For each patient, the following data were collected: HbA1C, fasting plasmaglucose, body weight, blood pressure, lipid profile, hypoglycemia episodes, kidney and liver function. Patient's medical records (both electronic and paper-based records) were used to collect required data. Data analyzed using descriptive & inferential analyses. The study was undertaken by ZJaspart of Masterin clinical pharmacy at Queen's University Belfast Results: 212 patients were included in the study (114 in exenatide group and 98 in liraglutide group). There were no significant differences in all of the patients’ demographics and characteristics between the two groups (table1). Around 73% of included patients were female and half them were aged between 50–59 years. There was in significant difference in mean HbA1C change between both medications at either 26 or 52 weeks (p = 0.23 and 0.40, respectively) (Fig. 1). Patients achieved HbA1C ≤ 7% were significantly higher in the liraglutide group at week 26 (Fig. 2). Liraglutide reduced the mean fasting plasma glucose more than exenatide did at week 26 ( − 1.099 vs. − 0.122 mmol/L; p = 0.15) and week 52 ( − 1.150 vs. − 0.616 mmol/L; p = 0.52). Both medication sex habited weight losses at 26 and 52 weeks; liraglutide − 1.24, − 2.54 vs. exenatide − 1.63, − 3.7 kg, respectively (figure 3). Although both medications were associated with some benefits in term so flipidprofile and blood pressure at a certain point, neither of them were able to show a significant change from baseline. No patients in either groups reported any GI side effects or episodes of hypoglycemia. There was no statistically significant difference between two groups in regards of liver and kidney functions except serum creatinine elevation in heliraglutide group at 52 weeks (p = 0.001). Conclusion: Exenatide and liraglutide resulted in similar glycemic effects (HbA1C and FPG changes) in patients with type 2 diabetes who were sub-optimally controlled with other anti-diabetic therapy. Weight reduction effectiveness was confirmed for both medications with noreported side effects or hypoglycemic episodes during the treatment perid. Future large scale prospective studies are needed to confirm these results.
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Antidiabetic and Toxicological studies of ethylacetate and nhexane fractions of Gymnema sylvestre
More LessGymnema sylvestre is a medicinal plant that is used in the folkloric treatment of diabetes mellitus and the management of its complications. This study was aimed at evaluation of antidiabetic and toxicological effects of ethylacetate and n-hexane fractions of Gymnema sylvestre whole plant. Diabetes was induced in Swiss albino rats by single intraperitoneal administration of 110 mg/kg bodyweight of alloxan monohydrate. Rats in their respective groups were orally administered 100, 300 and 600 mg/ kg bodyweight of ethylacetate and n-hexane fractions of the plant daily while the standard drug group received 100 mg/kg bodyweight of metformin. The treatment lasted for fourteen days and on the fifteenth day, animals were anaesthetized and euthanized. Blood samples were collected by carotid puncture for biochemical analysis. In the subchronic toxicological aspect of the study, rats in their respective groups were administered 100, 300, 600 mg/kg bodyweight of the extracts daily for twenty one days and the experiment was terminated on the twenty second day. All the extracts were able to reduce the blood glucose of diabetic rats with 300 mg/kg bodyweight of ethylacetate fraction having higher reduction at 82%. All diabetic rats showed decrease in bodyweight when compared with normoglycemic group. There was significant (p<0.05) reduction in the total cholesterol, triacylglycerol, low density lipoprotein and a concomitant increase in the values of high density lipoprotein in all treated groups when compared with diabetic untreated (negative control).The activity of serum liver enzymes (Ɣ- glutamine transpeptidase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase) and bilirubin significantly decreased (p<0.05) compared with the diabetic untreated group. Levels of total protein in the treated groups did not differ from the normoglycemic group, while there was a reduction in the concentration of albumin of ethylacetate fraction in a dose dependent manner. All treated groups gave urea and creatinine values that showed no significance differences (p>0.05) with the normoglycemic group. The electrolytes showed significant differences (p<0.05) in all treated groups when compared with the normoglycemic group. There was significant difference (p<0.05) in all the biochemical parameters carried out on the subchronic toxicity test with rats administered ethylacetate and n-hexane fractions of G.sylvestre. The hematological parameters (red blood cell, mean cell volume, mean corpuscular hemoglobin concentration, packed cell volume, hemoglobin) showed no significant difference but a non significant difference in the white blood cell of rats administered with the extract. Therefore, extracts of G. sylvestre might be useful for management of diabetes mellitus and other abnormalities associated with this metabolic disorder
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Generation of a Novel Population of Pancreatic Multipotent Progenitor Cells Expressing NKX61
Authors: Essam Abdelalim, Idil Aigha, Ahmed Elsayed and Bushra MemonDiabetes is a metabolic disease caused by the loss or impaired function of insulin-producing pancreatic β-cells. Different therapeutic strategies aim to restore the endogenous production of insulin rather than the cornerstone insulin injections treatment. Human pluripotent stem cells (hPSCs) have been proposed as an unlimited source for cell-based therapy of diabetes through the directed differentiation into functional pancreatic β cells. Step-wise differentiation protocols based on developmental biology of pancreas, have led to the generation of insulin-producing β cells. However, the majority of the cells produced were poly-hormonal as they expressed other hormones in addition to insulin and have failed to respond when challenged with glucose. The coordinate expression of particular transcription factors (TF) in distinct stages governs the differentiation of hPSCs into insulin β cells. Pancreatic and duodenal homeobox protein (PDX1) is a crucial TF required for pancreas development. On the other hand, homeobox protein NKX6.1 is a potent bi-functional TF that is essential for β cells maturation, proliferation and insulin metabolism. The dual expression of PDX1 and NKX6.1 during multipotent progenitor cell (MPC) stage is vital for guiding the cells towards functional β cells lineage. However, cells expressing PDX1 but lack NKX6.1 expression tend to take the poly-hormonal path. This guided the differentiation protocols to focus on enriching MPC population co-expressing PDX1 and NKX6.1. The aim of this study was to further explore different MPC populations in terms of PDX1/NKX6.1 expression. We used two different differentiation protocols to differentiate hESCs and hiPSCs into MPCs. The mRNA and protein expressions of the generated MPCs were analyzed using immunocytochemistry, RT-PCR, and flow cytometry. Our results showed that hPSCs were successfully differentiated into the conventional (PDX1+/NKX6.1+) and (PDX1+/NKX6.1-) MPC populations. The efficiency of differentiating hPSCs into PDX1+/NKX6.1+ MPCs has varied between the two used protocols. Immunofluorescence staining has unveiled the generation of a novel population that expressed NKX6.1 independently of PDX1 (PDX1-/NKX6.1+) in both hESCs and hiPSCs. This is surprising considering that PDX1 was reported to bind to the promoter of NKX6.1 gene and is needed for NKX6.1 expression. Furthermore, using our optimized protocol, this uncharacterized subset of MPCs was enriched and found to exhibit a pattern of three-dimensional (3D) aggregates that were consistently (PDX1-/NKX6.1+) and surrounded by either (PDX1+/NKX6.1+) or (PDX1+/NKX6.1-) MPCs. To understand and characterize this unique population, we examined the expression of other TFs including endocrine precursors markers Chromogranin A (CHGA) and NKX2.2. CHGA was found to be expressed in the same areas that were positive for NKX6.1 and PDX1. However, the 3D structures that were PDX1-/NKX6.1+ did not co-express CHGA. On the contrary, few cells of these 3D aggregates co-expressed NKX2.2, suggesting that this population may have an undefined role in the development of MPCs into endocrine progenitors. These findings showcase a novel population of NKX6.1 expressing MPCs that did not require PDX1 expression at this stage. Moreover, this population may retain an alternative path towards pancreatic islet cells development that is independent of PDX1. A thorough characterization of this population is needed to explore the regulatory gene network controlling their lineage specification.
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Nonphosphorylated Alphasynuclein at Serine 129: A Potential Biomarker for Parkinson's Disease
More LessAuthor and co-authors’ details: Muneera Fayyad1, Nour Majbour2, Mercy A. Thomas2, N. Vaikath2, and Omar M. A. El-Agnaf1,2 1Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar 2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. It is characterized by a progressive loss of dopaminergic neurons and an accumulation of Lewy Bodies (LBs) and Lewy Neurites (LNs), protein-rich inclusions that deposit in the neurons. These inclusions are mainly composed of alpha-synuclein (α-syn), a small protein with a propensity to aggregate. Evidence from genetic, biochemical, pathological and animal studies suggest that the aggregation of α-syn plays a key role in the pathogenesis of PD and related disorders, termed synucleinopathies. To date, the diagnosis of PD primarily depends on the clinical criteria, however, this approach has its limitations since the neurodegeneration starts several years before symptoms manifest. In other words, the onset of typical motor symptoms is mostly preceded by 50-70% loss of dopaminergic neurons, which explains the irreversibility of the disease at the time of diagnosis. This necessitates the development of reliable biomarkers that would allow early diagnosis of PD at the preclinical stage. Accumulative evidence suggests that the majority of α-syn from LBs in the brains of PD patients is found to be phosphorylated at Serine 129 (pS129-α-syn). It is unknown whether phosphorylation of α-syn promotes or prevents its aggregation and toxicity. Nevertheless, this highlights the importance measuring the levels of pS129-α-syn in biological fluids. We highlighted that CSF oligomeric-/total-α-syn and p-S129-/total-α-syn ratios improved the discrimination between PD and healthy subjects. However, little is known about a the potential diagnostic role for non- phosphorylated α-syn at S129 (npS129-α-syn). Recently, we developed a novel mouse monoclonal antibody (4B1) that is specific for npS129-a-syn (i.e. does not recognize pS129-α-syn). Here we describe the generation of 4B1 using hybridoma technology. The antibody purification was done using Protein-G agarose affinity chromatography, and later characterized using a wide range of biochemical assays. More importantly, we describe the development of the first ELISA assay to reliably quantify concentrations of npS129-α-syn in biological samples. We also assessed the usefulness of our assay using CSF samples from PD patients and age-matched healthy controls. We report that the discrimination power between PD and healthy controls was improved by including npS129-/ t-α-syn ratio. We highlight that the combination of multiple CSF biomarkers, improved the diagnostic accuracy of PD. Such immunoassays would not only aid in the discovery of ideal CSF biomarkers but may also serve as research tools to facilitate a better understanding of the underlying role of α-syn phosphorylation in PD and related disorders. In addition, our efforts to identify biomarkers for PD may reveal molecular species that could serve as therapeutic targets for the development of new disease modifying therapies.
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Screening for Novel Inhibitors of Beta Amyloid Aggregation and Toxicity as a Potential Drug for Alzheimer's Disease
Authors: Sanaa Ali Sharari, Nishant Narayanan, Magdalini Taskou, Kostas Vekrellis and Omar El-AgnafAlzheimer’s disease (AD) is one of the most common neurodegenerative disorders in which the disruption of brain cells causes impairment of cognition. The pathology of AD is still unclear, with different etiologies leading to formation of extracellular senile plaques, intracellular neurofibrillary tangle (NFT) and ultimately neuronal death. However, several investigations showed that the amyloid hypothesis in which the accumulation of the toxic amyloid beta (Aβ) protein in the central nervous system (CNS) is the main pathology of the AD. Currently, there is no proven medication to cure or prevent the disease, however, therapeutic approaches of AD show only relief of symptoms and mostly work on cognitive recovery. Moreover, herbal phenolic compounds grant more effective therapy due to its multifacted action. Based on the previous work targeted the amyloid hypothesis in disease other than AD by using Chinese medicinal compounds, our project is aimed to investigate the activity of Ginsenoside Rb1, Dihydromyricetin and Salvianolic acid on the process of aggregation of Aβ40 and Aβ42. The effect of these compounds on the Aβ aggregation was investigated using different biochemical assays. Our results showed that Salvianolic acid and dihydromyricetin inhibit Aβ40 and Aβ42 fibrilizations. Whereas, Ginsenoside Rbi1 showed no effect on the aggregation process of both amyloids. Overall, these compounds may be considered as starting point for designing new compounds that could be used as drugs for the treatment of AD and related disorders.
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Impact of a Collaborative Pharmaceutical Care Service among Patients with Diabetes in Qatar Petroleum Healthcare Center Dukhan: A Multiple Time Series Study
Authors: Ahmed Awaisu, Sara Abdulrhim, Rana Saleh, Mohamed Abdelazim, Hend Alraey, Ahmed Babiker and Nadir KheirAbstract Background: Diabetes mellitus is a highly prevalent non-communicable disease worldwide. The prevalence of diabetes in Qatar exceeds the prevalence of diabetes in the Middle East and North Africa region and the globe. Similarly, diabetes-related complications and mortality are dramatically increasing worldwide. Poor health outcomes and debilitating consequences can result from inadequate control of diabetes. Previous studies have demonstrated the benefit of pharmaceutical care services on outcomes of diabetes. No studies were done in Qatar regarding this issue. Therefore, the objectives of this study were to: (1) characterize the clinical profile of patients with diabetes attending an ambulatory care clinic at Qatar Petroleum (QP) Medical Center including diabetes-related comorbidities and complications; (2) evaluate the impact of a Comprehensive Pharmaceutical Care Service (CPCS) on glycemic control [glycated hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG)]; (3) evaluate the impact of the CPCS on diabetes comorbidities including lipid profile [low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), triglycerides (TG), and total cholesterol (TC)], systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) and; (4) classify the drug-related problems (DRPs) identified by pharmacists during the follow-up period. Methods: This was a multiple time series, observational, retrospective, pre-post study among patients attending diabetes clinic at QP Medical Center in Dukhan. Primary clinical outcome measures including HbA1c, FPG, weight, BMI, SBP, DBP, and lipid profile were measured at baseline, 6 months, and 12 months after receiving the CPCS through a retrospective chart review of electronic medical records for the year 2016. The secondary outcome measure, the types of DRPs identified by pharmacists, was collected over the period of 12 months of initiating the CPCS and categorized into a predetermined classification system. Data analyses were performed using IBM SPSS® version 23.0. Primary clinical outcome measures were analyzed inferentially using Repeated Measure ANOVA to determine the impact of the intervention. Sociodemographic characteristics, basic clinical characteristics, baseline and current medications regimens, and types of DRPs identified by pharmacists were analyzed descriptively using frequencies, percentages and means as appropriate. Results: A total of 96 eligible patients with diabetes were included in the study. CPCS significantly improved the following parameters from baseline to 6 and 12 months: HbA1c (8.5%, 7.4%, 7.1%, respectively; P <0.001), FPG (154.1 mg/dL, 115.4 mg/dL, 112.8 mg/dL, respectively; P <0.001), weight (79.9 Kg, 78.3 Kg, 76.9 Kg, respectively; P <0.001), BMI (29.1 Kg/m2, 28.5 Kg/m2, 28.1Kg/m2, respectively; P <0.001), SBP (140.2 mmHg, 129.1 mmHg, 125.3 mmHg, respectively; P <0.001) and DBP (84.7 mmHg, 79.5 mmHg, 76 mmHg, respectively; P <0.001). However, no significant reductions from baseline to 6 and 12 months were observed in LDL-C (2.7 mmol/L, 2.8 mmol/L, 2.7 mmol/L, respectively; P =0.702), HDL-C (1.2 mmol/L, 1.2 mmol/L, 1.3 mmol/L, respectively; P =0.551), TG (1.6 mmol/L, 1.7 mmol/L, 1.7 mmol/L, respectively; P =0.728), and TC (4.3 mmol/L, 4.3 mmol/L, 4.1 mmol/L, respectively; P =0.101). The most prevalent three DRPs identified were lack of understanding of the medication (39.8%), inappropriate dose, form, schedule, route, or method of administration (17.3%), and actual and potential adverse events (14.3%).
Conclusion: The provision of CPCS in a primary healthcare setting in Qatar improves clinical outcomes in patients with diabetes over a 12-month follow-up period. Future studies are needed to determine the long-term outcomes of CPCS.
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Does Weight Loss Through Means of Bariatric Surgery Reduce the Risk of Type 2 Diabetes in Obese Qatari Patient a Retrospective Analysis
Background: The use of bariatric surgeries such as Gastric Bypass and Sleeve Gastrectomy in managing obesity and associated diseases such as type 2 diabetes mellitus (T2DM) has been induced in clinical practice. Weight reduction through means of bariatric surgery has metabolic benefits and may improve the management of T2DM. Objectives: The aim of this study was to investigate if weight loss through bariatric surgery can reduce the risk of T2DM in patients without the onset of T2DM. Study design: A retrospective analysis was conducted on post-bariatric patients at the department of bariatric and metabolic surgery at Hamad General Hospital. Methods: Tow hundred and two eligible pre-diabetic Qatari patients who have undergone bariatric surgery in 2016 and satisfied the inclusion and exclusion criteria of the study were analyzed. Data on Glucose, Insulin and C-peptides levels at baseline and follow-up were extracted in order to compare the change of these variables at baseline, 6 and 10 months follow up before and after 10 months from the date of surgery. Results: Seventy one males with mean age of 32.73 ± 10.37 and one hundred and thirty one females with mean age of 33.90 ± 9.88 were included in the analysis. Change in weight was strongly and positively associated with change in insulin level (0.701, 95% CI: 0.027, 1.347, p = 0.042) also, as weight changes fasting glucose changes (1.993, 95% CI: 0.359, 3.627, p = 0.017). Follow-up period greater than 6 months was not found to be significantly associated with weight loss (2.049, 95% CI: − 2.249, 6.349, p = 0.313). Conclusion: Our study confirms results from international studies that weight loss through bariatric surgery can reduce the risk of developing Type 2 Diabetes Qatari obese patients. The results of the study suggest that post-surgery periods can be detrimental to the fate of fasting glucose and insulin levels and therefore compliance maybe of great importance to ensure success and sustainability of weight loss and diabetes prevention. Larger samples size and longer follow-up period is required to confirm these findings.
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Utilization of Date Pits in the Production of Functional Chocolates
Authors: Syed Zamzam, Eisha Rajab Nafiea, Huda Ahmed Al-hadhromi and Fida Anamangadan AliBackground: World production of dates was almost 9 million tons by 2010, with almost 960 thousand tons of date pits being discarded. This huge amount of waste has shown to be a rich source of carbohydrate, fiber, protein, fat, antioxidant, phenolic, and minerals like potassium, phosphorus, and magnesium. Not only is it an excellent source of nutrients, but has also shown to have health benefits such as reducing the side effects of certain therapeutic drugs, and otherfunctional uses, like used as a coffee substitute for a caffeine free drink. Like date pits, chocolate is a natural functional food which has shown to have a favorable effect on human health especially on the cardiovascular disease and it is one of the most consumed food among all age groups.Objectives: The main objective of this study is utilize date pits as an additive to chocolate and produce improved functional food product and evaluate its sensory features.Method: Commercial date pit powder, dark chocolate, and milk chocolate was purchased from local Qatari markets. The commercial powder was further grinded using a regular household blender and sieved into 300 and 150 μm using electronic sieve. The developed products had varying percentage of date pit powder: chocolate ratio (1:9, 3:7, 5:5 and 7:3). The chocolate products cross-section was observed under microscope, sensory analysis for 8 attributes and thenutrient content for each product was done using USDA food composition tables and food label of the commercial date pits purchased from Deyma.Results: Dark and milk chocolate with 150 μm date pits, (1:9) ratio had the highest homogeneity under the microscope. The quality test for functional chocolates of 1:9 and 3:7 ratio made with 150 μm rated as very good but 5:5 and 7:3 required lots of improvement. Sensory evaluation and hedonic test showed that dark chocolates made with 150 μm date pits was the most liked products. Finally, the calories and the fat content decreased as the date pits concentration increased.
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GameBased NonWeight Bearing Exercise to Improve Postural Balance in Diabetic Patients Underjoining Hemodialysis
Authors: He Zhou, Rania Ibrahim, Abdullah Hamad, Talal Talal, Fadwa Al-Ali and Bijan NajafiBackground: Poor balance, falls, and foot problems are serious detriments for the diabetic patients due to the obesity and diabetic foot ulcer. In addition, for the diabetic patients undergoing hemodialysis (HD) treatment, the HD process often leaves them too fatigued to engage in any physical activity or daily exercise, further deteriorating their motor functions and increasing risk of falling. Exercise would be effective for this population. However, due to the time availability, post-dialysis fatigue, as well as limitation of transportation to exercise facility, the conventional exercise for this population is impractical. Objective: We are developing an interactive foot and ankle exercise game that can be played during HD sessions to improve foot region blood flow, as well as reduce foot problems. In this study, we examined the feasibility and effectiveness of this innovative wearable sensor based non-weight bearing exercise (Exergame) to improve postural balance in diabetic patients undergoing HD treatment. Methods: Sixty diabetic subjects receiving HD treatment were recruited and randomized into an intervention group (IG: n = 29, age = 63.3 ± 7.9 years, BMI = 31.2 ± 6.5 kg/m2, female = 41%) and a control group (CG: n = 31, age = 66.5 ± 10.7 years, BMI = 32.3 ± 8.2 kg/m2, female = 55%). Both groups underwent a 4-week ankle and foot exercise program (30 minutes per session, two sessions per week) during HD process. The IG received exercise via the Exergame program, which uses wearable sensors attached on subject's feet. The subject's 3-dementional ankle and foot movements were visualized in real-time on a computer screen placed in front of him/her. The subject performed some game-like tasks by moving and rotating the foot and ankle. The difficulty level of the task was gradually increased depending on ability of the subject (like a game) from a simple flexion-extension movement to more complex movements including medial-lateral movement with different range of motion. The CG received traditional foot and ankle exercise without any technology. Postural balance was assessed in the semi-tandem test. Balance tests were performed at baseline and conclusion of the program, under both eyes-open and eyes-closed conditions. Balance parameters included ankle sway and hip sway in anterior-posterior (AP) direction (degree), medial-lateral (ML) direction (degree), as well as in area (degree2). Results: All IG subjects achieved to complete all exercise tasks indicating the feasibility of the Exergame platform. No adverse event or difficulty were reported indicating practicality of the exercise program. None subject in the IG was dropped out during the 4-week exercise program. Low dropout rate may indicate acceptability of the proposed Exergame platform. Under eyes-open condition, the IG had significant ankle sway reduction in the AP direction (Cohens’ d effect size = 0.55, p = 0.037), when comparing with the CG. At conclusion, the AP direction ankle sway reduced 18% in the IG, while in the CG it increased 58%. More significant improvements of postural balance were observed under eyes-closed condition. When comparing with the CG, the IG had significant ankle and hip sway reductions in both AP and ML directions, as well as in area (p < 0.050). The highest effect size contrasting changes between the IG and CG was also observed for ankle sway in ML direction (Cohens’ d effect size = 0.76, p = 0.005). Conclusions: This study demonstrated feasibility, acceptability, and effectiveness of an innovative Exergame program to improve postural balance in diabetic patients undergoing HD treatment. The key innovation of the proposed intervention is its practicality to be done during HD process, which could address the limitations of prior exercise interventions in HD patients, for example the low adherence of therapeutic exercise.
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Exergame: A Gamelike Exercise to Improve Motor Functions and Physical Activities in Diabetic Patients Undergoing Hemodialysis
Authors: He Zhou, Rania Ibrahim, Abdullah Hamad, Talal Talal, Fadwa Al-Ali and Bijan NajafiBackground: Balance, mobility, falls, and foot problems are serious detriments for the diabetic patients undergoing hemodialysis (HD) treatment. In addition, the HD process often leaves them too fatigued to engage in any physical activity or daily exercise, further deteriorating their motor functions. Exercise would be effective for this population. However, due to the time availability, post-dialysis fatigue, as well as limitation of transportation to exercise facility, the conventional exercise is impractical. Objective: We are developing an interactive foot and ankle exercise game that can be played during HD sessions to improve mobility and balance, as well as reduce foot problems. In this study, we examined the feasibility and effectiveness of this innovative wearable sensor based non-weight bearing exercise (Exergame) to improve daily physical activity in diabetic patients undergoing HD treatment. Methods: Thirty-three diabetic subjects receiving HD treatment were recruited and randomized into an intervention group (IG: n = 15, age = 62.2 ± 7.6 years, BMI = 29.1 ± 6.1 kg/m2) and a control group (CG: n = 18, age = 66.6 ± 8.7 years, BMI = 32.5 ± 9.0 kg/m2). Both groups underwent a 4-week ankle and foot exercise program (30 minutes per session, two sessions per week) during HD process. The IG received exercise via the Exergame program, which uses wearable sensors attached on subject's feet. The subject's 3-dementional ankle and foot movements were visualized in real-time on a computer screen placed in front of him/her. The subject played some game-like tasks by moving and rotating the foot and ankle. The difficulty level of the task was gradually increased depends on ability of the subject (like a game) from a simple flexion-extension movement to more complex movements including medial-lateral movement with different range of motion. The CG received traditional foot and ankle exercise without technology. Daily physical activity data was assessed for 48 hours (day and night) at baseline and post 4-week exercise, using a validated wearable sensor (PAMSysTM). Daily physical activity was quantified by duration spent in each main posture (i.e. lying, sitting, standing, and walking) and activities (e.g. postural transition, sedentary behavior, etc). Results: All IG subjects achieved to complete all exercise tasks indicating the feasibility of the Exergame platform. No adverse event or difficulty were reported indicating practicality of the exercise program. None subject in the IG was dropped out during the 4-week exercise program. Low dropout rate may indicate acceptability of the proposed Exergame platform. At the end of intervention, subjects in the IG were more active than subjects in the CG. In summary, the IG performed 53% more posture transitions to walking (Cohen's d effect size = 0.5) and 39% more posture transitions between sitting and walking (d = 0.5), when compared to the CG. Subjects in the IG also had significant less sedentary behavior than subjects in the CG. In summary, subjects in the IG spent 5% less time on sitting and lying (p = 0.049, d = 0.7), as well as 47% more time on standing and walking (p = 0.049, d = 0.7), when compared to subjects in the CG. Conclusions: This study demonstrated feasibility, acceptability, and effectiveness of an innovative Exergame program to improve daily physical activity in diabetic patients undergoing HD treatment. The key innovation of the proposed intervention is its practicality to be done during HD process, which could address the limitations of prior exercise interventions in HD patients, for example the low adherence of therapeutic exercise. Further studies should be addressed to confirm the observation with larger sample sizes.
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The epigenetic response to disease and environmental challenges: a deep multiomics phenotyping view
More LessEpigenetic regulation of human cellular functions allows temporal adaptation to changes in health, lifestyle and environment. Complex diseases and exposure to environmental challenges may lead to adjustments of the expression of certain enzymes, transporters, and metabolic regulator genes (1). The response to such challenges by adjustment of gene expression may be reflected through changes in the DNA methylome (2). A recent metabolomics epigenome wide association study showed that the methylation states of certain DNA cytosine-guanine (CpG) pairs are strongly associated with blood metabolite levels (3). Interestingly these metabolomics associated CpG sites are also associated with a related complex trait falling in one of two major groups, the first being disease driven i.e. diabetes and/or obesity that also associate with liver function or blood pressure, and the second being environmentally driven i.e. smoking. We performed deep molecular phenotyping covering approximately 4000 traits in blood, urine and salivary samples from a diverse population of 359 individuals from the Qatar Metabolomics Study of Diabetes (QMDiab), using array-based DNA genotyping and methylomics, NMR based lipidomics, mass-spectrometry based metabolomics, aptamer based blood-circulating proteomics, and total plasma protein N- and IgG-glycomics. We confirmed the established smoking and diabetes associations in QMDiab and identified novel multi-omics associations at these sites. In particular, we identified deep molecular phenotypes that are characteristic for the TXNIP-diabetes and the AHRR-smoking associations, including diabetes and smoking associated metabolites such as 1,5-anhydroglucitol (1,5-AG) and o-cresol sulfate respectively, diabetes and smoking associated proteins such as Sex hormone-binding globulin (SHBG) and Polymeric immunoglobulin receptor (PIGR) respectively, and diabetes and smoking associated glycans. We also replicated some of the novel associations, particularly the proteomics and glycomics associations, in two independent studies (KORA and TwinsUK). These observations show an interesting overlap between DNA methylation and pathways relevant to complex disorders and environmental challenges. We also addressed the direction of that involvement in this study. Motivated by a recent obesity Mendelian randomization study (4) that showed a causal effect of adiposity on the methylation levels of multiple CpG sites near obesity-related genes, we used Mendelian randomization to test the direction of association between the metabolite and methylation levels of selected obesity associated CpG sites in KORA. In line with that study, we also found a causal effect of metabolite levels on methylation of the given CpG sites, i.e. glycerophospholipid PC(O-36:5), glycine, and a very low density lipoprotein A (VLDL-A) on the methylation of the DHCR24, MYO5C, and CPT1A loci, respectively. The overlap of disease- and lifestyle-associated CpG sites with metabolite-associated CpG sites suggests that the organismal adjustment to disease or environmental challenges can be captured by measuring relevant intermediate molecular phenotypes (multi-omics) (5). Taken together, this study supports the hypothesis that multi-omics-associated CpG methylation can serve as a functional readout of the organism's response to the challenges induced by disease or environmental stress, serve as novel functional diagnostic and prognostic biomarkers, and indicate potential targets for therapeutic intervention.
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Development of Novel Diagnostic and Therapeutic Tools for Parkinson's Diseases and Related Disorders
Authors: Nour Majbour, Muneera Fayyad, Mercy Thomas, Nishant Vaikath and Omar El-AgnafParkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. The biggest roadblock we are facing for the cure of PD is the lack of reliable biomarkers for the disease diagnosis or progression. This is a problem, not only from a clinical standpoint, but also because it affects the integrity of clinical trials and epidemiological research. Thus, the development of simple diagnostic tests to aid the clinical diagnosis of PD and other neurodegenerative diseases is of great importance. PD is characterized with a long preclinical phase that might serve as a window for early therapeutic intervention once disease-modifying therapies are available, however, the lack of reliable biomarkers for PD diagnosis and progression represents a major obstacle to achievement of this goal. One of the most prominent pathological features of most neurodegenerative diseases is the deposition of specific protein aggregates in neuronal or glia cells. In PD, such deposits are named Lewy bodies (LBs) and Lewy neuritis (LNs), the pathological hallmarks of PD, in which α-syn is the main constituent. α-Syn is 140 amino acid, a pre-synaptic neuronal protein and its aggregation and dysfunction is linked to a number of neurodegenerative disorders, named Synucleinopathies. PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) all fall under the umbrella of Synucleinopathies. The aggregation of α-syn starts long before the onset of the clinical symptoms and continues to spread throughout the brain as the disease progresses, making these aggregates strong candidates for biomarkers development. α-Syn is subjected to several post-translational modifications (PTMs) such as phosphorylation, oxidation, nitrosylation, truncation and ubiquitination. However, whether these PTMs act to enhance or halt α-syn neurotoxicity remains poorly understood. Biochemical examination of LBs revealed the presence of full-length α-syn as well as different species of truncated α-syn. Truncated α-syn was found in the brains of patients as well as the brains of healthy subjects, suggesting that α-syn truncation takes place even under normal physiological conditions. A marked difference, however, in the amount of truncated α-syn in synucleinopathies’ patients was noted compared to control subjects. Truncated α-syn was also found to be abundant in the brains of PD and DLB patients, suggesting that truncated α-syn may play a normal physiological role as well as a pathological one. Our group has succeeded to develop several highly specific antibodies that were explored for diagnostic and therapeutic purposes. Here in this project, we describe the characterization and applications of our novel conformation-specific mouse monoclonal antibodies against different truncations of α-syn as diagnostic and therapeutic tools for synucleinopathies. The specificity and sensitivity of our antibodies were thoroughly assessed by an array of biochemical methods including slot blotting, western blotting, ELISA and immunohistochemical analysis in parallel with commercially available antibodies. Next, we explored the potential of these novel tools in biological samples (brain tissues and biofluids) from patients with different synucleinopathies. Our antibodies exhibited a specificity towards different conformations of α-syn that none of their commercial peers did. Our novel antibodies also stained a pathology in human brain tissues that was not captured by gold standard antibodies for Immunostaining of α-syn pathologies. Our antibodies are not only valuable for the discovery of ideal biomarkers but also for a better understanding of the underlying pathophysiological process in PD and related disorders.
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The Cellular Interplay Between CD44 and Na+/H+ Exchanger1 in Cardiac Remodeling
Authors: Fatima Mraiche, Muna Suleiman, Nabeel Abdulrahman and Jensa JosephBackground: Extracellular matrix (ECM) remodeling is a characteristic feature of cardiac remodeling which, if left untreated, progresses to heart failure. Cardiac fibroblasts, the major cellular component of the myocardium, are responsible for maintaining the ECM integrity. Upon cardiac injury, such as myocardial infarction (MI), pressure or volume overload, cardiac fibroblasts transdifferentiate into cardiac myofibroblasts. Cardiac myofibroblasts are capable, of secreting proinflammatory cytokines and collagen; initiating tissue repair mechanisms (i.e., cardiac fibrosis). Hyaluronan (HA), a major component of the ECM, which is synthesized by hyaluronan synthase enzyme-2 (HAS-2), was shown to contribute in ECM remodeling and myocardial fibrosis by interacting with its cell surface receptor; CD44. CD44 is a transmembrane glycoprotein involved in multiple physiological and pathological conditions. Na+/H+ exchanger isoform-1 (NHE-1) a cardiac specific intracellular pH regulator, which is triggered by neurohormonal stimulation of Angiotensin II (ANG II), phenylephrine (PE), endothelin-1 (ET-1) or inflammatory mediators has also been implicated in cardiac remodeling. A previous study has shown that HA-CD44 interaction enhanced ECM remodeling in a non-cardiac model through activation of NHE-1 and hyaluronidase-2 (HYAL-2), a low pH-dependent enzyme which degrades hyaluronan polymers into lower molecular weight fragments. Yet, the link between NHE-1 and CD44 interaction in cardiac setting has not been addressed.Methods: CD44 expression was measured by qPCR and immunohistochemistry in transgenic mice expressing cardiac specific NHE-1. In vitro, normal human ventricular cardiac fibroblasts (NHCF-V) were treated with either 0.1 or 1 μM ANG II for 6, 24 or 48 hours to induce myofibroblast phenotype. Cell lysates and culture media were collected and analyzed by immunoblotting. Standard CD44 (CD44s), soluble CD44 (solCD44), unmodified CD44, HYAL-2 and HAS-2 protein expression were measured following ANG II treatment.Results: mRNA expression and immunohistochemistry data demonstrated that CD44 expressions were significantly elevated in heart tissues from transgenic mice expressing cardiac specific NHE-1 compared to wild type. Immunoblotting analysis of NHCF-V cell lysates showed a significant increase of CD44s protein expression, appearing at 75 KDa, following 0.1 μM ANG II treatment for 24 hours (131.14 ± 3.18 % ANG II vs. 100% control; P ≤ 0.01). CD44s protein expression did not show a change at 6-hour or 48-hour time point. Unmodified CD44, appearing at 37 kDa, HAYL-2 and HAS-2 protein expressions did not change following stimulation with various concentrations of ANG II at various time points. However, a trend towards increase was observed with solCD44 protein expression, appearing at 75 kDa, in NHCF-V conditioned media (CM) following 24-hour treatment with both 0.1 and 1 μM ANG II. A similar trend was observed with HYAL-2 protein expression in NHCF-V CM following 24-hour treatment with 1 μM ANG II, whereas there was no observed protein expression for HAS-2 and the unmodified form of CD44. Conclusion: ANG II, a well-established stimulator of NHE-1, is able stimulate the CD44s in NHCF-V cell lysates. Similarly, a trend towards increase of solCD44 expression was demonstrated in NHCF-V CM when treated with ANG II. This maybe due to the expression of CD44 in the extracellular space. HYAL-2 protein was also expressed in NHCF-V CM, while no change was detected in cell lysates. The protein and gene expressions of CD44, HYAL-2 and HAS-2 in the presence and absence of a NHE-1 inhibitor and ANG II type 1 receptor (AT-1) inhibitor remain unknown and need to be addressed to further understand the cellular interplay of NHE1 and CD44 in cardiac remodeling. It is of a significant importance to identify the signaling pathways leading to CD44 activation for better understanding of etiologies behind cardiac remodeling; and to find new potential targets that would suppress or regress the progression to myocardial dysfunction and heart failure.
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Engineering antibodies for diagnostic and therapeutic approaches in neurodegenerative diseases
By Ahmad NajjarEngineering antibodies for diagnostic and therapeutic approaches in neurodegenerative diseasesA. Najjar1, N.N. Vaikath2, I, Hmila2, Nour Majbour2, O. El-Agnaf11Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar.Neurodegenerative diseases affect millions of people worldwide, with Parkinson’s disease (PD) ranked as the second most common age-related neurodegenerative disorder affecting over 1 million people in the United States alone. Common neurodegenerative diseases such as PD, Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) are characterised by progressive deposition of α-synuclein (α-syn) protein within inclusions referred to as Lewy bodies and glial cytoplasmic inclusions respectively. This has led to classifying these diseases under the umbrella term Synucleinopathies due to the pathological accumulation of this protein. The various diseases vary in where the protein is deposited and which regions in the brain become affected.α-Syn is a relatively small protein constituting 140 amino acid residues having an unfolded native state. α-Syn aggregation occurs in a stepwise manner where monomers lead to transient oligomers, which ultimately lead to proto and mature fibrils within neurons. Such accumulation seems to target dopaminergic neurons located in the substantia nigra pars compacta. Current treatment such as Deep Brain Stimulation (DBS) and L-dopa (the precursor for dopamine) does not focus on slowing disease progression; rather it focuses on symptomatic relief.Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, immunotherapy holds much promise. α-Syn antibodies could potentially block processes leading to the pathogenesis of such neurodegenerative diseases. The limitation of such antibodies is their inefficiency in crossing the Blood-Brain Barrier. The aim of our project focuses on using a fusion protein engineered to include the FAB region of an existing antibody, which is confirmation specific to α-syn pathology. This single-chain-fragment-variable is designed to have increased BBB penetration by virtue of its smaller size and its conjugation with a carrier. It is envisaged that with enhanced penetration there will be superior brain targeting results compared to conventional α-syn antibodies. Upon expression and purification of α-syn and various designs of the fusion protein, the two proteins will be extensively characterized by means of Dot Blots, ELISA assays and affinity experiments. Neuronal cell lines and primary neurons from rat or mice will be employed to test our fusion protein in vitro. This will ultimately be done using immunocytochemistry techniques. For in-vivo experiments, the fusion protein will be tested on transgenic mice overexpressing α-syn.The outcomes of this project are threefold. If successful, the fusion protein could be useful in passive immunization of Synucleinopathies, finding its use as a clinical diagnostic tool for such diseases. Further manipulation of the fusion protein, namely attachment of a Fluoro-radiolabelled isotope can be used for imaging using positron emission tomography (PET). This would allow for its use in medical imaging to track α-syn pathology in PD, DLB and MSA patients.
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Generation and Charactarization of Mouse Monoclonal Antibodies Against Phosphorylated Alphasynuclein at Serine 129
More LessGeneration and Charactarization of Mouse Monoclonal Antibodies Against Phosphorylated Alpha-synuclein at Serine 129. Author and co-authors> details: Heba Al- Tarawneh1, Muneera Fayyad2, Nishant Vaikath3, Nour Majbour3, and Omar M. A. El-Agnaf2,3 1Weill Cornell Medical College in Qatar, Education City, Qatar. 2Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar 3Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer>s disease and one of the leading causes of disability worldwide. It manifests itself through several clinical symptoms, with the most prominent being motor impairment, resting tremors, rigidity and bradykinesia. PD is mainly characterized by a progressive degeneration of dopaminergic neurons in a region within the midbrain known as the Substantia Nigra. The major pathological hallmark of Parkinson’s disease is the appearance of proteinaceous inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs). These depositions are largely composed of a small 140-amino acid protein called alpha synuclein (α-syn). In fact, aggregation of α-syn has been implicated in a number of neurodegenerative diseases, collectively termed synucleinopathies. These include PD, Dementia with Lewy bodies and multiple system atrophy. Given the central role of α-syn in PD and related disorders, extensive efforts have been devoted in investigating the mechanisms that may regulate the aggregation and toxicity of this protein. In the last decade, an increasing number of studies has indicated that while only a small fraction of α-syn (<4%) is phosphorylated in healthy brains, the majority of α-syn within LBs was found to be phosphorylated at Serine 129 (>90%), raising the possibility that phosphorylation may play an active role in α-syn aggregation. However, findings are contradictory and this hypothesis remains unclear. To date, there are no diagnostic tools that enable early detection of PD. The only Confirmed diagnosis is based on clinical criteria, which are preceded by a prolonged phase of neurodegeneration and an irreversible loss of dopaminergic neurons. This has driven several studies to explore α-syn as a candidate biomarker for PD. Given the strong association of pS129-α-syn with LB pathology, measuring levels of pS129-α-syn in biological samples could serve as a potential biomarker for diagnosis of PD. In this study, we describe the generation and characterization of three mouse monoclonal antibodies (5B9, 6H5, and 9G1) that are specific for pS129 α-syn. These were generated using hybridoma technology and purified by protein-G agarose chromatography. Using a wide range of biochemical assays, we demonstrated the specificity of our new antibodies to pS129 α-syn. This highlights that our highly specific antibodies represent excellent research tools to investigate the role of α-syn phosphorylation in LB pathology. Moreover, our antibodies can be used to develop different quantitative and qualitative immunoassays. Also our antibodies can help answer whether phosphorylation at S129 suppresses or enhances α-syn aggregation and toxicity, which is crucial for the understanding of synucleinopathies pathogenesis and thus the development of new disease-modifying therapies for PD and related disorders.
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Projecting the Burden of Type 2 Diabetes Mellitus in Qatar: Analytical Insights
Authors: Susanne F. Awad, Martin O'Flaherty, Julia Critchley and Laith J. Abu-RaddadBackground: Extracellular matrix (ECM) remodeling is a characteristic feature of cardiac remodeling which, if left untreated, progresses to heart failure. Cardiac fibroblasts, the major cellular component of the myocardium, are responsible for maintaining the ECM integrity. Upon cardiac injury, such as myocardial infarction (MI), pressure or volume overload, cardiac fibroblasts transdifferentiate into cardiac myofibroblasts. Cardiac myofibroblasts are capable, of secreting proinflammatory cytokines and collagen; initiating tissue repair mechanisms (i.e., cardiac fibrosis). Hyaluronan (HA), a major component of the ECM, which is synthesized by hyaluronan synthase enzyme-2 (HAS-2), was shown to contribute in ECM remodeling and myocardial fibrosis by interacting with its cell surface receptor; CD44. CD44 is a transmembrane glycoprotein involved in multiple physiological and pathological conditions. Na+/H+ exchanger isoform-1 (NHE-1) a cardiac specific intracellular pH regulator, which is triggered by neurohormonal stimulation of Angiotensin II (ANG II), phenylephrine (PE), endothelin-1 (ET-1) or inflammatory mediators has also been implicated in cardiac remodeling. A previous study has shown that HA-CD44 interaction enhanced ECM remodeling in a non-cardiac model through activation of NHE-1 and hyaluronidase-2 (HYAL-2), a low pH-dependent enzyme which degrades hyaluronan polymers into lower molecular weight fragments. Yet, the link between NHE-1 and CD44 interaction in cardiac setting has not been addressed.Methods: CD44 expression was measured by qPCR and immunohistochemistry in transgenic mice expressing cardiac specific NHE-1. In vitro, normal human ventricular cardiac fibroblasts (NHCF-V) were treated with either 0.1 or 1 μM ANG II for 6, 24 or 48 hours to induce myofibroblast phenotype. Cell lysates and culture media were collected and analyzed by immunoblotting. Standard CD44 (CD44s), soluble CD44 (solCD44), unmodified CD44, HYAL-2 and HAS-2 protein expression were measured following ANG II treatment.Results: mRNA expression and immunohistochemistry data demonstrated that CD44 expressions were significantly elevated in heart tissues from transgenic mice expressing cardiac specific NHE-1 compared to wild type. Immunoblotting analysis of NHCF-V cell lysates showed a significant increase of CD44s protein expression, appearing at 75 KDa, following 0.1 μM ANG II treatment for 24 hours (131.14 ± 3.18 % ANG II vs. 100% control; P ≤ 0.01). CD44s protein expression did not show a change at 6-hour or 48-hour time point. Unmodified CD44, appearing at 37 kDa, HAYL-2 and HAS-2 protein expressions did not change following stimulation with various concentrations of ANG II at various time points. However, a trend towards increase was observed with solCD44 protein expression, appearing at 75 kDa, in NHCF-V conditioned media (CM) following 24-hour treatment with both 0.1 and 1 μM ANG II. A similar trend was observed with HYAL-2 protein expression in NHCF-V CM following 24-hour treatment with 1 μM ANG II, whereas there was no observed protein expression for HAS-2 and the unmodified form of CD44. Conclusion: ANG II, a well-established stimulator of NHE-1, is able stimulate the CD44s in NHCF-V cell lysates. Similarly, a trend towards increase of solCD44 expression was demonstrated in NHCF-V CM when treated with ANG II. This maybe due to the expression of CD44 in the extracellular space. HYAL-2 protein was also expressed in NHCF-V CM, while no change was detected in cell lysates. The protein and gene expressions of CD44, HYAL-2 and HAS-2 in the presence and absence of a NHE-1 inhibitor and ANG II type 1 receptor (AT-1) inhibitor remain unknown and need to be addressed to further understand the cellular interplay of NHE1 and CD44 in cardiac remodeling. It is of a significant importance to identify the signaling pathways leading to CD44 activation for better understanding of etiologies behind cardiac remodeling; and to find new potential targets that would suppress or regress the progression to myocardial dysfunction and heart failure.
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Nutritional Value of Kid's Meal offered by International Fast Food Restaurants in Qatar
More LessBackground The nutritional content of kid's meal in Qatar has often been overlooked. In our efforts to reach out to children of Qatar, their nutritional intake could be a nice place to start. The objectives of this study are to assess nutritional value of kid's meal offered by some international fast food restaurants in Qatar and to determine density of international fast food restaurant offering kid's meal by municipality areas. Methods We selected 6 international fast food restaurants that offer kid's meals and the density of these restaurants by kids' population aged 1-9 years old in Qatar. The selected fast food restaurants were McDonald's, Burger king, KFC, Hardee's, Papa john's, and DQ Grill & Chill. We assessed the number of branches of the restaurants using phone applications zomato and talabat. These applications allow the user to know the location and the numbers of branches in different municipality areas. We used also the official restaurants websites to collect data of the nutrient content of kids' meals offered by the 6-international restaurant in Qatar. Moreover, we used GPS to make sure of the location and the number of these restaurants. The entire restaurant offered 4 combinations of kid's meals except Papa John's offered 4 meals. We selected from each restaurant chain 25% of the total branches available in Qatar. During our visit, we asked for nutrition fact table of meals offered for kid's. Only McDonald's provide nutrition fact with meals for all customers. The other restaurants provide it only on websites or management department. We calculated the density by dividing the number of international fast food over the total children population aged 1-9 years old multiplying by 1000. SPSS and ANOVA (at p < 0.05) were used to analysis data. Results McDonald»s has the highest number of outlets which represent 41 out of 133 international fast food outlets offering kids meals and the DQ grill and chill had the lowest number of outlet which represent 5 out of 133. The average nutrients content of 23 kid»s meal offered by international fast food restaurants in Qatar was 653.30 ± 31.5 calories for calories contents, 21.61 ± 10.24 g for protein content, 28.32 ± 9.07 g for fat content, 84.57 ± 19.22 g for CHO content, 4.40 ± 2.79 g for fiber content and 968 ± 372.31 mg for sodium content. The percentage of covering the RDA calories recommendation of children aged 1-9 years old by 23 kid»s meals offered by six international fast food in Qatar was 47.8 ± 0.02% for calories, 134.6 ± 0.13% for protein, 58.9 ± 0.04% for fat, 63.6 ± 0.03% for CHO, 20.2 ± 0.03% for fiber and 91.4 ± 0.08% for sodium. In general, international fast food kid's meals are rich in carbohydrates, fat, protein, sodium and low in fiber. Conclusion Nowadays children are eating foods prepared outside the home more frequently than ever; improving the nutritional quality of food items offered at fast-food restaurants can contribute to important gains in population health. In this study, we found that fast food meals are nutritionally inadequate and its regular consumption is associated with excess intake of calories, fat, sodium, protein, carbohydrate, and low fiber intake.
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Reducing Heart Failure Admissions through Improved Care Systems and Processes
More LessHeart failure (HF) is a complex chronic condition that can result from any cardiac disorder that impairs the ventricle's ability to fill with or eject blood. The American Heart Association predicts that there will be about 10 million HF patients in the US by 2037, with total hospitalization costs exceeding $70 billion. This represents a considerable burden to hospitals nationwide, including the Massachusetts General Hospital (MGH) – a leading medical center that has long grappled with patient overcrowding and capacity constraints. This research project covers an extensive mapping of the HF care pathway at MGH, followed by the results of a detailed retrospective analysis of the general behavior of HF patients admitted to MGH. We notice that the majority of HF admissions originate as self-referrals via the Emergency Department (ED) and take place on weekdays, between the hours of 9am and 6pm. Moreover, we find that about 57% of hospitalized HF patients often have no scheduled follow-up appointments with their providers in the two weeks leading up to their admissions and, similarly, about 43% have no scheduled appointments in the eight weeks post hospital discharge. These represent two critical time periods in the events of acute heart failure decompensation. In an effort to prioritize targeted outpatient care, we propose a predictive model which aims to identify patients at greatest risk of a first hospital admission following encounters with their primary care providers and/or cardiologists in any given year. We perform logit-linear regressions on multiple prior first admissions and use predictors that, among others, include clinical risk factors, socioeconomic features and histories of prior medications. Some of the model's most significant predictors, as identified by the Akaike information criterion (AIC), include patient's age, marital status, ability to speak English, estimated average income, previous administration of loop diuretics, and the total number of medications prescribed or administered. To assess the quality of our predictions, we turn to the receiver operating characteristic (ROC) and its resulting average area under the curve (AUC) of 0.712. As the team continues to focus on developing interventions that offer better care to HF patients, the value of our model lies in its ability to prioritize patient needs for outpatient care and monitoring, and to guide the allocation of limited care resources.
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Implementation and feasibility study of a tailored health education bot in Telegram for mothers of children with obesity and overweight
Authors: Luis Fernandez-Luque, Abdelkader Lattab, Santiago Hors and MOHAMED AhmednaINTRODUCTION Obesity is one of the major health risk factors behind the rise of non-communicable conditions. Understanding the factors influencing obesity is very complex since there are many variables that can affect the health behaviors leading to it. Nowadays, multiple data sources can be used to study health behaviors, such as wearable sensors for physical activity and sleep, social media, mobile and health data. In this poster we describe a system which uses an off-the-shelf messaging app coupled with a recommender system to provide tailored health recommendations in arabic and/or english to mothers with overweight children in Qatar. This is part of the ICAN project. The ICAN project was funded by the Qatar National Research Fund (a member of Qatar Foundation) under project number NPRP X- 036- 3–013 (Adapted Cognitive Behavioral Approach to Addressing Overweight and Obesity among Qatari Youth). Childhood obesity is a growing epidemic, and with technological advancements, new tools can be used to monitor and analyze lifestyle factors leading to obesity, which in turn can help in timely health behavior modifications. In this paper we describe developed Telegram bot coupled with a recommender system to send educational messages using health recommendations. This bot allows automatic sending of messages that can be answered by the user. The answers can be ratings in a 1 to 5 star scale, or plain text, depending on the message. In a trial held in Qatar, an educational intervention for mothers using Telegram was carried out for a twelve-week period, which overlapped with the holy month of Ramadan and the school summer break. The goal was to keep the mothers motivated to actively work towards keeping their children healthy. Our nutritional advice took into account the religious month of Ramadan. METHODOLOGY We defined a pool of motivational messages for the mothers associated to different topics and challenges regarding the nutrition and physical activity of their children. The messages were created in English and Arabic language. A total of 24 keywords were defined. These keywords linked each message to features that define it (i.e. deals about vegetables, fats, religious quotes regarding healthy eating, healthy recipes, etc). A special set of 9 messages were specifically designed to be sent when users start the intervention. The answers to these messages define the initial user profile. Once an initial user profile is known, the user will start receiving messages on a weekly basis, one tailored message per week up to a maximum of 77 messages. Users can rate the messages based on their perceived usefulness. This feedback is stored in the user profile - user-keyword vector - so that the following messages that she receives contain keywords - message-keyword vector- that were included in previously rated messages. A total of 38 mothers, with children between 9 and 12 years old, joined the program to receive tailored messages across the entire summer based on their personal preferences. After each week, the system asked them whether they had completed the challenge, and their opinion about their difficulty, and usefulness. RESULTS During the 4 months of the intervention, about 500 messages were sent to the participants. The participants had a total of 94 challenges included in the messages that they could promise to accomplish. Of these, 11 challenges were totally completed, 42 challenges were almost completed, and 7 challenges had been given up by the mothers after they initially promised to do them. The data also shows that over 59 messages, only 17 were found easy to do, however 39 challenges were found ‘just fine’ to do. Over 59 challenges, 47 messages were found useful by mothers. CONCLUSIONS In this study we have tested the feasibility of a recommender system tailoring health messages delivered by an off-the-shelf messaging app as Telegram, and we consider the results show that the system achieved to rise some motivation from mothers to give a healthy diet to their children. However, further work is needed to assess how we can increase the engagement so that more of the proposed challenges are accepted, and completed, and also if to compare with groups of mothers that do not have the app in a formal randomized control trial.
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Development and characterization of monoclonal antibodies for Parkinson's Diseases and Related Disorders
More LessDevelopment and characterization of monoclonal antibodies for Parkinson’s Diseases and Related Disorders Najlaa Al-Thani1, Nour Majbour2, Muneera Fayyad3, N. Vaikath2, and Omar M. A. El-Agnaf2,3 1 Carnegie Mellon University in Qatar 2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. 3Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar Neurodegenerative diseases are the leading cause for disability in the world and one of the biggest burdens on our societies. Parkinson's Disease (PD) is the second most common neurodegenerative disease after Alzheimer's Disease (AD). PD is a chronic, progressive and irreversible disorder. The social and economic burden imposed by PD is significantly increasing as populations age. PD was first described by James Parkinson, an English physician, in his assay “Shaking Palsy” over 100 years ago. Although in less than a year PD will enter its next century, many aspects of the disease remain to be elucidated. PD can be sporadic or inherited, and in both cases it is accompanied by degeneration of dopaminergic neurons in the SN. Lewy bodies (LBs) and Lewy nurites (LNs) are the main pathological features of PD, where alpha-synuclein (α-syn) is the main component. α-Syn is a pre-synaptic neuronal protein and its aggregation and dysfunction is linked to a number of neurodegenerative disorders named as “synucleinopathies”. Synucleinopathies mainly refer to PD, dementia with Lewy bodies and multiple system atrophy. α-Syn is 140 amino-acid protein that is highly abundant in the brain and can also be found in red blood cells, plasma, cerebrospinal fluid (CSF) and saliva. The protein is natively unfolded, however, accumulating evidence indicate that enhanced oligomerization and aggregation of α-syn is associated with increased toxicity. Extensive efforts have been put into the elucidation of the mechanisms responsible for the polymerization and aggregation of α-syn. α-Syn undergoes several post-translational modifications such as phosphorylation, truncation or ubiquitination. A better understanding of the role of α-syn post-translational modifications will help to elucidate the exact role of α-syn in the pathogenesis of PD, paving the way to develop new diagnostic and therapeutic strategies for synucleinopathies. Truncated α-syn was found to be abundant in the brains of PD and DLB patients, suggesting that truncated α-syn may play a normal physiological role as well as a pathological one. C-terminal truncated α-syn as it exhibited a higher propensity to fibrilize in comparison with WT full length α-syn. In vitro studies that truncated α-syn specifically C-terminal residues 109-140 promoted aggregation presumably through nucleation formation. Antibodies that recognize both full length and c-terminally truncated a-synuclein are available, however, to study the role of c-terminally truncated a-synuclein in PD, specific antibodies are needed. In this project, we have generated and thoroughly characterized monoclonal antibodies against c-terminally truncated α-syn at Asn122. Following the selection of the stable clones, only the clones of IgG antibody were selected. All the clones were passaged multiple times to identify stable clones and subjected to single cell cloning to achieve monoclonality. The selected clones were cultured on a larger scale, mass culture, and the culture supernatant was then purified using Protein G affinity purification. The mAbs were highly selective for 122 α-syn, and didn't cross-react with 140 α-syn. The specificity and sensitivity of these antibodies were assessed by an array of biochemical methods including slot-blotting, western blotting, ELISA and immunohistochemical analysis. The potential of our antibodies can be explored as diagnostic or therapeutic tools for PD and related disorders.
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Development and Exploration of a Novel Personalized Cancer Vaccine Based on Virus-Like Particles (VLPs) by Incorporating Melanoma Specific T-cell Epitopes
Authors: Mona Omar Mohsen and Martin F. BachmannCancer Immunotherapy has significantly advanced cancer treatment modalities. However, therapeutic cancer vaccine aiming at reactivating anergic or dormant T cells has so far shown modest immune responses. VLPs have made notable strides in the last three decades in vaccine development field, mainly as prophylactic vaccines against VPV and HBV. The current project aims to design and build a multi-step platform for developing personalized therapeutic melanoma VLP-vaccine by integrating immunopeptidomics and exome sequencing approaches using melanoma patient-specific T-cell epitopes. To obtain a proof of concept in murine models, we have performed immunopeptidomics and exome sequencing for B16F10 melanoma cell line. The central goal of this platform (combining immunopeptidomics and exome sequencing) is to integrate the obtained data to develop a pipeline enabling the identification of mutated peptides which would constitute a promising cancer target for vaccine development. Our preliminary data of immunopeptidomics (alone) has shown large number of peptides which we have then prioritize and filter using bio-informatics and via probing with tumorinfiltrating lymphocytes (TILs). Peptides from immunopeptidomics were assessed for number of physical characteristics, such as their length and MHC-class I affinity as well as biological characteristics, such as being melanocyte specific, oncogenic or mutated. One of the key candidate was PMEL (gp100), a 100kDa type I transmembrane protein expressed in pigmented cells of skin and eye (tissue-specificity). A second promising candidate was MTC-1, an anti-oncogene that plays role in cell cycle regulation. If constitutively expressed, MTC-1 increases CDK4/6 kinases in breast cancer and promotes angiogenesis by inhibiting apoptosis. Furthermore, the selected peptides were synthesized and used to stimulate TILs to search for pre-existing immunity. TILs were labelled with CFSE and stimulated with the respective peptides (PMEL, MTC-1, no peptide or a mixture) and proliferation was assessed by determining CFSE dilution of CD8+ T-cells. Both peptides and -in particular the peptide mix- induced proliferation of T-cells, indicating that the transplanted tumor spontaneously induced T-cells against the respective epitopes. In the next step, we have prepared the vaccine using the bio-orthogonal copper-free click chemistry to couple the peptides of interest. Bio-orthogonal copper free click chemistry coupling method has shown efficacy, specificity and safety in our previous experiments. Bacteriophage Qb-VLPs were used and loaded with type-B CpGs which are known to be one of the most potent adjuvants in mice. The prepared vaccine cocktail was tested in C57BL/6 mice bearing B16F10 melanoma tumors with/without checkpoint inhibitors. Our preliminary results have shown significant regression in tumor size as well as significant production of IFN-g. We have also carried out extra analysis to characterize myeloid cells in the tumor microenvironment for each group to assess the effect of vaccination on myeloid cells in the tumor. The results indicated a notable decrease in TAMs characterized by (CD11b+/F4/80+ cells) in the group treated with vaccine+anti-PD1. This multi-step process can later be translated into the clinics and can be applied to other types of solid tumors. We are in the process of integrating exome sequencing data with immunopeptidomics aiming to identify mutated antigens which will increase the efficacy of the developed vaccine.
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Qatar Steps up to Global Health Security: A Reflection on the Joint External Evaluation 2016
Authors: Mohamed Osman Bala, Mohamad Chehab and Nagah Abdel Aziz SelimSince the commencement of the International Health Regulations (IHR, 2005) in 2007, global public health security has been faced with numerous emerging and ongoing events. Moreover, the Joint External Evaluation (JEE) is a voluntary tool developed in compliance with the Global Health Security Agenda (GHSA) that represents a reaction by the international health community towards the increased incidence of emerging and re-emerging diseases. Against this background, between 29th May and 2nd June 2016, a team of WHO consultants arrived to the State of Qatar to assess, in collaboration with national experts, the country's capacity to prevent, detect, and rapidly respond to threats of public health aspect. They identified areas of strength, weakness, and recommendations for improving national health security of Qatar in anticipation of the 2022 World Cup event. Qatar has demonstrated a leading role in the region through its commitment to International Health Regulations (2005) and community. Similarly, the Qatar was the first Arab state and seventh volunteering country globally to undergo the JEE process. In this review, we highlighted Qatar's achievements and shortcomings of IHR core capacities to inform healthcare professionals and the scientific community about the country's contribution toward global health security.
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Bioinformatics and Virtual Metabolomics Core infrastructure to support omics research in Qatar
Authors: Anna Halama, Shaza Zaghlool, Aditya Bhagwat, Michal Kulinski, Aishah Latiff and Karsten SuhreMetabolic profiling assembles the information on the small molecule (metabolites) composition in the body. There are several factors like genetic makeup, protein abundance, environment, life habits or gut microbiota, which all contribute to “metabolic fingerprint” - specific for each individual. Metabolomics can be deployed to draw a map of metabolic interactions associated with diseased condition; characteristic metabolic alterations can be used to monitor disease onset, progression or response to the treatment. For instance, alteration in branch chain amino acids, aromatic amino acids and lipids was associated with prediction of type 2 diabetes (T2D). Furthermore, elevated level of 1,5-anhydroglucitol (1,5-AG) in plasma and saliva samples was determined by us and by others as biomarker, complementary to Hba1C, for detection and monitoring of T2D. Metabolomics study requires expertise in experimental design, sample measurements and data analysis to produce meaningful, reliable and reproducible data. Metabolomics data can be further integrated with other “omics” including transcriptomics, proteomics, glycomics and methylomics. Here we are presenting the “Bioinformatics and Virtual Metabolomics Core”, an infrastructure, which we established in Qatar to enable metabolic profiling as well as analysis of highly complex “omics” data. To perform metabolomics study the following components are required: 1) Technology providing reliable data with high quality; 2) Pipeline for data processing; 3) Expertise in data interpretation. 1) We were actively involved in establishment of targeted and non-targeted metabolomics platforms in Qatar. Targeted platform: In collaboration with Translational Research Institute (TRI) we established targeted metabolomics platform able to quantify 163 metabolites including amino acids, carnitines, phosphatidylcholines, lysophospholipids and sphingomyelins. This platform is deploying Absolute IDQ Biocrates Kit technology for sample preparation and processing. The measurements are performed using flow injection analysis (FIA) - mass spectrometry (MS) technology based on combined ExigentEksperLC100 and QTRAP4500 (ABSCIEX). Evaluation of the mass spectrometry data is performed with MetIDQ software package (integral part of the Absolute IDQ assay). Untargeted platform: We were involved in the process of untargeted platform establishment. This platform is hosted and operated by Anti-Doping Lab Qatar (ADLQ) and enables for semi quantitative profiling of metabolites in human biofluids, tissue samples and cell culture. The protocols for sample processing and preparation were optimized by Metabolon and are conducted in Qatar accordingly. These measurements are deploying Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer. Compounds identification is performed by comparison of peaks to library entries of purified standards based on retention index (RI). 2) The metabolomics study generates large amount of highly complex data, which requires further data processing. We have developed an internal pipeline for data processing, including quality control and data normalization. For visualization purposes we use Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures (OPLS). Statistical data analysis is tailored according to the experimental design and data structure. We are using web-tools to enable our customer direct data access. We also developed a tool called “AUTONOMICS” for automated data analysis, which can serve more advanced users. 3) Our team is extensively working with metabolomics data; we are using Gaussian graphical modeling to reconstruct metabolic pathways and to draw network of interactions between metabolites. For pathway analysis and data interpretation we are using KEGG database, Ingenuity® Pathway Analysis (IPA®) and our experience. To provide detailed overview on human disease including diabetes, kidney and liver disorders as well as cancer we are combining metabolomics with other “omics” data. Our expertise lies in processing and evaluation of data from human biofluids (urine, plasma, saliva). The team is also specialized in metabolomics in cell culture; we developed a pipeline for cell culture-optimized sample collection and processing. In conclusion, our platform was established to enable fully comprehensive metabolomics study in Qatar, which will serve local scientific community. We are supporting our collaborators and users over the whole process related to metabolomics study including experimental design, sample processing, metabolite measurements and data analysis.
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Adipose Tissue Derived-Mesenchymal Stromal Cell: Setting the ground for Clinical Grade Production at Sidra's GMP Facility
Adipose Tissue Derived-Mesenchymal Stromal Cell: Setting the ground for Clinical Grade Production at Sidra's GMP Facility1Asma Al-Sulaiti, 1 Moza Al- Khulaifi, 1Sara Al-Khawaga, 1Zohreh Tatari Calderone, 1Bella S. Guerrouahen, 1Chiara Cugno. 1 Clinical Research Center, Research Division, Translational Medicine, Sidra Medicine, Qatar. Correspondence: Dr. Chiara Cugno, MD, Director Clinical Research Center, Research Division, Translational Medicine Sidra Medicine, Qatar. Keyword list: MSC, Adipose tissue, SVF, GMP, ISCT criteria. Word count: 702Background: Among adult stem cells, mesenchymal stromal cells (MSCs) represent a highly examined population given their exceptional biological properties. The International Society for Cellular Therapy (ISCT) define human MSCs based on the following criteria: adherence to plastic in standard culture condition, expression of the surface molecules CD105, CD90, and CD73, and lack of expression for CD34, CD45, HLA-DR ( < 2%), CD14 or CD11b, CD79a or CD19, and finally capability to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. The US National Institutes of Health database has reported 493 MSC-based clinical trials as of June 2015. Most clinical trials were implemented towards evaluating the biomedical potential of MSCs in treating graft versus host disease, hematological and inflammatory diseases diseases, organ transplantation, diabetes, and diseases in the liver, kidneys, and lungs, cardiovascular, bone and cartilage, neurological, and autoimmune disorders. Although multipotent MSCs are more commonly isolated from bone marrow (BM), adipose tissue-derived MSCs (AT-MSCs) represent a valid alternative source for MSCs due to their greater abundance, simple accessibility, and better immunomodulatory properties. Aim: We ultimately aim to develop the clinical-grade production of human AT-MSCs for Cellular Therapy at Sidra Good Manufacturig Practise (GMP) Facility. Our study represent the stepping stone for developing an MSC bank of high quality, reliable and well-characterized cells timely available for potential future clinical applications, such as Type 1 Diabetes (T1D). Objective 1 focuses on the isolation, expansion and characterisation of AT-MSCs. Objective 2 focuses on optimizing AT-MSCs production protocols to comply with the requirements of producing clinical-grade AT-MSCs. Methods: A) Sample collection: Adipose tissue is collected and obtained as waste material after liposuction procedures carried at Plastic Surgicenter, Doha. Aspirated fat is transferred to the Clinical Research Center at Sidra Medicine in sterile containers. B) Isolation and expansion of AT-MSCs: Both fat and blood/saline portion of lipoaspirate are used. AT-MSCs are isolated from the fatty portion, using a collagenase-based enzymatic method and from the blood / saline portion, using a non-enzymatic digestion. The isolated AT-MSCs are seeded and cultured in 75 cm2 flasks for expansion till passage 5. C) Phenotypic characterization, multilineage differentiation capacity and clonogenic assay: After reaching more than 95% confluence in the flasks at passage 1 (around 14 days in culture), phenotypic characterisation of MSCs is verified using a multi-color flow cytometry for the following specific antibodies: CD45, CD34, CD14, CD19, HLA-DR, CD105, CD73 and CD90. The adipogenesis and osteogenesis differentiation potential of AT-MSCs is also assessed at low passage. AT-MSCs are cultured under differentiating conditions in separate culture vessels. Cultures are processed for Oil Red O staining to evaluate adipogenesis differentiation (from day 7 to 14). Osteogenesis differentiation can be visualized after 21 days, using an Alizarin Red staining. To measure the clonogenic ability of AT-MSCs, Colony Forming Unit (CFU) is carried out using crystal violet staining. D) Translating AT-MSCs production to GMP facility: all protocols are optimized and standardized to comply the requirement of producing clinical-grade MSCs at Sidra Medicine GMP facility. The isolators (Biospherix XVIVO System) provides a strictly controlled environment that assures manufacturing of sterile, potent and uncontaminated products. It consists of modular sets of closed incubators and closed hoods, all integrated together as co-chambers and sub-chambers. The system is completely closed, with aseptic conditions throughout, and advanced controls as for GMP international standards. Results: AT-MSCs displayed a fibroblast-like morphology. The ISCT-recommended specific cell surface signature was verified: AT-MSCs were positive for CD105, CD90 and CD73 and negative for CD45, CD34, CD14, CD19 and HLA-DR. The biochemical assessment of adipogenic differentiation showed induced adipocytes with vacuoles containing fatty acids positive for Oil Red O staining. Alizarin Red staining showed induced osteocytes with calcium phosphate deposits. The stemness property of AT-MSCs was also confirmed. Conclusion: Recent studies have indicated MSCs as a powerful tool for several clinical applications. We are developing AT-MSCs clinical grade production at Sidra GMP Facility. We established standard operating procedures following the highest GMP international standards. Sidra GMP Facility is the first of its type in Qatar for cell therapy purposes, bringing future perspectives for the establishment of an AT-MSC bank from third-party healthy donors, after adequate screening for allogeneic donation, and a clinical trial in T1D to tackle the unmet need of preventative studies in this area.
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Prevalence of Impaired fasting glucose in Qatari women between aged of 18 to 40 years old from the Qatari Bio Bank
Authors: Youssra Dakroury, Abeer Elshewehy, Soha Dargham, Eric Kilpatrick, Lina Ahmed and Stephen AtkinBackground Impaired fasting glucose (IFG), prediabetes, has a conversion to diabetes of 10% yearly, contributing to the prevalence of diabetes of 16.7% in Qatar. This high prevalence of diabetes will translate into serious vascular complications with considerable individual early mortality.Methods The Qatar Biobank is an ambitious national project that is collecting the demographic and biological data on an unselected Qatari population that currently has data from 1100 women aged 18-84 years. Data extraction of all women between the aged 18 to 40 years (inclusive) was undertaken by the Qatar Bio bank that identified 750 women in total. The number of women with and without impaired fasting glucose (IFG: plasma glucose 5.5-6.9 mmol/l; ADA diagnostic criteria) was determined and their demographic details compared. Bio statistical analysis was undertaken using SPSS. Results The prevalence of IFG was found to be 8.3% (62/750 women). Patients with IFG had a higher cardiovascular risk profile with a higher waist hip ratio (P < 0.01), lower HDL cholesterol (p < 0.01), higher insulin levels (( < 0.01), higher pulse wave velocity (a marker of arterial stiffness, p < 0.01), higher systolic and diastolic blood pressure (p < 0.01 and p < 0.03, respectively). C reactive protein, a marker of inflammation was also elevated (p < 0.04).Conclusion Whilst this is a small cohort, 8.3% of women had a ADA diagnosis of IFG with an adverse cardiovascular risk parameter profile. This study highlights that there is a need for early screening for IFG and intervention for the prevention of diabetes in young Qatari women below the age of 40. Acknowledgments We are grateful to the Qatar Foundation and the Qatar Biobank for their support and assistance.
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CYP2C19 Genetic Polymorphism Prevalence in Qataris
Authors: Hazem Elewa, Zainab Omer Ali and Loulia BaderBackground Clopidogrel is used by around 40 million patients worldwide to treat and prevent thrombotic events. Third of the patients have clopidogrel resistance which may lead to treatment failure. cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) is one of the key enzymes involved in the hepatic bio-activation of clopidogrel. Several studies have shown that polymorphisms in the gene encoding the CYP2C19 contribute to variability in response to clopidogrel. There is no available data on the distribution of CYP2C19 genetic polymorphisms in the Qatari population. Objective The objective of this study was to estimate the distribution of CYP2C19 genetic polymorphisms in Qatari population by determining the prevalence of CYP2C19*2, *3 and *17 alleles. Methods The study was conducted on a cohort of 129 adult Qatari individuals. DNA was extracted mainly from saliva samples by Oragene® self-collection kit and purified using the prepIT™√L2P purification protocol (DNA Genotek, Inc., Canada). For those who were unable to provide saliva, blood sample was collected. Five milliliters of peripheral blood were collected in test tubes containing EDTA and DNA was extracted using PureLink® genomic DNA extraction kit for purification of genomic DNA (Invitrogen life technologies, Germany). The amount and purity of the DNA samples were quantified using Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Genotyping was performed for CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), and CYP2C19*17 (rs12248560) using Fast 7500 Real-Time PCR System (Applied Biosystems™, USA) with TaqMan assays according to the manufacturer's instructions. Genotype frequencies were tested for deviations from Hardy–Weinberg equilibrium through chi-square analysis at P value of 0.05. Patients were categorized according to their CYP2C19 genotype into: ultra-rapid (*1/*17, *17/*17); extensive (*1/*1); intermediate (*1/*2, *1/*3, *2/*17); poor (*2/*2, *2/*3, *3/*3) metabolizers. Results This study included 129 Qatari individuals. Majority of our patients were 50 years and above (60%) and mostly females (63%). The allele frequencies were CYP2C19*1 (86 %), *2 (4%), *3 (0%) and *17 (10%). Fifty-eight subjects (45%) were homozygous for the *1/*1 genotype. Heterozygous genotypes were identified in 62 patients (48.1%), 42 carrying *1/*17 (32.6%), 12 carrying CYP2C19 *1/*2 (9.3%), and 8 carrying CYP2C19 *2/*17 (6.2%). Six patients were homozygous for the gain-of-function allele, CYP2C19 *17/*17 (4.7%). On the other hand, three patients were homozygous for the loss-of-function allele, CYP2C19 *2/*2 (2.3%). However, no subject was found to have *3 allele. All the genotypes were in Hardy–Weinberg equilibrium. The distribution of CYP2C19 phenotypes was divided into extensive metabolizers (*1/*1) (45%), ultra-rapid metabolizers (*1/*17, *17/*17) (37.2%), intermediate metabolizers (*1/*2, *1/*3, *2/*17) (15.5%), and poor metabolizers (*2/*2) (2.3%). Conclusion To the best of our knowledge, this is the first study to report the distribution of CP2C19 genetic polymorphisms in Qatari population. These findings have important clinical implications for the use of clopidogrel and other medications affected by CYP2C19 mutations in Qatari population. Thus, future association studies are needed to reveal clinical consequence of these genetic polymorphisms in Qataris.
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Combination novel modalities for enlightening global molecular elemental ultrastructure and morphological biochanges in subacute stroke in rat brain
By Mohamed AliBackground and Purpose: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Novel and advanced diagnostic modalities are required to improve treatment in order to reduce the social and economic burden of this disease. Understanding the post-stroke bio-chemical changes is critical for patient treatment and survival. Unfortunately, conventional diagnostic procedures are used to investigate global changes in the stroke brain, rather than understanding the bio-chemical changes at the molecular and cellular level. Therefore, in the current study we used combined approaches to investigate the molecular, elemental, ultrastructure and morphological changes in stroke brain. Methods: Application of Fourier transform infrared (FT-IR) spectroscopy; laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS); transmission electron microscope (TEM), Raman micro-spectroscopy and atomic force microscopy (AFM) were used to examine stroke brain and were compared with the standard approach of immunohistochemistry. The areas investigated, one-week post sub-acute photo-thrombotic ischemic stroke in male rats, were primary stroke lesion gray matter (PS-GM), perilesional gray matter (PL-GM), lesioned white matter (L-WM), contra-lesioned white matter (CL-WM), contra-lesioned gray matter (CL-GM) corpus callosum (CC) and dorso-ventral cortices (DVC). Results: The infrared spectroscopy results revealed that total lipid and protein and the lipid CH2 content decreased significantly in the (PS-GM) while the total aggregated protein increased significantly relative to total protein, in particular we detected an increase in Aβ1-42. In contrast, both (PL-GM) and (L-WM) regions experienced an increase in carbonyl esters, olefinic = CH, and the CH3 groups of lipids. In contrast, both (PL-GM) and (L-WM) regions experienced an increase of carbonyl esters, olefinic = CH, and CH3 groups of lipids which is associated with decrease in CH2 lipid groups and lipid contents. Amongst the novel findings of the study was that the aggregated protein detected in the L-WM was Aβ1-42. FT-Raman micro-spectroscopy showed a reduction in lipid content and iron accumulation around the stroke lesion in the form of heme. Elemental analyses for divalent cations: Ca, Cu, Fe and Zn realized major changes in the different brain structures that may underscore functionality within these regions. In the elemental maps high Fe intensity around the stroke region correlated well with the presence of heme shown by Raman spectroscopy. Conclusions: The complementary methodologies revealed, at multiple-levels, the effects of stroke: edema, lipid peroxidation, as detected by increased olefinic = CH and carbonyl ester content, and enhanced protein aggregation. For the latter, an example was the identification of Aβ1-42 in the L-WM. These data point to the possibility that amyloid plaque formation is not exclusive to cortical neurons, but also affects the WM. One conclusion being that application of strategies developed for AD-treatment may be fruitful in treating stroke. Alternative therapies would include targeting edema, antagonizing lipid peroxidation for the protection of the blood brain barrier (BBB) post-stroke by the application of antioxidants. Alternatively application of free Fe sequestering molecules, as FT-Raman micro-spectroscopy detected the reduction in the lipid content and the iron accumulation around the stroke lesion.
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MitochondrialDerived Peptide MOTSc promotes hepatic fatty acid metabolism and regulation by metformin
More LessMitochondrial-Derived Peptide MOTS-c promotes hepatic fatty acid metabolism and regulation by metformin. Ramanjaneya M1, Jerobin J1, Bettahi I1, Sivaraman SK1, ¬ Mohammad RM1, Skarulis MC1 and Abou-Samra AB1. 1Translational Research Institute, Academic Health System and Department of Medicine, Hamad Medical Corporation, Doha, Qatar. Objectives: Mitochondrial dysfunction contributes to the pathogenesis of metabolic disorders and is a leading cause for development of insulin resistance. This is promoted, at least partly, through lipid accumulation in ectopic tissues, including liver (non-alcoholic fatty liver disease) and skeletal muscle. The role of mitochondria as functional organelles, and the signaling molecules produced by them are critical for cellular energy homeostasis. Dysfunction in mitochondria contributes to the pathogenesis of metabolic disorders. MOTS-c (mitochondrial open reading frame of the 12S rRNA type-c) encoding a 16-amino-acid peptide is a recently identified peptide. The skeletal muscle appears to be the main target organ for MOTS-c and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, leading to AMPK activation. MOTS-c promotes glucose utilization, insulin sensitivity and metabolic homeostasis through activation of AMPK dependent mechanisms in skeletal muscle. MOTS-c protects rodents against diet and ageing induced insulin resistance. However the role of MOTS-c hepatocytes is unknown. In this current study we aimed to study the functional importance of MOTS-c on liver fatty acid metabolism in using HepG2 hepatocytes model. Methods: Analysis of MOTS-c gene expression in human tissue panel: Human tissue cDNA panel was obtained from Clontech. MOTS-c gene expression in various human tissues was measured by real-time PCR. Human hepatocytes (HepG2) cells were used for this study: Cells were maintained as subconfluent monolayers in DMEM (Invitrogen) with 10% fetal bovine serum and 100 units/ml penicillin plus 100 μg/ml streptomycin (Invitrogen) at 37 °C with 5% CO2. For all stimulation experiments cells were grown for overnight in 2% serum containing media. Fatty acid-induced steatosis: Approximately 80% confluence were pretreated with MOTS-c and further stimulated for 24 hrs with oleic acid and palmitic acid 1:2 ratio in 2%-serum medium for 24 hrs in 12-well culture plate. Following this incubated with nile red solution for 20 mins and then subjected to flow cytometric analysis to measure lipid accumulation in HepG2 cells. Control cells were treated with fatty acid-free medium containing albumin. Effects of antidiabetic drugs on MOTS-c expression: Approximately about 80% confluent cells were stimulated with commonly used antidiabetic drugs insulin 100 nM, rosiglitazone 2 mM or metformin 1 mM for 24 in 2%-serum medium 12-well culture plate. Following this MOTS-c secretion was measured using commercially available elisa kits. Results: MOTS-c gene transcripts are expressed in human liver and HepG2 cells. Stimulation of HepG2 cells with MOTS-c suppresses fatty acid synthase (FAS), sterol regulatory element-binding protein-1c (SREBP1c) and acetyl-CoA carboxylase (ACC) the key genes required for hepatic lipid production. Further MOTS-c reduced fatty acid induced hepatic lipid accumulation. Conclusion: Our primary result indicates that MOTS-c promotes hepatic fatty acid metabolism through down-regulation of multiple genes involved in fatty acid metabolism in liver. Further studies are in progress to assess the effects of anti-diabetic drugs such as metformin, insulin and rosiglitazone on MOTS-c secretion. The findings from this study are of clinical importance given that non-alcoholic liver disease (NAFLD) is highly prevalent in obese and diabetes subjects in Qatar. MOTS-c and its agonists have potential to be used as therapeutic agents for treatment of NAFLD.
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Foreskin DerivedMesenchymal Stromal Cell FSKMSC: Setting the ground for the Clinical Grade Production of MSC at Sidra's GMP Facility
Moza Al- Khulaifi1, Asma Al-Sulaiti1, Bella S. Guerrouahen1, Sara Al-Khawaga1, Chiara Cugno1, Zohreh Tatari Calderone1 1 Clinical Research Center, Research Division, Translational Medicine, Sidra Medicine, Qatar. Correspondence: Dr. Zohreh Tatari Calderone; Clinical Research Center, Research Division, Translational Medicine Sidra Medicine, Qatar. Email: [email protected] Keyword list: MSC, Foreskin, GMP, ISCT criteria. Abstract 1. Background: Mesenchymal stromal cells (MSCs) are known for their multipotent, immune-privileged and regenerative properties. MSCs can be obtained from different sources such as bone marrow, fat, umbilical cord, synovial fluid, tendons, muscles, periosteum, peripheral blood and nervous system. The International Society for Cellular Therapy (ISCT) has stated minimal characteristics for defining the MSCs as multipotent fibroblast-like cells highly adherent to plastic, positive for CD73, CD105 and CD90 but negative for CD45, CD34, CD14 or CD11b, CD19 or CD79a, and HLA-DR. The MSCs constitute a promising tool for regenerative medicine, due to their self-renewal capacity, multilineage differentiation potential, paracrine effects and immunosuppressive properties. They are extensively used in the treatment of several clinical settings including but not limited to, cardiovascular diseases, inflammatory bowel diseases, liver dysfunction, rheumatological disorders and diabetes. Bone Marrow-derived MSCs has been considered as the gold standard for cell-based therapy, but bone marrow aspiration is an invasive method that limits the access to a large number of MSC donor sources. Recently, the foreskin, which has been always retained as medical waste, has been recently shown as a source for MSCs. Sidra Medicine is performing 4000- 5000 circumcisions annually, therefore foreskin can be considered as a highly abundant and easily accessible source for the isolation of MSCs. Therefore, the aim of our study is: Specific Aim: To demonstrate that MSC isolated from foreskins (FSK-MSC) meet the high standard criteria of ISCT and to compare their phenotype, potency and multilineage potential with MSCs obtained from a classical source such as adipose tissue for the clinical application. 2. Method: Specimen Collection and MSC Isolation: Upon approval by the IRB committee, foreskins were collected after the circumcision from the Surgery Department at Sidra Medicine (age range: 1.5 -14 years old). Epidermis was manually separated from the dermis and was then subjected to enzymatic digestion by liberase. Cell suspension was cultured in T75 flasks containing low-Glucose media, complemented with 10% fetal bovine serum and 1% antibiotics for 3-5 days. After 3-5 days, the non-adherent cells were removed and fresh media was added to allow the adherent MSCs to grow. MSC Characterization:Cell Surface expression: Phenotypic characterization was determined using flow cytometry. Briefly cells were stained for the surface markers using fluorochrome conjugated antibodies for CD14, CD19, CD34, HLA-DR, CD45, CD73, CD105 and CD90 according to the ISCT criteria. Differentiation: FSK-MSCs were treated with osteogenic and adipogenic mediums in separate cultures for 7, 14 and 21 days and stained with Alizarin Red and Oil Red O staining respectively. The lineage differentiation was visualized using the inverted microscopy. Colony Forming Units (CFU): Colony formations Units measures the viable clonogenic cell numbers that is an important indication of the quality of cell preparation. The CFU assay was performed according to commonly used protocol and using crystal violet staining. 3. Results: FSK-MSC displayed a typical fibroblast-like morphology and an ISCT-compliant phenotype; FSK-MSCs were highly proliferative and had a marked clonogenic potential. FSK-MSCs displayed osteogenic and adipogenic differentiation capacities. 4. Conclusion: The isolated FSK-MSCs were shown to be compliant with ISCT criteria and can be considered as a reliable source of MSC. Their compliance allows considering the standardization of clinical grade FSK-MSCs production at Sidra Medicine GMP facility, and setting the ground for the potential establishment of a MSC biorepository.
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Effect of nonnutritive sweetener stevioside on regulation of adipogenesis and oxidative stress in adipocytes
More LessEffect of non-nutritive sweetener stevioside on regulation of adipogenesis and oxidative stress in adipocytes Jayakumar Jerobin, Manjunath Ramanjaneya, Ilham Bettahi, Siveen Kodappully Sivaraman, Ramzi M Mohammad, Monica Skarulis Young, Abdul Badi Abou Samra Interim Translational Research Institute, Qatar Metabolic Research Institute & Department of Medicine, Hamad Medical Corporation, Doha, Qatar. Introduction The metabolic syndrome which includes obesity, diabetes, polycystic ovarian syndrome and non-alcoholic fatty liver disease is a major health risk in Middle Eastern countries including Qatar. The metabolic syndrome is worsened by unhealthy diet and sedentary lifestyle. Both nutritive and nonnutritive sweeteners (NNS) are consumed by people worldwide, and the consumption of sweeteners above the recommended level contributes to metabolic syndrome. In adipocytes, artificial NNS such as saccharin and acesulfame potassium promotes adipogenesis and suppresses adipose tissue lipolysis. The consumption of artificial NNS saccharin modulates the gut microbiota and promotes glucose intolerance. Currently the artificial NNS are replaced with natural NNS stevioside in foods and beverages since it has low caloric value. Studies have shown that stevioside possess glucose uptake properties which may have a potential role in type 2 diabetes mellitus treatment. This proposed study mainly focuses on the effect of NNS stevioside on regulation of adipogenesis and oxidative stress in matured adipocytes. Methods The 3T3L1 preadipocytes cells were cultured in DMEM medium, and the preadipocyte cells were differentiated in to mature adipocytes by the addition of IBMX, Insulin and Dexamethasone on 0th day. It was replaced with maintenance medium containing insulin on 2,4 and 6th days of differentiation. The stevioside at different dosage (10, 100 and 200 μM) were treated on 0, 2, 4 and 6th days of differentiation in matured adipocytes. Cell viability was analyzed using MTS solution. Oil red¬ O staining was carried out to study the accumulation of lipids in the undifferentiated and matured adipocyte cells. The different oxidative stress markers such as Reactive oxygen species (ROS) and reduced Glutathione were measured using flow cytometry. Western blot and gene expression studies were carried out to study the adipogenesis and insulin sensitivity markers. Results The stevioside was found to be non-toxic to both preadipocyte and adipocytes. On eighth day of differentiation, a significant increase in the accumulation of lipids was observed in the differentiated adipocytes compared to the undifferentiated preadipocytes as analyzed by Oil Red O. The generation of oxidative stress was higher in adipocytes compared to the undifferentiated adipocytes. This was confirmed by increase in the level of reactive oxygen species radicals and reduced glutathione in untreated adipocytes. Treatment with stevioside resulted in inhibition of oxidative stress markers in a dose dependent manner. The protein expression level of early adipogenesis marker such as PPAR-γ and C/EBP-alpha was significantly decreased in stevioside treated adipocytes. Adiponectin, an adipokine found to have association with insulin sensitivity was significantly increased in stevioside treated adipocytes. The protein and gene expression levels of GLUT4 was also found to be significantly increased in stevioside treated matured adipocytes. Conclusion Stevioside reduces oxidative stress by modulating the level of free radicals in maturing adipocytes. Adiponectin an insulin sensitizer which was found to be lower in obese individuals was significantly upregulated in stevioside treated adipocytes. Similarly, the increase in the expression of insulin regulated glucose transporter GLUT-4 in adipocytes indicates that stevioside may possess insulin sensitivity properties in matured adipocytes.
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Generating an imagingbased approach for enhanced structural and functional analysis of zebrafish cardiovascular systems
Authors: Huseyin Yalcin, UmmaySalma Abuhabib, Naila Kitaz, Ala Mohamed and Zain ZakariaCardiovascular diseases (CVDs) have latterly become one of the leading cause of death and impairment worldwide. According to the World Health Organization (WHO), data estimation in 2008 were about 17.3, 7.3 and 6.2 million death cases for CVDs, heart attacks, and strokes, respectively [1]. Congenital heart defects (CHDs) are important forms of CVDs affecting about 1% of the population. According to Dr. Ahmad Sallehuddin (Consultant and Chief of Pediatric Cardiac Surgery at HMC), CHD incidence in Qatar is about 6-8 in every 1,000 births and about 100 new patients with CHDs require surgery every year [4]. Both genetic and environmental factors were shown to contribute to CVDs. Human genetic studies are not sufficient alone to explain the genetic basis of CVDs due to disease heterogeneity, inconsistent penetrance, and predominantly a delayed onset of symptoms. Therefore, animal models are necessary to investigate and distinguish novel genes that contribute to the pathology of CVDs and also to unravel environmental factors that play role [5]. More recently, the Danio rerio (Zebrafish) has emerged to become an intriguing vertebral model in medical science research. This model has several advantages, such as an almost entirely sequenced genome that is highly preserved with humans: approximately 70% of the human genes are estimated to have orthologue genes in the zebrafish genome [2,8]. The features of the zebrafish over mammals as a vertebral model include its simplicity of genetic manipulation, a large quantity of offspring, and their external and fast development [5,8]. Transparency of zebrafish embryos provide precise observation of the heart beats, heart chambers and circulating blood in vivo via light microscopy [5]. Additionally, passive diffusion is sufficient for oxygen delivery during early stages of zebrafish embryos development because they are independent of circulatory system [7]. Therefore, embryos having intensive cardiac faults survive early development, which enables investigation of mutations whereas mammalian models are not appropriate. There are many genetic cardiovascular models for zebrafish that allow researchers to investigate cardiovascular diseases. Novel techniques are needed for these models to assess comprehensive and quantitative phenotyping of mutant hearts and blood vessels. These analyses involve determination of the cardiac atrial and ventricular shortening fractions, examination of the blood flow velocities and recording of the electrocardiograms. In addition to genetic studies, cardiotoxicity investigations for drugs or teratogens necessitate heart function and morphology assessment. For the current study, the aim is to adapt previously established experimental and image analysis techniques to zebrafish studies, which will enhance structural and functional analysis of the zebrafish cardiovascular systems in biomedical research Methodology: This project involves generation of two main protocols, which are for structural and functional analysis of zebrafish embryos. Both these analyses are required to be performed to assess the severity of induced heart defects in zebrafish embryos. For the structural analysis, we will perfuse zebrafish (3 -5 days post fertilization (dpf)) hearts with microfilm, a CT- dense agent. Once it polymerises, a cast is generated for heart lumen. The embryos are then fixed with paraformaldehyde and scanned via micro-CT. This procedure provides 3D images for the cardiovascular systems of the animals enabling measurement of the volumes of the heart chambers. This technique enables us to measure the effect of induced heart mutations on the heart morphology during the embryonic growth of the animal. We have previously used this technique for visualization of cardiac chambers for embryonic chicken successfully [3,10]. The approach is adapted for zebrafish studies in the current work. For the functional analysis, image sequences of the beating ventricles and the red blood cell (RBC) movements of the zebrafish larvae (3 -5 dpf) are recorded at a camera speed of 70 fps and above. This imaging is done by taking videos using Micromanager Program that is connected to a stereomicroscope. To take image sequences, zebrafish larvae are placed on its lateral side, in which the right side will be on top. A variety of measurements and calculations are performed using Image J Program to assess heart functions. The parameters that are calculated include myocardial wall velocities for assessment of severity of induced heart muscle defects, and measurement of RBC blood flow velocities for assessment of rhythmic beating defects of the heart. Other parameters are fractional area change, fractional shortening, heart rate, stroke volume, cardiac output and ejection fraction [9]. Stroke volume, is the blood volume that is ejected from the heart in each beat, cardiac output, is the amount of circulating blood across the heart in each minute, and ejection fraction, is the measurement of blood that is ejected from the ventricle with each beat. This will provide more precise and accurate evaluation on the heart function for genetically mutated animals to be compared with the normal ones. Several zebrafish mutants display phenotypic features resembling human cardiac diseases. These mutants whose molecular damages have been determined like mutations in regulatory myosin light chain, titin, cardiac troponin T, essential myosin light chain, and beta-myosin heavy chain, have been linked with cardiomyopathy in humans [6]. Therefore, reduced contractility, myocardial wall velocities, and cardiac output can be studied as a sign of having cardiomyopathy. Accordingly, opening ways to apply drugs for morphological and structural evaluation of the heart. In conclusion, in this study, we are developing imaging-based techniques for accurate structural and functional analysis of the zebrafish hearts through adapting previously established methods on another animal systems. Once we establish our approach, analysis methods will be readily available for other Zebrafish researchers.
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