Qatar Foundation Annual Research Forum Volume 2013 Issue 1

Various betaine materials - single molecules or polymers - represent one of the best tools for prevention of biofouling on surfaces due to their biomimetic character and unique properties [1]. Betaines consist of internal salts in a single unit between positive quaternary ammonium and negative sulfo, carboxy or phosphate groups. This contribution highlights our progress in preparation and application of betaine materials to resist non-specific interactions on a surface of biosensors and membranes. Various betaine derivates with sulfo- [2] or carboxylbetaine groups were synthetized from lipoic acid as a natural precursor. Disulfide moiety from lipoic acid structure assists in attachment of a betaine derivative to a gold surface applied for preparation of biosensors. Biosensor devices were characterized by a set of tools including electrochemical impedance spectroscopy, FTIR, AFM, XPS, QCM, etc. Betaine derivatives introduced to the surface of various biosensors allowed their effective work in complex samples such as human plasma with a high reliability of detection and with a detection limit down to a femtomolar level for detection of specific glycoproteins. Carboxybetaine derivate provided a dual function for preparing biosensors i.e. a non biofouling surface and introduction of functional groups for covalent immobilization of sensoric biomolecules after performing a NHS/EDC coupling chemistry, as well. Moreover, formation, characterization and application of nanofibres and hydrogel from sulfo and carboxybetaine polymer as a membrane precursor will be discussed. Optimal formation of nanofibres was performed via an electrospinning process from trifluoroethanol solution. Characterization of membranes prepared was done by water sorption, FTIR, AFM and SEM techniques. Preliminarily proteins and cells attachment tests showed a dramatic decrease in adsorption and adhesion. Keywords: betaines, biomimetic, biosensor Acknowledgement: The financial support from the Slovak research and development agency APVV 0282-11 is acknowledged. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement No. 311532 References: [1] P. Sobolciak, I. Lacik, P. Kasak Chem. Papers, 2011, 105, 918-925. [2] T. Bertok, L. Klukova, A. Sediva, P. Kasak, V. Semak, M. Micusik, M. Omastova, L. Chovanová, M. Vlcek, R. Imrich, A. Vikartovska, J. Tkac Anal. Chemistry 2013, 85, 7324-7332.

Background: The efficacy of medical male circumcision (MMC) in reducing female to male HIV transmission has been established by three clinical trials. Programs are being developed for massive roll-out of MMC as an HIV intervention across multiple countries in sub-Saharan Africa (SSA). We aimed in this study to explore generic results of the effectiveness of MMC as an HIV intervention. Method: A population-level deterministic model was constructed to describe a prototype heterosexual HIV epidemic in SSA. The model consists of a set of coupled nonlinear differential equations that stratifies the population into compartments according to sex, circumcision status, age group, sexual risk group, and HIV status and stage of infection. The model was parameterized by state of the art empirical data of HIV natural history and transmission. Sensitivity analyses with respect to different effects were conducted. Results: The effectiveness of MMC, defined as the number of MMCs needed to avert one HIV infection, was inversely proportional to HIV prevalence with as little as about 10 MMCs needed for each infection averted in settings at high HIV prevalence. The effectiveness also varied substantially with each of the 5-years age groups targeted, with the highest effectiveness found by targeting the 20-24 years age group. Risk targeting strongly affected the estimated effectiveness. MMC effectiveness was an order of magnitude higher by targeting the high-risk groups as opposed to the general population. The speed of scale-up influenced also the estimated effectiveness in the short-term, but was less influential for long-term time horizons. MMC was found to be generally cost-effective, with the cost per infection averted being less than $1000 for most settings. Conclusion: Our findings demonstrate that MMC is an effective HIV prevention intervention, and likely to be cost-effective in most settings in SSA. The highest MMC effectiveness is attained by targeting young sexually active males and high-risk groups. These generic results of MMC effectiveness suggest the need for custom-designed assessments for each country separately, taking into account the likely temporal evolution of the epidemic in the next few decades, the age and geographic distribution of HIV infection, population pyramid, and the country-specific cost for each MMC performed.

Back ground The volume of cardiac diagnostic procedures involving the use of ionizing radiation has increased rapidly in recent years. Coronary computed tomography angiography (CCTA) has been increasingly used in the diagnosis of coronary artery disease (CAD). The CT is an excellent technique to make a diagnosis of disorders in many different parts of the body, however, because the heart is constantly beating, CT scan could never be used to image it in the past. The main advantages of coronary CT is non-invasiveness, rapid acquisition of high-resolution images and high diagnostic accuracy .With rapid improvements in spatial and temporal resolution, CCTA not only visualizes coronary anatomy and characterizes plaque components, but also allows for quantitative analysis of coronary stenosis. OBJECTIVE. The purpose of this study was to assess and compare the radiation doses of different coronary CTA (CTA) protocols: second-generation dual-source 128-MDCT, and single-source 64-MDCT. MATERIALS AND METHODS. CT Systems and Protocols Coronary CTA protocols used with two different systems were evaluated: a single-source 64-MDCT scanner with adaptive section collimation (Somatom Definition 64 AS), and a second-generation dual-source 128-MDCT scanner (Somatom Definition Flash). DISCUSSIONS This study highlights two important findings in the radiation dose associated with the prospective ECG-triggered CCTA. Firstly, a low radiation dose can be achieved in CCTA between different generations of CT scanners with the application of the prospective ECG-triggering protocol. Secondly, BMI affects the radiation dose significantly. RESULTS. Regardless of coronary CTA protocol and CT system, imaging at 100 kV lowered the ED 40-50%. In retrospectively gated 120-kV coronary CTA, the ED ranged from 4.4-21 mSv and in prospectively triggered 120-kV step and shoot coronary CTA, the ED ranged from 3.08- to 7.8 mSv. The lowest ED of all protocols (1.2 mSv) was observed in prospectively triggered high-pitch 100-kV coronary CTA performed with dual-source 128-MDCT. Patient measurements showed similar dose reductions for prospective triggering and low voltage settings without an influence on signal-to-noise ratio or image quality. CONCLUSION. In conclusion, a low radiation dose can be achieved in a low and regular heart rate with a prospective ECG-triggering protocol, regardless of the CT scanner generation. Although there is no significant difference in the effective dose between genders, BMI is identified as the main factor that significantly affects the radiation dose in prospective ECG-triggered CCTA in this study. A combination of prospective triggering with low voltage settings and high pitch is an effective measure for reducing the ED of coronary CTA to values of 1.2-4.8 mSv. it offers a dose reduction potential of more than 80%, which to our knowledge has not been achieved with any other coronary CTA technique. the High-pitch helical coronary CTA is associated with a very low radiation dose is one of the good option for any general coronary screening with the limitation of single phase images and the prospective sequencial mode is almost (95%) suit for all patients with bit more radiation dose than high pitch with the advantage of multiphase and diagnostic accuracy.

Heart failure (HF) is one of the most common causes for death, to prevent HF the understanding of the (patho-)physiology is key and the development of efficient tools is an important contribution for cardiac research. Cardiac tissue slice is an increasingly popular model for cardiac electrophysiology research and pharmacological compound testing. We use optical mapping to investigate cardiac tissue slices. In this study, we perform dual imaging of trans-membrane potential (Vm) and the intracellular free calcium concentration transient (CaT) in tissue slices from the rabbit, using a relatively high spatio-temporal resolution. We detail our method for processing of the data to extract relevant characteristics of the action potential (AP) and CaT. We found that slices needed a recovery time of about 40-70 minutes after cutting, before the AP reaches a steady-state. To characterise the CaT, AP and conduction properties, we used a combination of multi-point and field stimulation, to avoid results biased by source-sink mismatches. The tight control of experimental conditions (e.g. slice 'recovery protocol' and stimulation method), leads to reproducible results, this was shown in our other studies based on using this standardised methodology [1,2]. The monitoring of multiple parameters (Vm, CaT) simultaneously at high spatial resolution (compared to patch clamp, sharp electrode, or multi-electrode arrays) allows one to study not only the properties of each parameter in a spatially detailed manner, but also the spatio-temporal interrelation between them. Especially for drug safety studies this method is superior to lower resolution methods. References: 1. Lee P, Klos M, Bollensdorff C, Hou L, Ewart P, Kamp TJ, Zhang J, Bizy A, Guerrero-Serna G, Kohl P, Jalife J, Herron TJ (2012) Simultaneous Voltage and Calcium Mapping of Genetically Purified Human Induced Pluripotent Stem Cell-Derived Cardiac Myocyte Monolayers. Circ Res 2. Lee P, Wang K, Woods CE, Yan P, Kohl P, Ewart P, Loew LM, Terrar DA, Bollensdorff C (2012) Cardiac electrophysiological imaging systems scalable for high-throughput drug testing. Pflugers Arch 464 (6):645-656. doi:10.1007/s00424-012-1149-0

Cystic Fibrosis (CF) is a genetic disorder that is caused by mutations in the gene for the CF Transmembrane Conductance Regulator (CFTR). CFTR is an ABC transporter chloride channel containing five domains: two membrane-spanning domains (MSD1 & 2) connected by two nucleotide-binding domains (NBD1 & 2) and linked by a regulatory domain. The most common CF causing mutation is the deletion of phenylalanine 508 (?F508) in NBD1. It was shown that ?F508 thermodynamically destabilizes NBD1, as a result, misfold and degrade CFTR channel in the endoplasmic reticulum and prevents its processing and translocation to the plasma membrane. An effective drug that rescue the channel enhance its folding will increase its concentration on the plasma membrane. Here, we show stabilization of ?F508-NBD1 with second site mutations was not sufficient to increase the ?F508-CFTR folding efficiency and membrane concentration to that of the wild-type level. However, the introduction of additional mutations that stabilizes the interaction between NBD1 and MSD2 of CFTR in the presence of ?F508-NBD1 stabilization mutations increased the ?F508-CFTR folding efficiency and biogenesis from ~2% to 80% of the wild-type. As a result, a two-component drug that energetically stabilizes ?F508-NBD1 and maintain the NBD1-MSD2 interface interactions are required for wild-type like folding, processing, and transport function, suggesting a two step correction process.

Qatar Biobank (QBB), the first very large scale, long-term public biorepository in Qatar, is designed to build a powerful research infrastructure for future investigations of the lifestyle, metabolic and genetic risk factors by collecting comprehensive phenotypic baseline data among healthy volunteers, including ECG, blood pressure, anthropometry, spirometry, retinal imaging, carotid 3D ultrasound, arterial stiffness, total body iDXA, in addition to detailed personal lifestyle and clinical data. QBB collects and stores blood samples subdivided in 68 aliquotes for different future research purposes. Qatar Biobank understands that building a successful biobank depends on the willing participation of the public to come forward to contribute. The recruitment of participants requires insight into the public's existing knowledge of biobanking, level of willingness and an understanding of the motivators and barriers to participation. From December 2012 to June 2013, 503 participants completed anonymously a feedback form to evaluate their experience. The aim of this specific survey is to gain insight into recruitment methods, incentive to participate and satisfaction with various aspects of the visit. This will enable assessment of the processes of registration, scheduling, checking in, consent and reporting back results. Around 75% of those who completed the feedback form participated in QBB to improve the health of future generations and to have a comprehensive health checkup. 95% were willing to participate again if needed and 85% would recommend participation to friends and family. 87% and 95% thought the length of the questionnaire was appropriate and the questions not too intrusive, respectively. Finally, 91% found the staff welcoming, 89% rated the check-in process as "good" and 76% found scheduling an appointment straightforward. Further comments from participants suggested appreciation of the protection of confidentiality and privacy that was displayed throughout the process and that the process was smooth and enjoyable.

Purpose: The main objective of this study was to develop and validate a simple, rapid and stability-indicating ultra-performance liquid chromatography (UPLC) method for the determination of Letrozole in pharmaceutical gels. Methods: Chromatographic separation was achieved using Aquity UPLC BEH C-18 (2.1 x 50 mm, 1.7µm particle size) with an isocratic mobile phase consisting of acetonitrile: water (35:65, v/v). The flow rate was set to 0.3 ml/min, injection volume was set to 1µl, and the UV detection was carried out at λ= 240 nm. The method was validated as per ICH guidelines. Letrozole was subjected to different stress conditions (acidic, basic, oxidative, thermal, UV-radiation) to assess the stability indicating power of the developed UPLC method. Letrozole was also analyzed in different emulgel formulations containing a mixture of oil, surfactants, co-surfactants and gelling polymers. Furthermore, the stability of letrozole was assessed in the presence of different emulgel ingredients , where the mixtures of the saturated solubility studies were kept at 50°C, and then analyzed by the proposed method at predetermined time intervals. Results: The retention time of Letrozole was 1.80 min (RSD=0.12%). The method was linear over the concentration range of 2.5-200 µg/ml (R2 = 0.9999). The limit of detection (LOD) and the limit of quantitation (LOQ) were found to be 0.025 µg/ml and 0.125 µg/ml, respectively. The inter-day and intra-day precisions (% RSD) were less than 2%. All validation parameters were in the acceptable range. Neither the degradation products, nor the emulgel components interfere with the analysis of letrozole peak. Percentage recovery in emulgels were in agreement with the labeled spiked amounts. Letrozole was found unstable in the presence of PEG400, diethylene gycolmonoethyl ether, oleic acid, glycerylmonooleate when stored at high temperature of 50°C. Conclusions: The proposed method is simple, rapid, accurate, and can be successfully used for the analysis of letrozole's content and its stability in the pharmaceutical emulgels. Acknowledgements: This work has been financially supported by a grant to HM Younes and AA Sallam provided by Qatar National Research Foundation through the National Priorities Research Program grant # 4 - 496 - 3 - 157. Ahmed Abu Helwa is a research assistant financially supported by the same grant.

Cardiovascular diseases are a major cause of global disability and death worldwide. The prevalence of heart disease is quite high in the Unites States and similar trends are becoming apparent in various Middle Eastern and Gulf region countries such as Qatar. It has been suggested that within 10 years, atherosclerosis and its complications, mainly coronary heart disease, will become the leading cause of death and loss of productive life years worldwide. The statin therapy is one of the most effective strategies for lowering plasma low-density lipoprotein, thereby preventing atherosclerosis and reducing the incidence of heart attacks. The statin group of drugs, which include Zocor, Lipitor, and Crestor, competitively inhibit the activity of the endoplasmic reticulum (ER)-localized enzyme HMG CoA reductase. The reductase catalyzes the reduction of HMG CoA to mevalonate, a rate-determining step in the synthesis of cholesterol as well as essential nonsterol isoprenoids such as dolichol, heme, and the farnesyl and geranylgeranyl groups that are found attached to many cellular proteins. Despite the successful application in curbing cholesterol levels, statin therapy is limited because they disrupt feedback regulation of reductase causing the enzyme to accumulate to high levels. One mechanism for feedback regulation of reductase involves sterol-induced ubiquitination and subsequent degradation of the enzyme from ER membranes. Thus, a complete understanding of molecular mechanisms for reductase degradation will provide targets for novel cholesterol lowering drugs that will aid statins or be a better alternative to prevent atherosclerosis and coronary heart disease. An unresolved aspect of HMG CoA reductase degradation is how the enzyme is removed from ER membranes through a reaction called dislocation and degraded in the cytosol. To answer this, we reconstituted the cytosolic dislocation of reductase in a cell-free system. Characterization of this system revealed that dislocation of reductase from membranes required the in vitro additions of the oxysterol 25-hydroxycholesterol, the nonsterol isoprenoid geranylgeraniol, an energy source, and rat liver cytosol. We were also able to show that dislocation of reductase in the cell-free system was stimulated by two compounds, Apomine and SR-12813 that mimic sterols in stimulating reductase degradation in intact cells. This finding illustrates the utility of our cell-free system in the identification of new drugs that directly stimulate degradation of reductase. Moreover, establishment of the cell-free system positions us to identify proteins in rat liver cytosol required for reductase degradation. Modulating expression of these newly identified proteins may augment reductase dislocation and subsequent degradation, rendering them new therapeutic targets for coronary heart disease.

Background & Objectives: Sprint races are extremely popular in athletic competitions. Although those events have hitherto mainly been studied from a split-time perspective, at present, the nature of the adjustments in sprint mechanics and the extent to which stride parameters are altered are yet to be fully elucidated. This study was undertaken to test the hypothesis that single, maximal treadmill sprints (100 m, 200 m and 400 m) modify running kinetic-kinematic variables and spring-mass characteristics, with larger magnitude changes as sprint distance increases. Methods: Eleven physically active males performed 100-, 200- and 400-m running sprints on a validated instrumented sprint ergometer (ADAL3D-WR, Medical Development - HEF Tecmachine, Andrézieux-Bouthéon, France), which allowed subjects to run and produce speed ''freely'', i.e. with no predetermined belt speed imposed. Vertical and horizontal ground reaction forces were measured continuously (averaged every 50 m distance intervals) and used to determine stride parameters and spring-mass behavior. Results: Compared with the initial 50 m, running speed decreased (P<0.001) by 8±2%, 20±4% and 39±7% at the end of the 100, 200 and 400 m, respectively. All sprint distances (with the exception of stride length in the 100 m) induced significant (P<0.05) increments in contact (+7±4%, +22±8% and +36±13%) and swing times (+12±15%, +16±15% and +16±9%) and decrements in stride lengths (-1±4%, -5±5% and -41±2%) and frequencies (-6±3%, -13±7% and -22±8%) at the end of the 100, 200 and 400 m, respectively. Running kinetics all significantly decreased (P<0.001): mean vertical and total ground reaction forces for each step were reduced by about 2, 7 and 17% in the 100, 200 and 400 m, while net horizontal forces decreased by 14±17%, 41±21% and 121±35% on average. On the 100 m, the only spring-mass parameters to change (P<0.001) were an increased center of mass vertical displacement and a decreased vertical stiffness (+13±7% and -12±6%, respectively), with no change in leg stiffness. All the leg-spring variables significantly changed (P<0.05) over the 200 and 400 m: peak vertical forces: -6±3% vs. -14±9%; center of mass vertical displacement: +36±20% vs. +48±23%; leg compression: +8±8% vs. +8±9%; vertical stiffness: -30±11% vs. -63±20%; leg stiffness: -12±8% vs. -23±18%, respectively. Multiple linear regression analysis with best subset approach showed that 96% of the variation in speed was explained by stride length (ß=1.7) and frequency (ß=4.5) during 100 m. Stride length (ß=3.7) was also important predictor for speed during 200 m along with flight (ß=-24.9) and contact (ß=-25.6) times (r2=0.98), while leg stiffness showed a negative association (ß=-0.08) for predicting speed in the 400 m sprint model together with stride length (ß=-24.8) and contact time (ß=1.9) (r2=0.92). Conclusion: Along with speed, the magnitude of changes in running kinematics-kinetics and spring-mass behavior over short (100 m), intermediate (200 m) and long (400 m) treadmill sprint increased with sprint distance. Stride length was positively associated with speed in all sprint distances. Future research should evaluate the effects of various individualized training interventions on optimizing musculo-skeletal stiffness regulation, and their effects on sprinting speed development and maintenance.

Introduction The CXC-chemokine CXCL4 is released from alpha granules of activated platelets and is involved in platelet aggregation. This protein is a chemo-attractant for numerous other cell types and also functions as an inhibitor of hematopoiesis, T-cell function, tumor-induced angiogenesis and modulation of host inflammatory response. Due to its multiple functions, CXCL4 is a potential clinical anti-tumor target. We examined the effects of the CXCL4 gene on biological properties of a colorectal cancer cell line by in vitro functional tests. RNA interference (RNAi) methodology by means of gene specific stealth siRNA technology was used to control the expressional level of the CXCL4 gene. Aim of study * To determine the effect of CXCL4 knockdown by RNAi methodology on biological properties of CC531 cells in vitro. * To evaluate the role of CXCL4 gene in colorectal cancer and later on its metastasis in animal rat liver model. Materials and Methods * Rat colorectal CC531 cancer cells were inplanted into the rat liver. After selected time intervals, CC531 cancer cells were re-isolated and their expression profile was studied by mRNA micro-array analysis. In vitro cultured CC531 cells were used as control in this study. * Transient knockdown of the CXCL4 gene was achieved in CC531 cells by using Lipofectamine 2000 transfection reagent along with 100nM gene specific stealth siRNA. * After 24, 48 and 72 h of treatment with siRNA, the extent of knockdown of CXCL4 was determined at mRNA level by RT-PCR and qPCR. * Functional effects of CXCL4 knockdown on CC531 cells were investigated by proliferation, colony formation, migration and scratch assays. Results In vivo Expression of CXCL4 Implanted colorectal CC531 cancer cells exhibited 3-4 times lower expression in the rat liver for an initial period of 3 weeks and came back to normal level after week 4. Knock-down of CXCL4 Exposure to siRNA species reduced the expression of CXCL4 at mRNA level after 24-72h with maximum reduction of 70% after 24h. MTT Assay Following the post-transfection period, we found a moderate decline in cell proliferation by 20, 26 and 8% for 24, 48 and 72h, respectively, in knockdown CC531 cells. Colony Formation Assay We observed reduced ability of knockdown CC531 cells to produce small and large colonies. Migration Assay Significant reduction in directional migration was observed in the knockdown CC531 cells especially after 48 and 72hr. Scratch Assay Photomicrographs, obtained after 12 and 24 hours of scrape wounding showed a significant reduction of knockdown CC531 cells to cover the scratched area as compare to control CC531 cells. Conclusion Higher expression of CXCL4 contributes to enhanced cell proliferation, migration, colonization and wound healing abilities of colorectal CC531 cancer cells in vitro. Based on these preliminary results, we will also study the effect of CXCL4 in a liver cancer metastasis model to evaluate an anticipated correlation with colorectal cancer progression.