Qatar Foundation Annual Research Forum Volume 2011 Issue 1

Obesity is a global epidemic and has been implicated as a risk factor for end-stage renal disease. In this study, we investigated the impact of obesity in the absence of hypertension, on renal lipid accumulation, oxidative stress, mitochondrial function, and endoplasmic reticulum (ER) stress, which could play a major role in the development of obesity-induced renal dysfunction. We compared two genetic mouse models of obesity which we have shown to be normotensive, the leptin-deficient ob/ob mice and a hyperleptinemic melanocortin 4 receptor knockout mice (LoxTB MC4R-/-), to lean wild type (WT) C57BL/6J mice and littermate controls (WT-LoxTB) from LoxTB MC4R-/-breeding colony respectively. We measured urinary albumin excretion, creatinine clearance, renal triglycerides, ATP levels, state-3 mitochondrial respiration, protein carbonylation (a marker of oxidative stress) and C/EBP homologous protein (CHOP) expression (a marker of ER stress) in these mice. Our results indicate that the ob/ob mice and LoxTB MC4R-/-mice exhibit significant albuminuria, increased creatinine clearance (693±61.1 vs. 534±31.5 and 752.3±50.6 vs. 488.9±81.2 μL/min) and renal triglyceride accumulation (8.1±0.8 vs. 4.8±0.2 and 3.9±0.5 vs. 2.2±0.3 mg triglyceride/g tissue) expressed as ob/ob vs. WT and LoxTB MC4R-/- vs. WT-LoxTB respectively. Despite significant decreases in renal ATP levels (6±0.3 vs. 7.9±0.4 and 5±0.2 vs. 8±1.1 pmol/mg) in both obese models, only the LoxTB MC4R-/-mice kidneys showed an impaired state-3 fatty acid oxidation, increased protein carbonylation and 3-fold induction of CHOP protein compared to WT-LoxTB control mice. Taken together, our data suggest that obesity in the absence of hypertension cause only mild renal dysfunction, and unveils the potential involvement of oxidative stress, impaired fatty acid oxidation and ER stress in obesity-induced renal injury associated with MC4R deficiency.

Imatinib is currently the standard drug used in the treatment of CML patients. However, imatinib is not curative since most patients who discontinue therapy will relapse. Interferon alpha (IFN) induces hematologic and cytogenetic remissions and interestingly, prior exposure to IFN allowed Imatinib discontinuation in some CML patients. Arsenic trioxide inhibits the proliferation of BCR-ABL-expressing cells.

We have investigated the effects of the combination arsenic/IFN on the proliferation of CML cell lines. We found that IFN alone had minimal effect. Arsenic alone significantly decreased their proliferation in a time and dose-dependent manner. Interestingly, the addition of IFN to arsenic was synergistic in AR230 and additive in K562. This synergistic effect between IFN and arsenic was accompanied by dose-dependent apoptosis as evidenced by annexin V staining, TUNEL positivity and caspase activation. Colony-forming assay was performed on bone marrow and CD34+ cells collected from CML patients. Interestingly, arsenic and IFN produced a synergistic decrease in myeloid colony formation, especially when compared to Imatinib.

Preliminary results of an in vivo study using the retroviral transduction CML mouse model showed prolonged survival of secondary recipients that received cells from primary leukemic mice treated with arsenic/IFN, as compared to those that received cells from untreated controls. These results suggest that arsenic and IFN synergize to inhibit proliferation, induce apoptosis and target dormant CML stem cells that are spared by Imatinib.

In addition to physical conditioning, successful sporting performance requires a level of perception coupled with efficiency and accuracy in movement. The FIFA World Cup is usually played during the summer months; therefore the aim of this study was to investigate the effects of heat exposure on cognitive performance.

Three experiments were performed in an environmental chamber under hot (HOT: 50°C, 30% rH) and control (CON: 24°C, 30% rH) conditions. Exposure time ranged from 15 minutes to 4.5 hours. Subjects performed simple and complex cognitive tasks under different levels of thermal strain. Neuromuscular assessments were performed to assess neural drive and muscular force production. Central (Tcore) and skin (Tskin) temperatures were recorded along with subjective measures of thermal comfort (TC) and thermal sensation (TS).

Hyperthermia had no impact upon attention tasks, however, impulsivity increased during sustained attention over prolonged periods. Significant increases in Tcore (>38.30°C) were associated with impaired complex cognitive task performance. The regular application of cold packs to the head limited the detrimental effects of hyperthermia upon short-term memory. However, rapid and substantial variations in Tskin of ∼3°C, independent of changes in Tcore, had similar detrimental effects upon cognitive task performance.

Results suggest that reductions in complex cognitive performance with heat exposure occur via a decrement in motor cortical excitability when Tcore increases to ∼38.3°C, but they also originate from an additional cognitive load imposed by thermal strain and the resulting allesthesial Tskin variations; with both these factors acting as competing variables to the cognitive processes.

The increase in Tcore with prolonged heat exposure impairs cognitive performance; however applying cold packs to the head can preserve these. Increases in Tskin during brief heat exposures appear to be a sufficient physiological response to alter the emotional state of individuals and impair effective decision-making. Hydration protocols and reducing exercise intensity and load can reduce the physiological strain. Strategies to minimise the ‘thermal shock’ of entering a warm environment should be developed to minimise the impact of subjective feelings of thermal discomfort upon cognitive performance.

Background: Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is a highly effective antitumor drug. However, tumors can acquire resistance to CDDP. CDDP elevates the intracellular calcium concentration ([Ca²⁺]i), in various cell lines, leading to the activation of apoptotic pathways and cell death. Using cultured breast cancer (MCF-7) cells we: (1) investigated the effects of CDDP on [Ca²⁺]i, (2) compared these results to a cell line that has been desensitized to CDDP (“resistant”), (3) investigated the source of [Ca²⁺]i by modulating calcium channels and transport mechanisms, and (4) correlated these findings to cytotoxicity. Methods: Changes in the [Ca2+]i were recorded using fluorescence microscopy and Ca2+-binding fluorescent dye, Fluo-4AM. CDDP (1nM-10μM) was administered via a bath perfusion system to the sensitive and CDDP-”resistant” MCF-7 cells over a period of 1.5–2.5h. The [Ca²⁺]i modulators, (caffeine; 10mM, nimodipine; 10μM, ionomycin; 10μM, thapsigargin; 500nM, and 2-APB; 50μM) were administered. MTT assays and trypan blue cell viability tests were performed using CDDP at concentrations of 100pM, 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, and 10mM after 4, 8, and 24h of incubation. Results: CDDP induced a concentration-dependent increase of [Ca²⁺i. A concentration of CDDP 0.1μM triggered the largest elevation of [Ca²⁺]i with 120% increase (n=19). Induction of cytotoxicity was most likely directly correlated to the increase of [Ca²⁺]i, and was significantly lower in CDDP-”resistant” cells (P←0.05). Preapplication of the calcium channel blocker, nimodipine as well as the IP3 receptor blocker 2-APB significantly reduced this elevation (46.6%; n=26, 71.4%; n=52, increase, respectively) (p←0.05). Surprisingly, when [Ca²⁺]i was elevated due to the pre-application of caffeine, ionomycin or thapsigargin, the subsequent application of CDDP was also significantly reduced compared to control conditions (37.8%; n=15, 34.9%; n=32, 53.7%; n=21, increase, respectively) (p←0.05). Conclusion: CDDP concentration-dependently elevates [Ca²⁺]i by Ca²⁺ entry and Ca²⁺ release from the stores. These mechanisms, however, seem to be less effective in CDDP-”resistant” cells since they show increased restriction on Ca2+ elevation. The pre-elevation of [Ca²⁺]i, through releasing Ca²⁺ from the stores, reduces this elevation significantly. The exact mechanisms remain unclear and further investigations are required to determine the mechanisms and pathways that are involved in the disruption of [Ca²⁺]i.

Introduction: The application of therapeutic proteins and cytokines like Interleukin-2 (IL-2) for long-term, localized delivery has been hindered by a lack of a delivery device that releases active protein at a concentration within their therapeutic window. The purpose of this oral presentation is to report on the osmotic-driven, controlled-release from novel visible-light photocrosslinked biodegradable elastomeric devices recently designed in an attempt to overcome this limitation.

Materials and Methods: All chemicals were purchased from Sigma-Aldrich and used as received. Novel biodegradable and biocompatible poly (decaneco- tricarballylate) [PDET] elastomers were synthesized by polycondensation reaction between tricarballylic acid and alkylene diols, followed by acrylation and photo-curing. IL-2 loaded micro-cylinder and disk-shaped elastomeric devices were prepared by intimately mixing IL-2 lyophilized powder with the acrylated prepolymer prior to photocrosslinking. IL-2 release was analysed using IL-2 ELISA system and the in vitro bioactivity of released IL-2 was assessed using C57BL/6 mouse cytotoxic T lymphocyte. The influence of various parameters such as the elastomer crosslinking density, the volumetric drug loading percentage and the incorporation of osmotic excipients like trehalose on the release kinetics of the drug was also examined.

Results and discussion: The disk-shaped specimens showed faster IL-2 release profiles than microcylinders, with drug release proceeding via typical zero-order release kinetics. The increase in the device's surface area and the incorporation of trehalose in the loaded lyophilized mix increased the IL-2 release rate. As well, it was shown that the decrease in the degree of prepolymer acrylation of the prepared devices increased the IL-2 release rate. Cell based bioactivity assays showed that IL-2 released over a period of 28 days, retained more than 94% of its initial activity. These bioactivity results represent a highly significant improvement over the other previously published data provided with a quantitative analysis of the actual percentage of bioactive IL-2 released during the period of the release study.

Conclusion: The novel PDET elastomeric drug delivery systems demonstrated to be promising as protein drug delivery vehicles for localized and sustained IL-2 immunotherapy.

Acknowledgements: Work is supported by QNRF through the NPRP grant # 09-969-3-251 awarded to HM Younes.

T-cells genetically modified to express a chimeric antigen receptor (CAR) against a tumor associated antigen are attractive anti-cancer therapeutic agents. The 5T4 cell surface oncofoetal antigen is an attractive target antigen for cancer immunotherapy as it is expressed by a wide spectrum of cancers including gastric, ovarian and colorectal while showing limited expression in normal adult tissues. Previously it was shown that human and murine T-cells engineered to express human 5T4 specific CAR can specifically lyse human 5T4-expressing tumor targets in vitro and in vivo, respectively. This study aimed to isolate single chain variable fragments (scFvs) specific for murine 5T4 (m5T4) and to examine their efficacy in the context of CAR in a fully autologous model. Screening four novel hybridoma cell-lines producing anti-m5T4 monoclonal antibodies to clone the scFv yielded one functional m5T4-specific scFv from the hybridoma cell-line P1C9. The P1C9 scFv expressed as a fusion protein with the Fc domain of human IgG significantly labels m5T4-expressing targets. Murine T-cells modified to express the P1C9 scFv fused to the CD3? molecule can specifically lyse target cells in vitro and result in IFN-? cytokine release, while T-cells expressing the non-signalling CAR derivative; P1C9 scFv fused to the murine MHC-I transmembrane domain, were unable to lyse m5T4-expressing tumor targets. On-going experiments aim to test the efficacy and assess the toxicity of m5T4-specific CAR against m5T4-expressing tumor models in vivo. This model will allow further understanding of how gene-modified T-cells function in an autologous setting with the aim to improve human T-cell based cancer immunotherapy.

Background: Caring for an autistic child places significant stress on the lives of family members. To date, no study has evaluated how looking after a child with autism affects quality of life (QoL) of caregivers in Qatar or the Gulf country region. Our study is aimed at quantifying the health-related QoL of caregivers of children with autism in Qatar.

Methods: We recruited two groups of caregivers of children between 3 to17 years old. The first group was formed of caregivers of children with autism from two developmental paediatric rehabilitation clinics, and the second group was formed of caregivers of typically-growing children and who were visiting a family clinic for routine medical check-up. Demographic information of all caregivers and children were collected. The Arabic version of the Short Form-36 (SF-36), which is a generic type questionnaire, was administered to caregivers in both groups to assess QoL.

Results: A total of 98 participants consented to take part in the study. 56 were caregivers of children with autism, and 42 were caregivers of a typically-growing children. There was no statistically significant difference in QoL domains between both groups, but caregivers of autistic children rated their health as poor and likely to get worse (p=0.003). Mental health components were consistently poorer than the physical components in the autistic group, and female caregivers had poorer mental health than males in this cohort of participants (p<0.05).

Conclusion: This study provided evidence for the impact of caring for a child with autism on the life of the caregiver. However, there seems to be a need to develop condition-specific QoL questionnaires that can specifically measure QoL in caregivers of autistic children more accurately than generic questionnaires. The findings should still help health policy-makers provide more focused support to the children with autism and their families.

Background: Cigarette smoking is one of the preventable causes of ill health in Qatar. Qatar community pharmacists are in an ideal position to play an important role in smoking cessation. This role necessitates adequate smoking cessation knowledge and education. The study objectives were to assess Qatar community pharmacists' smoking cessation knowledge and to gauge their perceptions of which aspects of smoking-related education would be most interesting.

Methods: A pretested survey was used to solicit community pharmacists' anonymous responses. The survey was designed after reviewing relevant smoking cessation literature. A phone call was made to all community pharmacists in Qatar to request their participation. Interested pharmacists were sent the survey link by email or by fax. Data was descriptively analyzed using the Statistical Package of Social Sciences software version 18.

Results: Over 20 weeks, we collected 112 surveys (35% response rate). Smoking cessation knowledge was evaluated using 8 true or false questions. Thirty seven percent of respondents scored less than 60% and 13% scored more than 80%. The mean score was 61% with a standard deviation of 17%. Eighty-nine percent of respondents indicated that they have not received before any smoking cessation education. Nevertheless, at least 70% indicated that they were interested in receiving additional smoking cessation education. Respondents were mostly interested in receiving education on motivating smokers to quit and on counseling on behavioral techniques (89% and 86% respectively). Sixty nine percent indicated a preference for mailings of printed materials as method of information delivery.

Conclusion: Despite their low smoking cessation knowledge, Qatar community pharmacists are interested in receiving additional smoking cessation education. A smoking cessation education program should be offered to these pharmacists to give them the knowledge they need to be competent smoking cessation counselors.

Introduction: Controlled drug release from implantable silicone elastomers was reported to occur mainly via the osmotic rupture release mechanism. In this work, we are attempting to determine the effects of drug volumetric loading, particle size, device shape, and dissolution media on the release rate of the water-soluble osmotically active drug, Papaverine Hydrochloride (PH) from silicon elastomers.

Materials and Methods: Medical grade silicone (Sylgard kit) was purchased from Dow Corning. All other chemicals were purchased from Sigma-Aldrich and used as received. The release profiles from devices of three geometries (cylinders, cubes and tablets), of different drug particle sizes (< 45μm, 63–125μm or 125–250μm) in different dissolution media (phosphate buffered saline, distilled water, and 3% sodium chloride solution) were investigated. The effect of degree of elastomer crosslinking and the percentage of volumetric loading of the osmotic excipient and pore forming agent, Trehalose, on the release rate were also investigated. The drug fraction released of PH was analyzed using ultraviolet spectrophotometric analysis. Data obtained was then plotted as cumulative percentage release against time and then further analyzed.

Results and Discussion: Contrary to previous reports, devices formulated with the same volumetric loading and smaller drug particles sizes released drug faster than those devices with a larger particle size. The drug release from tabular implants was of faster rate when compared to cylindrical and cubic devices. Contrary to the expected, higher drug release rate was also observed from cubic elastomeric devices with higher crosslinking density. In addition, the release profiles demonstrated that osmotic release was the predominant mechanism governing the release of PH from silicone elastomers.

Conclusion: Our results show that osmotic rupture is the predominant mechanism of release of PH from silicone elastomer. Further studies must be conducted to confirm the effect of the elastomer crosslinking density on drug release rate.

Acknowledgements: This report was made possible by a UREP award [UREP 07-120-3-027] to HM Younes from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors.

Background: Fetal movements are clinically correlated to fetal wellbeing. Ultrasounds are the most accurate measurements of the fetal movement but expensive and intrusive. To avoid these constraints, Fetal activity is captured through data acquired using low cost and nonintrusive accelerometer . Time-frequency distributions (TFDs) are often used to represent the energy, temporal and spectral characteristics of non-stationary signals in the time and frequency plane.

Many quadratic TFDs were proposed in the literature such as Wigner-Ville distribution, Spectrogram, B-Distribution, Choi-Williams , etc. The drawbacks of the majority of these techniques is that only a few parameters can be modified in the kernel, generally the lag and Doppler parameters, so that they cannot be easily adapted to the data.

Objectives: This work aims at designing a new kernel with several parameters that leads to a higher resolution time frequency representation (TFR) of the signal, therefore improving the characteristic of the fetal movement.

Methods: The proposed TFD can be considered as an extension of the Gaussian TFD (also called Choi-Williams distribution). The kernel function of the proposed kernel is given by the sum of the weighted derivative Gaussian TFD (refer to the equation).

Results and Conclusion: The weighted parameters in the above formula can be estimated by maximizing the concentration of the instantaneous frequency. The resulting TFD is compared with other methods and applied to the analysis and classification of the fetal movement data recorded by the accelerometers. The results obtained indicate that the proposed time-frequency methodology allows the detection of fetal movement data recorded by accelerometers.

Introduction: Autism spectrum disorder (ASD) affects children at around the age of 3 years old and last throughout the person's lifetime. ASD imposes great burden on the family, and forces family members to considerably modify their daily lives to suit their reality with autistic child(ren). To our knowledge, no previous research assessed the burden of ASD on the lives of parents/caregivers of autistic children in Qatar or the Gulf region.

Methods: Caregivers of a child with autism between 3–17 years were recruited from children rehabilitation clinics. The control group was represented by caregivers of a typically-growing child visiting a primary health care facility for a routine medical examination. Data collected from both groups included demographic information of caregivers and children and quality of life information. The Lebanese Arabic version of the Short Form-36 (SF-36) was used to assess quality of life.

Results: Children in the AG spent more time indoors, watching TV, or sleeping than children in the control group (p←0.05). Around 40% of caregivers in the AG said they would encourage their child to get married and become parent when s/he grows up. Half of the sample in the AG utilizes special education classes and other facilities, and the remaining half has access problems. There was no statistically significant difference between quality of life domains between the two groups of caregivers, but caregivers of autistic children rated their health as poor and likely to get worse (p=0.003). However, mental health components were consistently poorer in the AG compared to the physical components, and female caregivers also had poorer mental health than males in this cohort of participants (p<0.05).

Conclusion: This study provided evidence for the impact of caring for a child with autism on the life of the caregiver. The findings should help health policy-makers provide more focused support to the children with autism and their families.

Background: Anterior cruciate ligament (ACL) tears are common injuries occurring upwards of 250,000 times annually only in the U.S. These injuries create an annual billion dollar expense. It is important to understand these injury mechanisms as these injuries continue to be one of the largest problems in orthopedic sports medicine. Improved understanding of the injury mechanisms may improve prevention, rehabilitation and surgical procedures.

Objective: This study used a 3-Dimensional finite element (FE) knee joint model to investigate the combinations of movement, which cause ACL injury.

Method: Digital bone structures were created from magnetic resonance images (MRI). Ligament bundles were modeled based on the origins and insertion sites determined from MRI. Bone was modeled as rigid, and a transversely isotropic Mooney-Rivlin material was applied to the ligament structures. This study incorporates a novel approach for developing bundle specific prestrain within 3-D ligament structures. The bundles were stretched from their zero load lengths to their reference lengths, producing a strain field mimicking the in vivo strain conditions of the ACL at full knee extension. A failure locus was created by performing multiple FE simulations of knee joint motion combinations until ACL failure.

Result: The relationship between knee joint orientation and ligament rupture was plotted providing a spectrum for the propensity of ACL injury based on knee joint orientations, known as a failure locus. The locus shows which combinations of internal/external femoral rotation and varus/valgus angle cause ACL failure. The results show the posterolateral bundle more susceptible to rupture than the anteromedial bundle in 17 of the 22 simulations. The results also show 45% less varus angle needed for ligament failure relative to valgus angle. The results highlight femoral external rotation as an important factor for ACL injury as it decreases the failure angle by an average of 23% compared to femoral internal rotation.

Conclusion: These results have various clinical applications. In sports where ACL injuries are prevalent, training programs can be adapted to address the avoidance of harmful knee orientations.

Transient Receptor Potential (TRP) ion channels are formed by either homomeric or heteromeric assembly of four TRP subunits that have six transmembrane domains (TM) and a P-loop located between TM5 and TM6 defining the channel pore. So far 30 different subunits classified in 6 families sharing as low as 20% homology, have been identified in mammals and about 20 in Xenopus. Although heterotetramers can be formed within a family, the assembly of subunits from members of different families was thought to be unlikely. We here propose that TRPV6 and TRPC1 subunits can co-assemble in the Xenopus oocyte. Western blots performed on naive oocytes lysates revealed that they express both TRPV6 and TRPC1 proteins. Furthermore, co-immunoprecipitation of tagged xTRPV6 and xTRPC1 proteins expressed in oocytes suggested a heteromeric assembly. In mammals, TRPV6 is mainly expressed in intestinal epithelia and in the placenta where it forms a homomeric channel with high calcium permeability whereas TRPC1 is a ubiquitous protein forming cationic channels with an unclear gating mechanism.

In oocytes, over expression of xTRPC1, as previously reported, did not induce any resting ionic current. Conversely, oocytes overexpressing xTRPV6 alone did show an inward rectifying cationic current that could be blocked by La3+ ions and Ruthenium red. As described for mammals, the xTRPV6 channel was largely permeable to calcium ions and displayed an anomalous mole fraction effect. A striking distinction with its mammalian counterpart is the high permeability of the xTRPV6 channel to Mg2+ over other divalents (Mg2+>Sr2+>Ba2+>Ca2+), the Mg2+/Ca2+ ratio being 2.08.

This high permeability to Mg2+ could be abolished by a point mutation in the P-loop (D525N) lowering the ratio to 1.18. Co-expression of xTRPC1 and xTRPV6 lead to a very strong reduction in the current amplitude and in cationic selectivity, the Mg2+/Ca2+ ratio also dropping to 1.18. Further investigations using heterologously expressed xTRPV6 and xTRPC1 in HEK293, allowing whole-cell recordings, will help us clarify the selectivity and the regulation of the channel. The potential interaction between TRPV6 and TRPC1 in mammals will also be examined using tissue extracts and selected cell lines.

The oscillation of cytoplasmic calcium concentration exists in a large number of non-excitable cells such as astrocytes in the brain, intestinal cells and oocytes (Fig.1). This rhythmic activity carries information into the cell, regulating various processes such as gene expression or transmitter release. In some cells, such as Xenopus oocytes, the oscillations can rely only on calcium being released in the cytoplasm from intracellular stores after stimulation of inositol triphosphate (IP3) receptors located on the endoplasmic reticulum (ER). To refill the intracellular stores in the ER, the cell has to import extracellular calcium. This is achieved through the STIM/Orai pathway where an ER calcium sensor (STIM) is activated by store depletion and in turn interacts and opens a calcium channel in the plasma membrane (Orai) leading to calcium influx, a process known as Store Operated Calcium Entry (SOCE). In the present work we evaluated the interactions between SOC and intracellular calcium oscillations in Xenopus oocytes. Intracellular calcium levels were monitored using the amplitude of the endogenous calcium-activated chloride currents. Following injection into the cells of a non-hydrolysable analog of IP3 (IP3-DF), long lasting calcium oscillations could be triggered as well as SOCE ( Fig. 1 ). The calcium oscillations were more efficient in promoting SOC than store depletion by ionomycin (2.34 ± 0.55 nA, n=13 vs 0.05 ± 0.02 nA, n=8), the mechanisms involved are currently under study. We also observed that activation of SOC could trigger a calcium wave in a dose-dependent manner following a “Calcium Induced Calcium Release” pattern. This suggests that SOC might not only be involved in replenishing the calcium stores but also in the regulation of the amplitude and timing of calcium oscillations. The well known down-regulation of SOC during oocyte maturation should therefore have important functional consequences on intracellular calcium oscillations.

Recently, a speculative model is proposed that tumor tissue is continuously regenerated by a small population of self-renewing cancer stem cells responsible for initiation, growth and propagation of tumor. Tumor cells reside in a “niche” and have constant interaction with the niche components including tumor vasculature, bone marrow mesenchymal stem cells (BM-MSCs) and the components of the extracellular matrix. It is believed that the growth advantage acquired by tumor cells as well as the ability of cancer stem cells (CSCs) to maintain tumor propagation is partly the outcome of this interaction.

Our study intends to better characterize this crosstalk by looking into the possibility of BM-MSC/endothelial cell-mediated activation of Notch pathway in breast cancer cells through production of certain cytokines aiming at proposing novel therapeutic approaches for targeting this disease.

The CSCs were enriched by culturing breast cancer cells (BCC) in 3D media to obtain multicellular spheres for further analysis. BM-MSC and Akt-activated endothelial cells were co-cultured with BCC and spheres to assess their capability in promoting cancer cell growth. RNA interference approach was applied to Notch3 to determine its role in BCC survival by the niche players. A human cytokine array was used to identify the cytokines that showed different expression patterns in the BCC co-cultures with the niche residents.

BM-MSC or Akt-activated Endothelium was able to increase BCC growth up to 4- and 5-fold, respectively, in contact and under serum-free condition. Blocking Notch3 in BCC or spheres by siRNA or inhibition of notch pathway by GSI reduced the proliferation rate of both entities to 2.5-fold, which might underline the role of notch in the interaction between tumor cells and the niche components. Also, several cytokines were identified that were differentially regulated in co-cultures system among which IL6, IL8, and GROa showed significant up-regulation.

Our preliminary data suggest the role of BM-MSC and endothelial cells in breast cancer growth and survival. Our results propose the idea of notch involvement in this interaction through stimulation by certain cytokines. Further investigation is required to elucidate the exact mechanism that is triggered by these cytokines in tumor propagation and maintenance.

Background: The reasons behind the differences in the levels of discordancy (that is one partner testing HIV positive while the other testing HIV negative) among spousal and cohabiting partnerships affected by HIV across sub-Saharan Africa (SSA) remain inadequately understood. Recently, many randomized clinical trials have shown substantial efficacies for several prevention interventions among these partnerships.

Moreover, there has been an intense debate about the priority of HIV prevention interventions among discordant couples relative to other prevention approaches such as among commercial sex networks.

Objective: To describe and explain patterns of HIV infection among spousal and cohabiting sexual partnerships, across a range of settings in SSA.

Methods: Demographic health survey (DHS) data for 20 countries in SSA were used to estimate the prevalence of HIV sero-discordancy among stable partnerships with at least one HIV-infected individual in the partnership (P), prevalence of discordancy among all stable partnerships in the population (S), prevalence of discordancy among the entire sexually active population (A), and prevalence of discordancy among HIV infected individuals (I).

Results: Two distinct patterns of HIV among stable partnerships were observed. Countries with low HIV prevalence had a high discordancy prevalence among P and I ranging from 48.4–87.8% and 30.6–60.0%, respectively, but low discordancy prevalence among S and A ranging from 0.4–6.4% and 0.2–3.8%, respectively. Conversely, countries at hyper-endemic HIV epidemics had a low discordancy prevalence among P and I ranging from 36.3–58.5% and 17.2–46.0%, respectively, but high discordancy prevalence among S and A ranging from 9.3–17.2% and 5.8–8.3%, respectively.

Conclusions: Two distinct patterns of HIV in stable partnerships were observed. In high prevalence settings, many partnerships were affected by HIV but relatively few were discordant, whilst the opposite was true for low prevalence settings. This pattern may be arising from variations in the HIV transmission probability which is dependent on biological and behavioral factors and might also be affected by the frequency of infection from external partners especially in high prevalence countries. These findings may complicate considerations for rolling out prevention interventions among stable discordant partnerships in SSA.

Background: Empirical evidence suggests that HIV incidence rate within stable discordant sexual partnerships in sub-Saharan Africa (SSA) varies between 1.2 and 19.0 per 100 person-years. Estimating HIV incidence rate within stable discordant partnerships is critical for determining the contribution of HIV sero-conversions among these partnerships to total HIV incidence.

Objective: To estimate HIV incidence rate within stable discordant partnerships using nationally representative data and explore potential underlying factors contributing to their variability across a range of epidemic settings in SSA.

Methods: We constructed a mathematical model based on competing-hazards formalism to estimate HIV incidence rate within stable discordant partnerships across 20 countries in SSA. We also used the model to analyze the patterns of HIV discordancy in SSA. The model was parameterized using Demographic Health Survey data and analyses were conducted at endemic equilibrium. Sensitivity analyses were performed to explore the dependence on the dynamical drivers of discordancy.

Results: Our model fitted well the empirical epidemiological measures of HIV discordancy and yielded an estimate for HIV incidence rate among discordant partnerships of 14.7 per 100 person-years (95% CI 9.9–19.4 per 100 person-years). HIV incidence rate ranged between 3.9 and 34.9 per 100 person-years across the countries. We also identified HIV incidence rate within stable discordant partnerships and HIV incidence rate from sources external to the partnership (or equivalently HIV prevalence) as key determinants of the variability in discordancy measures across SSA.

Conclusions: Our estimate for the HIV incidence rate among discordant partnerships agrees well with empirical estimates for this measure. There is however considerable variability across the countries. Biological and behavioral factors including differences in transmission rates such as due to male circumcision, may have contributed to the variability in HIV incidence rates among discordant partnerships across SSA. More research is needed to elucidate the determinants of this variability in incidence rates.

Background: Hepatitis C virus (HCV) currently infects around 2% of the world's population. Among all nations, HCV prevalence ranges from 0.01% in Scandinavia to 3% in North Africa, with one exception: Egypt. Egypt has the highest prevalence of HCV in the world, estimated nationally at 14.7%. Numerous HCV prevalence studies have published various estimates from different Egyptian communities, suggesting that Egypt, relative to the other nations of the world, might be experiencing an intense ongoing HCV transmission.

Objectives: To review all the evidence on the epidemiology of HCV transmission among different population groups in Egypt.

Methods: This was a systematic review following the PRISMA guidelines of all prevalence data on HCV infection in Egypt. Sources of data included PubMed, international organizations' reports and databases, and country-level reports and databases. Measures were classified into different population categories according to risk of infection.

Results: Seventy-four studies have measured HCV prevalence in Egypt in populations at varying levels of risk. Among Egypt's general population, HCV prevalence in pregnant women was 8.6%, and among blood donors it ranged between 9.0% and 23.2%. A nationally representative survey reported a prevalence of 14.7%. Among populations at high risk of infection, HCV prevalence was found to be as high as 58.3% in multi-transfused children, and 87.5% in adult dialysis patients. Among populations at intermediate risk, diabetic children had a prevalence of 3.1%, barbers a prevalence of 12.3%, health care workers a prevalence of 15.7%, and prisoners a prevalence of 31.4%. Common risk factors appear to be parenteral anti-schistosomal therapy, frequent transfusions, injections or surgical procedures.

Conclusion: Egypt has experienced, and possibly continues to experience, a large HCV epidemic. Prevention measures need to be implemented targeting HCV transmission routes such as better infection control practices in health and dental care facilities, hemodialysis centers, and reducing the excessive numbers of non-therapeutic injections.

Urinary bladder cancer is one of the most common cancers worldwide and has a high recurrence rate. It has the highest lifetime cost of care per patient due to long follow-up cystoscopic surveillance after surgery to detect the high risk of recurrence.

This research develops a portable custom cystoscopic procedure to improve the efficiency and accuracy of the bladder cancer surveillance. The system uses a segmented bending mechanism that is inserted into the bladder via the urethra to steer a flexible imaging probe to provide a comprehensive diagnostic tool for review by an urologist as illustrated in Fig. 1. The position and orientation of the camera locating at the tip of the probe can be automatically controlled remotely to scan the entire bladder surface.

The structure of the bending segment is shown in Fig. 2. The segment bending is tendon driven. Four distributed small wires are connected to the segment body via guiding rings. The wires to control the distal segments are also guided through the mechanism via the rings of proximal segments. The bending angle and bending direction of a segment can be controlled by pulling and releasing its four wires accordingly. The design of the mechanism and the forward/inverse kinematics simulation were finished. A mockup model is under construction to verify the proposed design.

Images abstracted from the video are used to reconstruct a 3D panorama of the whole bladder surface. Our 3D and image mosaicking software is under developed by our collaborator at University of Washington, USA.

Background: Chronic hypertension is a deadly disease that affects nearly 30–36% of the adult population in Qatar and the regional. Inositol 1,4,5-triphosphate receptors (IP3R) are intracellular calcium (Ca²⁺) channels that mediate the release of Ca2+ from sarcoplasmic reticulum in response to IP3 binding. A rise in cytoplasmic Ca²⁺ mediated by voltage-dependent L-type Ca²⁺ (CaL) channels and IP3-dependent Ca²⁺ release can enhance vascular smooth muscle cells (VSMC) contractility and determine peripheral vascular resistance.

Objective: The goal of this study is to elucidate the role of IP3R in hypertension.

Methods: Two rat models of hypertension and the A7r5 rat embryonic aortic cells are used in this project. Proteins for western blot were isolated from small mesenteric arteries (SMA) from hypertensive rats and A7r5 cells. Contraction measurements were performed in isolated arterial rings mounted for tension-recording assays. Contraction was induced with KCl and the phospholipase C (PLC) activator m-3M3FBS. The modulation of the vasoconstrictor responsiveness of SMA by IP3R and nitric oxide (NO) was depicted in a pressurized SMA incubated in the presence or absence of 2-APB (IP3R blocker) and L-NAME (NO synthase inhibitor). A7r5 basal Ca²⁺ levels were assessed using a fluorescence Ca²⁺ imaging system.

Results: Our preliminary results show that IP3R is up regulated in SMA from two different hypertensive rat models and following membrane depolarization in A7r5 cells. This up regulation is associated with an enhanced myogenic tone in response to activation of the PLC-IP3 pathway in SMA and with an increased basal Ca²⁺ levels in A7r5 cells. In contrast, pharmacological inhibition of IP3R alters the vasoconstriction response of pressurized vessels. Furthermore, IP3R upregulation and basal Ca²⁺ increase are lost in A7r5 cells treated with Nifedipine (CaL channels blocker) and drugs that block the Calcineurin-NFAT pathway before depolarization with KCl.

Conclusions: These findings improve our basic understanding of the etiology of hypertension by defining the abnormalities of IP3-dependent Ca²⁺ signaling in VSMC. This may provide novel insights into the pathogenesis of hypertension and set the groundwork for developing novel therapeutic targets for the treatment of hypertension.

Each olfactory sensory neuron in the mammalian nose selects just a single member of the large odorant receptor (OR) gene family. The core processes regulating OR selection may involve mechanisms that limit initial OR transcription and once a single OR is chosen, repress subsequent activations. We have used a genetic strategy to monitor the transcriptional permissiveness of the OR gene P2 by inserting an exogenous promoter, the tetracycline-dependent transactivator responsive promoter (teto), into its start site through homologous recombination. We observe that the OR locus limits the expression of the teto: repressing it outside of the wild type P2 zone while allowing sporadic activation from within its zone. Staged conditional expression experiments reveal that the receptor locus becomes fully repressed over time and that this repression does not require the receptor's open reading frame. Further, the exogenous promoter is inhibited by an OR transgene that similarly suppresses the endogenous receptor repertoire. Neurons in which both P2 alleles bear the teto-modified insertion show predominantly monoallelic expression, despite the genetic potential to express both. Finally, we observe that OR genes are expressed from the edge of pericentromeric heterochromatin. These data support a model of initiation of OR choice limited by non-permissive OR chromatin and maintained by repression of the non-selected OR genes.

Background: Single nucleotide polymorphisms (SNPs) may be causally related to breast cancer risk or be indirectly associated with breast cancer risk through linkage disequilibrium with a causal sequence variant. Risk-associated SNPs will have different frequencies among women with or without breast cancer and can be detected using genetic association studies. Recently, several genome-wide association studies (GWAS) have identified novel risk alleles for breast cancer including those related to FGFR2, TNRC9, MAP3K1, LSP1 genes and other locus. Replication in independent population samples is essential for validation of the results of any genome-wide association. Since the genetic variants (SNPs) are common, they are likely to be shared across different populations with diverse ancestry backgrounds. It would be of interest to determine and investigate the potential implications of these novel markers revealed by genome-wide association studies to predict the “sporadic” breast cancer risk and progression in MENA populations.

Methods: Using TaqMan® SNP genotyping assays, we characterize the variation of 9 SNPs (include rs1219648, rs2981582, rs8051542, rs12443621, rs3803662, rs889312, rs3817198, rs13387042 and rs13281615) for 520 patients with sporadic breast cancer and 360 healthy controls in the Tunisian population. The association between the genotypes and breast cancer susceptibility and tumors characteristics was estimated by computing odds ratio (OR) and 95% confidence levels from logistic regression analyses. Association of the genetic marker with the rates of breast cancer overall survival was assessed using univariate analysis.

Results: Two genetic variants in FGFR2 are significantly associated with the risk of breast cancer: rs1219648 AG/GG (OR=1.23, P=0.002) and rs2981582 AG/ AA (OR=1.33, P=0.003). Two significantly increased risks of breast cancer were respectively associated with T allele of rs8051542 in TNRC9 (OR=1.43, P=0.0003) and C allele of rs889312 in MAP3K1 (OR=1.33, P=0.006). The AG and GG genotypes of rs2981582 in FGFR2 have a significant association with a high risk of lymph node metastasis and a decreased overall survival in breast cancer patients.

Conclusion: Our results for the first time replicated the results of breast cancer GWAS in the Arabic population and indicated that some polymorphisms are associated with increased breast cancer risk and disease progress in the Tunisian population.

Background: Prevalence of metabolic syndrome is directly correlated with increasing occurrence of obesity characterized by accumulation of fat in visceral, lower body, and upper body subcutaneous depots. In a number of species, brown adipose tissue (BAT) converts triglycerides into heat by non-shivering thermogenesis, thus controlling the amount of white adipose tissue. Until recently, BAT was thought to be mostly absent in adult humans but now sizeable BAT depots have been visualized by Positron Emission Tomography (PET) and Computed Tomography (CT) scanning. Relatively little is known about the signaling pathways differentially regulated in brown adipocytes compared to white adipocytes. However, our progress of understanding is hindered by the paucity of well-characterized reliable in vitro model systems.

Objectives: The aim of this study is to characterize the only in vitro model of human BAT cell line, and to identify signature genes for BAT, and compounds implicated in the enhancement of brown phenotype.

Methods: A human BAT cell line, PAZ6, was previously created by immortalizing somatic cells using the large T antigen of Simian Virus 40. The expression of BAT associated markers has been verified by real time RT-PCR done on RNA and immunoblot using specific antibodies performed on cell lysates and supernatants, before and after differentiation and treatment with various compounds such as adrenergic receptor agonist (isoproterenol) and PPAR agonist (rosiglitazone).

Results: We have identified several BAT associated markers, including PDRM16 and the 3 adrenergic receptor. This marker is highly expressed in PAZ6 cells compared to the white adipocyte. In addition, we demonstrate that these cells share a common precursor with myocytes but not with white adipocytes. Our results show that several genes are up-regulated after PPAR gamma activation. Moreover, we confirm that these cells respond to Beta adrenergic agonist treatment. Finally, we identify that the transcription factor farnesoid X Receptor (FXR) induces the expression of BAT associated marker following treatment by its agonist : CDCA.

Conclusions: The PAZ6 cells constitute a readily available surrogate for human brown adipocytes to develop pharmacologic strategies to promote BAT expansion and activation.

The prevalence of insulin resistance and diabetes has shown a dramatic worldwide increase. In Qatar, the prevalence of diabetes is twice that of the United States today, possibly due to lifestyle and dietary changes. Diabetes has an enormously adverse impact on the afflicted population leading to patient mortality, morbidity and staggering health care costs to the nation. Untreated or inadequately treated diabetes results in serious health complications including eye, heart, kidney and nerve damage.

Mass spectrometry based molecular profiling is a powerful tool for interrogation of the underlying molecular alterations which define the etiology and patho-physiology of a complex disease such as diabetes. As a part of the QNRF funded study, we have used ultra performance liquid chromatography (UPLC) in conjunction with high resolution quadrupole time of flight mass spectrometry (Q-TOFMS) for proteomic and metabolomic profiling of bio-fluids derived from diabetic and healthy individuals recruited at the Qatar University Hopsital. The overall goal of this project was to investigate the changes in the molecular profiles in the two groups (normal and diabetic) at the protein and small molecule metabolite level and correlate these changes to define putative biomarkers with potentially diagnostic or prognostic clinical value. We have used a “smart pooling strategy” for bio-fluids from each group to enhance disease associated differences. LCMS/MS based metabolomic analysis of the diabetic and normal pre and post meal metabolome was followed by bioinformatics analysis to identify dysregulated metabolites. Additionally, we have used iTRAQ based UPLC-QTOFMS/MS for quantitative proteomic profiling.

These data were taken together for integrated functional pathway analysis to correlate the changes. Furthermore, we have used a validation cohort for targeted quantitation of candidate markers using multiple-reaction monitoring mass spectrometry. The results will be discussed in context of the state of art technology used for studying pathway perturbations resulting in a diabetic phenotype. A part of this study reported at the HUPO2010, won a “young investigator award” for Dr. Cheema. The ARF presents a unique opportunity to present results from this exciting study, deliberate upon the challenges faced and elaborate on the plans for future studies aimed at a mechanistic insight.

Background: The functional relevance of strength and power measures as an index of functional performance (i.e., sprinting speed) in soccer remains unclear.

Objective: The aim of this study was to investigate the relationships among mechanical variables related to strength and power tests and their influences on sprinting speed in professional soccer players across their life-span.

Methods: Isokinetic measures (quadriceps (Q) peak torques and power at 60°/s and 300°/s), kinetic outputs of the counter-movement jump (CMJ), and sprinting speed (the first 5 m split time (i.e., initial speed) and the 15 to 20 m split time (i.e., leading sprint) of a 20 m sprint), were measured in 224 professional soccer players (age 23.7 ± 4.4 y, body mass 71.4±8.8 kg and height 1.75±0.06 m).

Results: The values for each variable for the fast and slow groups are displayed in Table 1. All the mechanical variables were significantly different between the fast and slow players regardless of the results were ranked according to 5 m or 15–20 m times. The results of the multiple regression analyses identified CMJ height as the best predictor of sprinting performance for the 5 m (R=0.39, P=0.000) and 15–20 m (R=0.51, P=0.000) sprint.

Conclusions: All the mechanical variables could discriminate between the fast and slow players. However, a great degree of unexplained variance still remains indicating there may be better mechanical predictors of sprinting speed in soccer players.